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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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Suggested Citation:"Appendix B Workshop Papers." Institute of Medicine. 2006. Mineral Requirements for Military Personnel: Levels Needed for Cognitive and Physical Performance During Garrison Training. Washington, DC: The National Academies Press. doi: 10.17226/11610.
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B Workshop Papers Concerns About the Effects of Military Environments on Mineral Metabolism and Consequences of Marginal Deficiencies to Performance Karl E. Friedl U.S. Army Research Institute of Environmental Medicine, Natick, Massachusetts OVERVIEW Statement of the Problem Mineral requirements to sustain soldier performance in stressful conditions have been considered in military nutrition studies for many decades but rarely as the primary focus of the studies (Johnson, 1986; Sauberlich, 1984). These stud- ies, which were often underpowered, only descriptive, or not designed to address specific hypotheses in this area, raised questions that remained unresolved. The primary concern is the inability to-date to determine if there is a problem related to mineral metabolism (especially iron, calcium, zinc, and magnesium) that im- pairs health and performance in some soldiers in any field conditions. This should be considered from at least the following two perspectives: (a) does the military environment produce somewhat unique derangements or specifically change min- eral intake requirements that affect performance? and (b) are marginally defi- cient individuals (regardless of the reason for their deficiency) impaired relative to the demands of military performance? Answers to these questions are also 240

APPENDIX B 241 important as they will help the Army address persistent suggestions about using mineral supplemention for soldiers that arise from health advocates and inter- ested entrepreneurs. Military Stressors Key stressors in the military environment include thermoregulatory chal- lenges, hard work and exercise, inadequate rest and energy intakes, and psycho- logical stress. Inadequate food intake is a problem in field environments, where soldiers typically underconsume by 25 percent of their energy requirements, and it can be worsened by the loss of appetite in hot or hypoxic environments; underconsumption is also a consequence of strictly enforced body fat standards, possibly with a larger effect on service women than men because women are more likely to exceed the standards and restrict their food intake. High sweat rate and water turnover is an important feature of hot work environments. Psycho- logical stressors range from trauma (e.g., exposure to traumatic injuries and death, exposures to populations living in poverty and ruin, and feelings of help- lessness in some peacekeeping operations) and anxiety (e.g., worry about per- sonal safety and family separation), to information overload (e.g., managing complex data from multiple sources). The extent to which these exposures are manifested in various stress responses depend on the resilience of the individual as well as leadership, unit cohesion, and other stress mitigators. Some unique environments and exposures (e.g., cold, altitude, enclosed en- vironments, blast overpressure in field artillery units, noise and toxic chemicals around military vehicles and aircraft, radiofrequency radiation in communica- tions centers) may also have to be considered when recommending optimal min- eral intakes. A previous Committee on Military Nutrition Research (CMNR) concluded that none of these special environments had been adequately charac- terized as producing higher oxidative stress burdens to soldiers than that for the healthy active U.S. population (IOM, 1999). Health and Performance Outcomes Soldiers are likely to be involved in demanding physical tasks that require strength and endurance; most physical tasks require lifting and carrying heavy loads. There are also significant mental demands that come with increased speed, complexity, and lethality of modern warfare; in this regard, every individual soldier may be called upon for rapid decision making and judgment calls, may require fine psychomotor performance (e.g., marksmanship), spatial mapping ability, pattern recognition, etc. Mood and motivation are important underlying aspects of soldier mental performance at all times. Even momentary lapses in mental and physical performance may have catastrophic effects in today's mili- tary environment.

242 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Practical tests that adequately reflect militarily-relevant performance have been elusive but various test paradigms have provided useful measures in mili- tary studies, such as repeated box lifts (strength endurance), simulated sentry duty (vigilance, judgment, psychomotor performance), and simulated mission control center (multiple cognitive domains). The Army is increasingly turning to realistic training simulators that include electronic "combat" games and new markmanship trainers; these systems can also be converted to research tools that unobtrusively assess performance. In addition to cognition abilities, a fully responsive immune system is criti- cal to maintain the health and optimal health needed to face the demands of military lifestyle. Regardless of how good our vaccines may be, soldiers are not likely to be protected against all the endemic and deliberate infectious threats that may determine success or failure of a mission, nor will they have equipment and drugs to fully prevent inflammatory responses to physical demands and other physical and chemical threats. This highlights one more broad outcome of interest to the Army, optimizing a soldier's resistance to disease and injury. Some of the host defense systems that determine immunological and inflamma- tory responses appear to be importantly affected by mineral status. RANGER TRAINING AS A STRESS MODEL In the early 1990s, the Army had concerns about the high prevalence of infectious illnesses (pneumococcal pneumonia and soft tissue infections such as cellulitis) occurring in healthy young men undergoing 65 days of Ranger train- ing with multiple stressors, including extreme food and sleep restriction and hot humid conditions. This concern led to a request to determine if we could just provide "an iron pill or a vitamin" to prevent soldiers from getting sick while subjected to the same high level of stressful training. Initially we conducted a descriptive study to quantify the stressors (1,000 kcal/day energy deficits; 3.6 hr/ day sleep) and their effects; this was followed by a study that increased feeding by only 400 kcal/day, which was associated with marked improvement in im- mune function and reduction in infection rates (Kramer et al., 1997). Most of the men lost all of their fat reserves and the main adverse outcomes due to the semistarvation were in the cognitive and immune function (Friedl, 2003; Friedl et al., 2001). In addition, soldiers were hyperphagic and had sleep disturbances for several weeks after the course but were fully recovered six months later. During the initial planning of these experiments, we worried repeatedly about what would happen to iron and hematological parameters in association with the extreme privation. It was assumed that at some point hemoglobin and hematocrit would become low enough that it would not be safe or ethical to continue to draw blood from the test volunteers. This never happened and the significant changes in iron status that were observed occurred in the first few

APPENDIX B 243 weeks of the course and corrected by the end of the course (Figure B-1). The observed changes were ascribed to acute phase responses, with no changes in iron, zinc, or copper status between baseline and the end of the course (Moore et al., 1993; Shippee et al., 1993). One possible explanation for the absence of a progressive decline in iron status is that periodic re-feeding that occurred at the end of each two week phase through the four training phases of this course replenished any deficiencies (sampling was done at the end of each phase of restriction and before the re-feeding began). The loss of muscle mass in this study would have also provided a steady supply of minerals and nutrients into the circulation. These findings matched those in the 1973 biomedical studies of Ranger training, with comparable weight loss and also no significant changes in hematological parameters and iron status across the training period. Thus, we did not detect overt mineral deficiencies in one of the most stressful models we could ethically study. However, only iron was targeted for study a priori and periodic re-feeding provided a less challenging intermittent replenishment. MILITARY QUESTIONS Is There a Problem (#1)?: Stress of Initial Entry Training and Iron Status in Young Women Iron became a subject of interest after a 1979 study identified deficiencies in female West Point cadets; this was observed again in the decennial 1989 study, despite the high prevalence of mineral and vitamin supplement use (Friedl et al., 1991; Kretsch et al., 1986). The observation was attributed to "just part of the stress of West Point," rather than being considered a medical concern, a treatable condition, or an important performance issue. However, in 1993, a comprehen- sive study of womens' health and performance in the last gender-segregated Army basic training class also suggested a high incidence of iron deficiency anemia compared to the U.S. population (Westphal et al., 1995). We noted inad- equate intakes of several minerals compared to established RDAs, a slight wors- ening of iron status through the course, and a correspondence between poor iron status and physical fitness test run time (Westphal et al., 1995). Several subse- quent studies attempted to followup on these findings as part of the 1994 De- fense Women's Health Research Program (DWHRP). One followed a select population of new female officers, comparing iron and hematological status to treadmill measured aerobic performance at the beginning and end of their officer basic course. This group was uniformly well nourished and fit, and offered no significant correlation between measures of iron and hematological status and aerobic performance (Cline et al., 1998); this sample was probably not represen- tative of the majority of female soldiers. The second study was a cross sectional examination of three populations of female soldiers, with iron and hematological

244 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Hemoglobin Hematocrit 160 40 140 35 120 (g/L) 100 30 80 25 Hemoglobin 60 40 20 Baseline wk 2 wk 4 wk 6 wk 8 Baseline wk 2 wk 4 wk 6 wk 8 Ferritin Iron 240 14 12 190 10 (ug/L) 8 140 (umol/L) 6 Ferritin Iron 90 4 2 40 0 Baseline wk 2 wk 4 wk 6 wk 8 Baseline wk 2 wk 4 wk 6 wk 8 FIGURE B-1 Changes in iron status indicators status during Ranger training under severe food restriction and other stressors. SOURCE: Moore et al. (1993). B-1 new data that suggested a worsening status through their initial training (IOM, 1995). The cross sectional design presented a significant weakness in this study, and because of different locations, feeding regimens, and stressors, it was difficult to draw conclusions about the nature of this apparent decline in health status. A clinical study investigated the prevalence of iron deficiency in women referred to a military gastroenterology clinic, concluding that the majority of asymptom- atic iron deficiency anemia cases, including several already diagnosed by spe- cialists as having excess blood loss related to menstrual flow, actually had a high prevalence of significant endoscopy findings (Kepzyk et al., 1999). A fourth study funded in the DWHRP examined the benefits to neurocognitive perfor- mance from zinc and iron repletion in marginal deficiency. In preliminary analy- ses, many cognitive and psychometric tests were apparently improved (Sandstead, 2001); however, the results have not yet been reported.

APPENDIX B 245 Is There a Problem (#2)?: Calcium Requirements and Bone Health Calcium intakes have been given more attention in military studies in the past decade because of research initiatives on prevention of stress fractures, particularly in female recruits. As part of the DHWRP, the CMNR convened a special review of women's body composition, nutrition and health which in- cluded a major review of stress fractures in the military (IOM, 1998). The subse- quent Bone Health and Military Medical Readiness (BHMMR) research pro- gram was shaped by the recommendations from this review and from the DRI report on calcium, vitamin D, and related nutrients (IOM, 1997). One important DWHRP project demonstrated that energy deficit, not in- tense exercise, was a key determinant of menstrual disturbances (Loucks and Thuma, 2003), refuting earlier concepts of a "female athlete triad" of intense exercise, menstrual abnormalities and osteoporosis. This was consistent with findings that high functioning women, such as Olympic athletes, exercising in- tensively but not restricting their diet had normal rates of oligomenorrhea. As a follow up effort supported by the BHMMR, experimental manipulations of the energy deficit that exceeded thresholds and caused alterations in LH (luteinized hormone) pulsatility, produced changes in bone mineral metabolism consistent with demineralization (Ihle and Loucks, 2004). Another important study, currently underway, is testing the hypothesis that 2,000 mg of calcium along with 800 IU of Vitamin D can substantially reduce the acute occurrence of stress fracture in a study of 5,200 young women during eight weeks of recruit training at the Great Lakes Naval Base. Part of the hypoth- esis and assumptions are that young women still have not attained peak bone mass, recruit training stimulates new bone formation, calcium intakes are nor- mally low in this population, and substantial sweat calcium losses occur in this training (Lappe, 2003). Special populations such as submariners living for extended periods in closed environments and away from sunlight have reduced plasma levels of 25-(OH) Vitamin D and special challenges in calcium metabolism (Sack et al., 1986). Two studies in progress in the BHMMR program are defining Vitamin D needs in relation to ethnicity and skin pigmentation, following up on a research gap identified in the review of calcium, vitamin D, and related nutrients (IOM, 1997). Are We Missing a Performance Enhancing Benefit (Or Are Some Supplements a Health or Performance Risk)? Some military studies have raised questions about the effects of stressors on mineral requirements and/or effects of supplementation on mitigation of stress responses. For example, as part of an important series of starvation and limited intake studies to determine minimum requirements for 10-day patrols, the 1968

246 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Panama study measured intakes and losses for sodium, potassium, calcium, and magnesium, including sweat and urine excretion rates (Consolazio et al., 1979). The authors concluded that magnesium intakes were deficient but there was no biomedical evidence of a decline in magnesium levels. In a study at the Uni- formed Services University of Health Sciences (USUHS), magnesium balance in anaerobic exercise was considered. Another study tested specific performance benefits of zinc combined with Vitamin E on exhaustive running in women, based on hypothesized antioxidant actions, but found no effect of acute dosing (Singh et al., 1999). However, there has been no concerted effort through a carefully planned program of studies to determine benefits and risks of mineral supplements in military populations. Most service members report using supplements of some kind; this is cur- rently being resurveyed to improve estimates of specific supplement use. Young soldiers are at high risk for the use of supplements with putative performance enhancement because of the ready availability of these products in military com- missaries and stores, and because of perceptions that military training and opera- tional demands somehow drive a higher intake requirement, as well as the strate- gically seeded suggestions of performance enhancement in popular fitness magazines and the prevalent belief that such claims could not be made if they were not true (Friedl et al., 1992). A potentially important question that has never been addressed in military research studies is whether or not higher than usual intakes of certain supplements, including minerals, produce a deficiency state from withdrawal that is likely to occur when soldiers leave them behind for a field exercise or operational deployment. Another concern would be individual and population bases of upper limits of intakes, such as the obvious problem of iron overload if supplements are provided across military groups, for men and women or to all women regardless of their iron status. SUMMARY Constant discoveries of new roles of minerals in integrated physiological processes, where a mineral deficiency may have far reaching consequences on stress responses and susceptibility to disease makes this an important and rel- evant line of inquiry for military medical research. The key question that has never been properly addressed in military or other relevant studies is "can we achieve a sizeable improvement in mental or physical functioning, especially in stressful operational or training conditions, by improving the mineral status of young men and women in the military?" This area of investigation has been hindered by the absence of (1) practical and validated tools to definitely assess outcomes including neuropsychological outcomes and changes in disease sus- ceptibility, (2) adequate indices of mineral status, not confused by stress states including acute phase responses, and (3) clear indications of health and perfor- mance deficiencies.

APPENDIX B 247 Research conducted under the special performance demands and conditions in which soldiers have to operate will not be addressed elsewhere and needs to be conducted by the Army. In fact, there is no other federal agency with a primary focus on biomedical aspects of performance; more typically the research is focused purely on health outcomes. This work is primarily centered at the U.S. Army Research Institute of Environmental Medicine in the Military Nutrition Division in collaboration with the Pennington Biomedical Research Center and with scientific guidance from the Institute of Medicine's CMNR. The key question(s) can also be framed by a pragmatic question: Should the Department of Defense consider the addition of a mineral supplement pack in every ration with instructions on use for "health and performance optimization," or perhaps other strategies on the use of whole foods? DISCLAIMER The opinions and assertions in this paper are those of the authors and do not necessarily reflect the official views of the Department of the Army. REFERENCES Cline AD, Patton JF, Tharion WJ. 1998. Assessment of the relationship between iron status, dietary intake, performance, and mood state of female Army officers in a basic training population. Technical Report T98-24, September 1998. AD A351 973. Natick, MA: U.S. Army Research Institute of Environmental Medicine. Consolazio CF, Johnson HL, Nelson R. 1979. The relationship of diet to the performance of the combat soldier. Minimal calorie intake during combat patrols in a hot humid environment (Panama). Letterman Army Institute of Research Report No. 76. October 1979. Friedl KE. 2003. Military nutritional immunology. In: Hughes DA, Darlington LG, Bendich A, eds. Diet and Human Immune Function. Totowa, NJ: Humana Press, Inc. Pp. 381­396. Friedl KE, Marchitelli LJ, Sherman DE, Tulley R. 1991. Nutritional Assessment of Cadets at the U.S. Military Academy: Part 1. Anthropometric and Biochemical Measures. Technical Report No. T4-91, November 1990. ADA231918. Natick, MA: U.S. Army Research Institute of Envi- ronmental Medicine. Friedl KE, Moore LJ, Marchitelli LJ. 1992. Physiology of nutritional supplements. "Steroid replac- ers": Let the athlete beware! Natl Strength Conditioning Assoc J 14:14­19. Friedl KE, Mays MZ, Kramer TR, Shippee RL. 2001. Acute recovery of physiological and cognitive function in U.S. Army Ranger students in a multistressor field environment. In: The Effect of Prolonged Military Activities in Man--Physiological and Biochemical Changes--Possi- ble Means of Rapid Recuperation. Technical Report RTO-MP-042. Neuilly-sur-Seine Cedex, France: North Atlantic Treaty Organization. Ihle R, Loucks AB. 2004. Dose-response relationships between energy availability and bone turn- over in young exercising women. J Bone Miner Res 19:1231­1240. IOM (Institute of Medicine). 1995. A Review of Issues Related to Iron Status in Women during U.S. Army Basic Combat Training. Letter Report to BG Russ Zajtchuk, December 19, 1995. IOM. 1997. Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluo- ride. Washington, DC: National Academy Press.

248 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL IOM. 1998. Reducing Stress Fracture in Physically Active Military Women. Washington, DC: Na- tional Academy Press. IOM. 1999. Letter Report to the Office of the Surgeon General United States Army on Antioxidants and Oxidative Stress in Military Personnel. Washington, DC: Institute of Medicine. Johnson HL. 1986. Practical military implications of fluid and nutritional imbalances for perfor- mance. In: Predicting Decrements in Military Performance Due to Inadequate Nutrition. Wash- ington DC: National Academy Press. Pp. 55­68. Kepczyk T, Cremins JE, Long BD, Bachinski MB, Smith LR, McNally PR. 1999. A prospective, multidisciplinary evaluation of premenopausal women with iron deficiency anemia. Am J Gastroenterol 94:109­115. Kramer TR, Moore RJ, Shippee RL, Friedl KE, Martinez-Lopez L, Chan MM, Askew EW (1997). Effects of food restriction in military training on T-lymphocyte responses. Int J Sportsmed 18:S84­S90. Kretsch MJ, Conforti PM, Sauberlich HE. 1986. Nutrient intake evaluation of male and female cadets at the United States Military Academy, West Point, New York. Technical Report LAIR-218. April 1986. ADA168120. San Francisco, CA: Letterman Army Institute of Research. Lappe JM. 2003. Efficacy of calcium and Vitamin D supplementation for the prevention of stress fracture in female Naval recruits. Annual Report. Grant # DAMD17-01-1-0807. ADA419678. October 2003. Omaha, NE: Creighton University. Loucks AB, Thuma JR. 2003. Luteinizing hormone pulsatility is disrupted at a threshold of energy availability in regularly menstruating women. J Clin Endocr Metab 88:297­311. Moore RJ, KE Friedl, RT Tulley, EW Askew. 1993. Maintenance of iron status in healthy men during an extended period of stress and physical activity. Am J Clin Nutr 58:923­927. Sack DM, Holick M, Bondi KR. 1986. Calcium and Vitamin D metabolism in submariners--carbon dioxide, sunlight, and absorption considerations. Technical Report NSMRL-1037. January 1986. ADA166292. Groton, CT: Naval Submarine Medical Research Laboratory. Sandstead HH. 2001. Repletion of zinc and iron deficiencies improve cognition of premenopausal women. Final Report. Grant DAMD17-95-C-5112. December 2001. Galveston, TX: University of Texas Medical Branch. Sauberlich HE. 1984. Implications of nutritional status on human biochemistry, physiology and health. Clin Biochem 17:132­142. Shippee RL, Friedl KE, Tulley R, Christensen E, Arsenault J. 1993. Changes in plasma iron, copper, and zinc concentrations of young males during 8 weeks of extreme physiological and psycho- logical stress. J Am Coll Nutr 12:614 (abstract). Singh A, Papanicolaou DA, Lawrence LL, Howel EA, Chrousos GP, Deuster PA. 1999. Neuroendo- crine responses to running in women after zinc and vitamin E supplementation. Med Sci Sports Exerc 31:536­542. Westphal KA, Friedl KE, Sharp MA, King N, Kramer TR, Reynolds KL, Marchitelli LJ. 1995. Health, performance, and nutritional status of U.S. Army women during basic combat training. Technical Report No. T96-2. May 1995. ADA302042. Natick, MA: U.S. Army Research Insti- tute of Environmental Medicine.

APPENDIX B 249 Derivation of the Military Dietary Reference Intakes and the Mineral Content of Military Rations Carol J. Baker-Fulco U.S. Army Research Institute of Environmental Medicine, Natick, Massachusetts This paper presents the nutritional standards for military rations and the mineral composition of military field rations. In addition, it summarizes the findings of a few studies that have estimated mineral intakes of soldiers in the field and in garrison. This paper focuses on operational rations, which are rations intended for military operations, whether combat or field training. A ration is one day's food supply for a group or an individual. The type of ration provided is based upon the unit's mission, tactical scenario, location, and availability of food service equipment and personnel. The operational rations examined in this paper are the Meal, Ready-to-Eat (MRE); Meal, Cold Weather (MCW); one of the Unitized Group Rations (UGR), the Heat-and-Serve (H&S); and the First Strike Ration (FSR). DESCRIPTIONS OF OPERATIONAL RATION The MRE is the standard individual operational ration and consists of heat- processed entrees and other food components that require no preparation. Each meal contains an entrée/starch, crackers, a spread (cheese, peanut butter, jam or jelly), a dessert or snack, beverages, and an accessory packet that contains cof- fee, tea or cider and condiments. The MRE is issued at three menu bags per day for a complete ration with an average of 3,900 kcal. For variety, there are twenty- four different meal menus in the inventory. The MCW is a higher calorie ration intended for arctic feeding. This ration contains freeze-dried, cooked entrees and other low-moisture foods that will not freeze. Meal bags for each of the twelve menus contain the entrée and a variety of spreads and crackers, cookies, sports bars, nuts, candy, and powdered drink mixes. The MCW is issued at three menu bags per day for a provision of roughly 4,500 kcal. The MCW menus are identical to those of the Food Packet, Long Range Patrol (LRP) which is a restricted ration issued as one menu bag per day during special operations when weight and volume of the ration are critical factors. Another restricted calorie ration considered in this report is the FSR. The FSR is a new, individual combat ration developed for forward deployed ground forces engaged in high-intensity operations. This ration is smaller and lighter than a full day's ration of MREs and is comprised of foods that can be eaten "on the move." The FSR as currently configured provides about 2,800 kcal.

250 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL The UGR is a group of rations in which all the components for 50 complete meals are packaged as one unit to streamline the ordering and delivery process. There are seven breakfast and fourteen lunch and dinner menus for each type of UGR; however, the lunch meal is often a MRE. The UGR-Heat & Serve (UGR- H&S) is comprised of shelf-stable, ready-to-eat entrees, starches, vegetables, and desserts packaged in short, rectangular plastic trays. It is the more common (hot) group feeding ration for the field and is used when neither cooking nor refrigeration are possible. Each meal, including the mandatory supplement of milk, provides an average of 1,400 kcal. UGR menus may be enhanced with cold breakfast cereal, bread, fresh fruits and salad. There are other UGR options that comprise perishable and frozen ingredients (UGR-A) or are cook-prepared from canned and dehydrated foods (UGR-B), but these will not be discussed in this paper. MILITARY DIETARY AND RATION STANDARDS Nutritional standards for operational and restricted rations (NSOR), i.e., what the rations must contain, are presented in the tri-service regulation, Nutritional Standards and Education, which for the Army is Army Regulation (AR) 40-25 (U.S. Departments of the Army, Navy, and Air Force, 2001). The NSORs are based on the Military Dietary Reference Intakes (MDRI) presented in the same regulation (see Table B-1). The MDRIs are, in turn, based on the Dietary Refer- ence Intakes (DRIs) (IOM, 1997, 1998, 2000a) or earlier Recommended Dietary Allowances (RDA) (National Research Council, 1989). Since the current regula- tion was prepared prior to the 2001 DRI publication (IOM, 2001)--which up- dated the RDAs for iron, iodine and zinc, and established DRIs for chromium, copper, manganese and molybdenum--current MDRIs are based on the 1989 RDAs for iron, iodine, and zinc, and there are no MDRIs for chromium, copper, manganese and molybdenum. A soon to be drafted change to the regulation will update the MDRIs and include additional MDRIs based on the more recent IOM publications. The MDRIs are applicable to healthy, 17 to 50 year old, physically active military men and non-lactating, non-pregnant women. This age range covers the majority of military personnel on active or reserve duty. The 17 to 50 year age range incorporates three of the age classes for which DRIs have been set (14 to 18, 19 to 30, and 31 to 50 years) and three of the age classes used in the 10th edition of the RDA (15 to 18, 19 to 24, and 25 to 50 years). For most nutrients, the MDRI is the highest gender-specific reference value or RDA. However, the MDRIs for calcium, phosphorus, and iron for males and calcium, phosphorus, and magnesium for females, are not based on the highest reference intake or allowance, which is for 14 to 18 year old individuals. Only 2­3 percent of the military population is 17 to 18 years old; thus, inflating the MDRIs to meet the needs of relatively so few individuals is not warranted. The regulation advises

APPENDIX B 251 TABLE B-1 Military Nutritional Allowances and Ration Standards for Minerals MDRIs NSORs Nutrient Unit Men Women Operational Restricted Energy: Light activity kcal 3,000 2,200 Moderate activity kcal 3,250 2,300 Heavy activity kcal 3,950 2,700 3,600 1,500 Exceptionally-heavy kcal 4,600 3,150 Calcium mg 1,000 1,000 1,000 500 Fluoride mg 4.0 3.1 4.0 2.0 Iodine µg 150 150 150 75 Iron mg 10 15 15 8 Magnesium mg 420 320 420 210 Phosphorus mg 700 700 700 350 Potassium mg 3,200 2,500 3,200 2,000 Selenium µg 55 55 55 28 Sodium* mg 5,000 3,600 5,000­7,000 2,500­3,500 Zinc mg 15 12 15 8 NOTE: MDRI = Military Dietary Reference Intake; NSOR = Nutritional Standard for Operational Rations. *Sodium recommendations are based on 1,400­1,700 milligrams of sodium per 1,000 kcal of energy consumed. The MDRI values in the table represent the midpoints of the ranges calculated using energy intakes for moderate activity of 3,250 kcal for men and 2,300 kcal for women. SOURCE: U.S. Departments of the Army, Navy, and Air Force (2001). persons planning menus or diets for groups with a large proportion of 17 to 18 year olds to consider the higher requirements of that age group. The military population in general tends to be a little heavier and more active then the Ameri- can population, thus the military energy allowances are higher than the DRI values. In addition to prescribing the dietary allowances and standards for opera- tional rations (full rations and restricted rations), AR 40-25 prescribes the length of time that certain rations can be used as a sole source of nutrition. The MRE can be fed for up to 21 days, while restricted rations are limited to 10 days. There are nutritional standards for nutritionally complete and restricted operational rations. The full ration standards are based directly on the MDRIs. For most nutrients, the ration standard is the higher of the two gender-specific MDRIs. The energy standard is based on the estimated average energy require- ment for moderate to heavy physical activity, to provide for the extended work- days and high levels of activity typical of operational settings (combat or train- ing). Although there are groups that will have energy requirements much higher than 3,600 kcal, this is a practical standard, given that many warfighters have

252 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL lower energy needs and that there is less time or inclination to eat as the intensity and duration of activity increase. The NSORs are minimum content standards at the time of consumption except for sodium. The MDRIs and NSORs for sodium are much higher than the DRIs (1,500 mg) (IOM, 2005) or other dietary guidelines for the general popula- tion because of the potential for military personnel to sustain high sodium losses in sweat in hot environments or strenuous activity (CMNR, 1991). Restricted rations are used when operational conditions and missions re- quire troops to subsist for short periods on nutritionally-inadequate rations. Such scenarios include long-range patrol, assault and reconnaissance, and other situa- tions where resupply is tactically unfeasible. The restricted ration standards for most nutrients are set at 50 percent of the respective standards for operational rations, rounding up to the same number of digits used to express the NSOR. This convention is mainly based on the work of Consolazio and colleagues from the 1960s that suggests that nutrient intakes at 50 percent of RDA levels for no more than 10 days do not measurably affect health or performance (Consolazio, 1976). Intakes at these levels maintain bodily functions and prevent rapid deple- tion of body stores. MINERAL CONTENT OF OPERATIONAL RATIONS Calculated Menu Data The ration composition data presented here are from the Combat Feeding Directorate, Natick (i.e., the ration developers) and derive from a variety of sources. Most of the data are values calculated using Genesis R&D nutrition and labeling software and database (ESHA Research, Salem, OR). The ration devel- opers in Combat Feeding calculate the nutrient composition of their prototype formulations, while the contracted manufacturers calculate the nutrient content of their products according to their, often-different, proprietary formulations. Chemically analyzed data are mostly limited to macronutrients or select nutri- ents (such as sodium) that may be stipulated in the procurement contracts. Data for the commercial items in the ration are taken from the USDA Survey Nutrient Database as maintained in the Genesis or Food Processor applications or the USDA National Nutrient Database for Standard Reference. Ration composition data for some minerals (e.g., copper or selenium) are missing or very incomplete and do not provide reasonable estimates of ration provisions and, thus, are not presented here. The data presented in Table B-2 and Table B-3 are estimated average values of all menus in a particular ration system and do not reflect the variability due to different manufacturers or to ingredient sources and growing conditions. There are currently 24 menus in the MRE line, 12 menus in the MCW, and 7 breakfast menus and 14 lunch and dinner menus in the UGR. For the MRE, the values are averages of the three contracted ration assemblers which are also the manufacturers of the major ration components.

APPENDIX B 253 TABLE B-2 Calculated Mean Mineral Content of Meals in Meal-Based Rations Ca I* Fe Mg P K Na* Se Zn (mg) (µg) (mg) (mg) (mg) (mg) (mg) (µg) (mg) 1 /3 NSOR 333 50 5 140 233 1,067 <2,333 18.3 5 MRE XXII 511 -- 7.9 114 643 1,048 2,046 10.1 4.2 MRE XXIII 527 -- 8.6 130 646 1,027 2,051 11.9 4.2 MRE XXIV 557 -- 9.0 141 691 1,084 2,181 12.5 4.7 MCW 475 62 10.9 201 -- 1,666 3,078 53.2 6.1 UGR B'fast 467 49 7.2 111 456 1,747 2,580 9.2 6.9 UGR Dinner 472 -- 9.7 190 590 1,873 2,465 16.0 7.6 NOTE: The UGR menu data is for the 2003 production year. Ca = Calcium; I = Iodine; Fe = Iron; Mg = Magnesium; P = Phosphorus; K = Potassium; Na = Sodium; Se = Selenium; Zn = Zinc. NSOR = Nutritional Standard for Operational Rations; MRE = Meal, Ready-to-Eat; MCW = Meal, Cold Weather; UGR = Unitized Group Ration; B'fast = Breakfast. *Values for iodine and sodium do not include content of salt packet. Table B-2 presents mineral contents of average meals in current meal-based rations to include the past three production years of MREs, the MCW, and UGR menus for 2003. The UGRs are typically provided as a hot breakfast and a hot dinner with a MRE for "lunch." The first row of the table presents the ration standards for the corresponding minerals divided by three as comparison figures for a meal. Although calcium was a nutrient of concern in earlier versions of field ra- tions, it is not so in current rations. The levels of calcium, as of phosphorus, TABLE B-3 Mean Mineral Content of First Strike Ration Compared to Standards Ca I* Fe Mg P K Na* Se Zn (mg) (µg) (mg) (mg) (mg) (mg) (mg) (µg) (mg) NSOR 100 150 15 420 700 3,200 >5,000 55 5 Restricted 500 75 8 210 350 2,000 >2,500 28 8 Ration FSR 679 53 17.4 393 1,167 2,356 4,244 115 12.1 NOTE: Ca = Calcium; I = Iodine; Fe = Iron; Mg = Magnesium; P = Phosphorus; K = Potassium; Na = Sodium; Se = Selenium; Zn = Zinc. NSOR = Nutritional Standard for Operational Rations; FSR = First Strike Ration *Values for iodine and sodium do not include content of salt packet.

254 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL surpass the requirement by substantial amounts. The mineral content (especially calcium) of the UGR menus reflects the contribution of the mandatory milk at both meals but not the optional dry cereal or bread. The wheat snack bread, crackers, cheese spreads, and dairy shakes in the MRE each provide more than 150 mg calcium per serving. The iron content of all meals is relatively high, yielding nutrient densities of 6.9 mg/1,000 kcal, 7.1 mg/1,000 kcal, and 7.5 mg/ 1,000 kcal for MRE XXIV, MCW, and UGR dinner, respectively. The iodine values do not include iodine provided by the salt packet. The salt in the acces- sory pack is required to be iodized; however, salt used as an ingredient in ration components is specifically not iodized due to concerns about taste and accept- ability. Table B-2 shows that the magnesium content of MREs has increased and now meets the NSOR. This improvement is due to the inclusion of more bean and nut items. The apparently low magnesium content (compared to /3 NSOR) 1 of the UGR breakfast would be offset by the UGR dinner plus a MRE. The potassium level of the MRE is just meeting the NSOR while the sodium content of the MRE is about twice that of potassium, still within the range of the NSOR. The average UGR meals contain much higher levels of potassium and sodium than MREs. The sodium content of both the estimated average UGR breakfast and dinner exceeds the maximum sodium content standard (see Table B-2). Much of the sodium is contained in the entrées. In the MRE, 39 percent of the sodium comes from the entrée, while 44 percent of the sodium in the UGR dinner is in the entrée. Selenium in the MREs appears to be lower than the NSOR (Table B-2), but this is mostly because there is no selenium data for many MRE components. The selenium data for the MCW and UGR are much more complete and show that these rations would be expected to provide ample amounts of this mineral. The very generous content of selenium in the MCW is from the egg dishes that are present in 3 of the 12 menus. The zinc content of the MRE has improved but remains below the NSOR (see Table B-2). The calculated zinc content of MRE XXIV is at 94 percent of the NSOR, while the zinc level of the MCW is 122 percent and that of the UGR is 168 percent of the NSOR. The zinc provision of rations is predominately from the beef containing entrées, which in the MRE are in 8 to 9 of the 24 menus. If beef-based entrées were removed from the MRE, the average for the menu line would be closer to three and a half milligrams of zinc per meal. The MCW is relatively high in sodium and potassium (as well as protein), compared to the NSOR and its predecessor, the Ration, Cold Weather (RCW), which was specifically designed to contain modest amounts of these nutrients to moderate water requirements. The mineral content of the MCW is so generous because this ration is composed of three restricted rations, the LRP. As the NSORs for restricted rations for most nutrients are half the standards for full operational rations, three LRP ration packets per day provide minerals well above the full ration standards.

APPENDIX B 255 TABLE B-4 Mineral Content of Additional Ration Components Ca I Fe Mg P K Se Na Zn Amt (mg) (µg) (mg) (mg) (mg) (mg) (µg) (mg) (mg) MRE Salt 4g 0.96 40 0 0.08 0 0.32 -- 1,550 0 Pouch Bread 51g 75 0 1.64 13 53 67 -- 593 0.38 NOTE: Ca = Calcium; I = Iodine; Fe = Iron; Mg = Magnesium; P = Phosphorus; K = Potassium; Na = Sodium; Se = Selenium; Zn = Zinc. MRE = Meal, Ready-to-Eat. Table B-3 shows the estimated levels of minerals in the First Strike Ration in comparison to the nutritional standards for full operational rations as well as the standards for restricted rations. Although the First Strike Ration is technically a restricted calorie ration because it provides less than 3600 kcal, as currently formu- lated it provides 2800 to 2900 kcal. Because this ration is composed of mostly nutrient-dense foods, it greatly exceeds the nutritional standards for restricted ra- tion and, in many cases, meets the standards for full operational rations. The ration standards for and the ration contents of sodium presented do not include sodium provided in salt packets. There is a salt packet in the accessory packet of every MRE, FSR, and LRP menu. The mineral composition of this individual salt packet is shown in Table B-4. The packet contains four grams of salt; in contrast, most commercial salt packets contain less than one gram of salt. The large salt packet provides for the preparation of antiseptic salt water solu- tions and for high sodium intakes during periods of heat acclimation, if needed. Field study data indicate that consumption of the salt packet is limited. Although no longer a mandatory supplement, bread is a frequently provided enhancement to the UGR. Table B-4 also shows the estimated mineral contents of shelf-stable white bread which is called pouch bread. Other UGR enhance- ments (data not shown) are assorted, bowl-pack cold cereals at breakfast (poten- tially providing significant amounts of copper, iron, magnesium, and zinc) and fresh fruit and salad at dinner (significant sources of iron, magnesium, potassium and other trace elements). When dry cereal is served, an additional /2 pint of 1 milk must be provided, adding significant quantities of calcium and phosphorus as well as magnesium, potassium, and zinc to the menu. The milk may be ultra- high temperature (UHT), soy, or fresh. Laboratory Analyses of Ration Components The data discussed in the previous section were calculated values provided by the Combat Feeding Directorate. In order to assess whether those values are over- or under-estimates of actual content, we had select components of MRE

256 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL XXIII chemically analyzed. We purchased MREs from each of the three assem- blers and sent 32 items (18 of the 24 entrées plus 14 side dishes and snacks--see Table B-5 for list) to a food analysis laboratory (Woodson Tenet Laboratories Division, Eurofins Scientific Inc, Des Moines, IA). The selection of ration items to analyze was based on consideration of the quality of existing data, the popu- larity of the item, the number of times the item appears in the entire menu line, and an expected significant mineral composition. In addition, we did not analyze components that were slated to be dropped from the MRE menu line within the next two procurement cycles. The analyses included proximates (data not shown) and the minerals calcium, iron, magnesium, phosphorus, potassium, and zinc. The selection of minerals to assay was limited to nutrients for which Combat Feeding was compiling data. Thus, we did not assay copper because Combat Feed- ing does not report values for this nutrient. Although Combat Feeding has started to request selenium data, we did not assay selenium because we would have had to analyze almost all menu components to fill the gaps in missing data. Figures B-2 to B-7 present the comparison of the lab values with the calcu- lated or estimated values as per Combat Feeding. For calcium (Figure B-2), although there are some significant differences for individual products, on aver- age there is no significant difference between the lab data and Combat Feeding data. The average of the analytical values for iron content was significantly less (28 percent less) than the average of the estimated values (Figure B-3). This was unexpected given that iron is one of the nutrients required on nutrition facts labels. Per U.S. food labeling laws, nutrients such as minerals that are added as TABLE B-5 MRE Components Chemically Analyzed Entrées Side Dishes and Snacks BBQ Pork Rib Western Beans Beef Enchiladas Beef Snack Strips Beef Patty Minestrone Stew Beef Ravioli Cheese Spread (2 flavors) Beef Stew Dairy Shake (3 flavors) Beefsteak w/ Mushrooms Mashed Potatoes Meat Loaf w/ Gravy Crackers (2 flavors) Roast Beef Clam Chowder Chicken w/ Salsa Wheat Snack Bread Chili and Macaroni Pound Cake (composite of flavors) Grilled Chicken Breast Manicotti w/ Vegetables Spaghetti w/ Meat Sauce Jambalaya Chicken w/ Cavatelli Cheese Tortellini Chicken Tetrazzini

APPENDIX B 257 CALCIUM Entrees Entrées -100 -50 0 50 -50% 0% 50% 100% 150% Side &&Snacks Side Snacks -50 0 50 100 -200% 0% 200% 400% 600% Absolute Difference, mg Percent Difference P = 0.5 FIGURE B-2 Differences between analytical values and calculated values for the cal- cium content of 18 entrees and 14 side dishes or snacks in MREs. Each bar represents an individual ration component. The two panels on the left show absolute differences; the two right panels show percent differences. Values negative if calculated > analytical; values positive if analytical > calculated. NOTE: Axes are not to same scale. IRON Entrees Entrées -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 -75% -50% -25% 0% 25% 50% 75% Side &&Snacks Side Snacks -75% -25% 25% 75% 125% 175% 225% 275% -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 Absolute Difference, mg Percent Difference P = 0.043 FIGURE B-3 Differences between analytical values and calculated values for the iron content of 18 entrees and 14 side dishes or snacks in MREs. Each bar represents an individual ration component. The two panels on the left show absolute differences; the two right panels show percent differences. Values negative if calculated > analytical; values positive if analytical > calculated. NOTE: Axes are not to same scale.

258 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL MAGNESIUM Entrees Entrées -60 -40 -20 0 20 40 60 -100% 100% 300% 500% 700% Side &&Snacks Side Snacks 1869% -60 -40 -20 0 20 40 60 -100% 0% 100% 200% 300% 400% 500% Absolute Difference, mg Percent Difference P = 0.13 FIGURE B-4 Differences between analytical values and calculated values for the mag- nesium content of 18 entrees and 14 side dishes or snacks in MREs. Each bar represents an individual ration component. The two panels on the left show absolute differences; the two right panels show percent differences. Values negative if calculated > analytical; values positive if analytical > calculated. NOTE: Axes are not to same scale. enrichments or fortificants must be present at levels at least equal to the value declared on the label. For naturally occurring minerals, except sodium, the actual nutrient content must be at least equal to 80 percent of the value declared. There- fore, we would expect the ration manufacturers to provide iron content values that support their label declarations (which are usually minimum content claims) and, thus, find the actual contents to be greater. Given that the calculated iron content of MRE menus is quite generous (7.9­9.0 mg per meal), an actual con- tent 25­30 percent less than expected is not of concern. For magnesium (Figure B-4), although there is no statistically significant difference between the aver- ages of the lab values and the calculated values, most of the measured contents of the individual items were higher than calculated. The average potassium con- tent (Figure B-5) of the components analyzed was much higher than expected by the calculated values, while the measured content of sodium (Figure B-6) was much lower than the calculated values indicated. For zinc (Figure B-7), the lab values were on average higher than the calculated values. Entrées are the great- est contributors of zinc in the ration and these are the items for which we found the greatest differences between analytical and calculated values. In general, the

APPENDIX B 259 POTASSIUM Entrées Entrees -100 0% 100 200 300 400 500 600 700 800 -300 -200 -100 0 100 200 300 400 500 % % % % % % % % % Side & Snacks -100 -50 0 50 100 -25% 0% 25% 50% 75% 100% Absolute Difference, mg Percent Difference P = 0.001 FIGURE B-5 Differences between analytical values and calculated values for the potas- sium content of 18 entrees and 14 side dishes or snacks in MREs. Each bar represents an individual ration component. The two panels on the left show absolute differences; the two right panels show percent differences. Values negative if calculated > analytical; values positive if analytical > calculated. NOTE: Axes are not to same scale. SODIUM Entrees Entrées -600 -500 -400 -300 -200 -100 0 100 200 -50% -25% 0% 25% 50% Side &&Snacks Side Snacks -400 -300 -200 -100 0 100 -50% -25% 0% 25% 50% Absolute Difference, mg P = 0.007 Percent Difference FIGURE B-6 Differences between analytical values and calculated values for the so- dium content of 18 entrees and 14 side dishes or snacks in MREs. Each bar represents an individual ration component. The two panels on the left show absolute differences; the two right panels show percent differences. Values negative if calculated > analytical; values positive if analytical > calculated. NOTE: Axes are not to same scale.

260 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL ZINC Entrees Entrées -2 -1 0 1 2 3 4 -100 50% 200% 350% 500% 650% 800% Side &&Snacks Side Snacks -2 -1 0 1 2 3 4 -75% 75% 225% 375% 525% 675% 825% Absolute Difference, mg Percent Difference P = 0.043 FIGURE B-7 Differences between analytical values and calculated values for the zinc content of 18 entrees and 14 side dishes or snacks in MREs. Each bar represents an individual ration component. The two panels on the left show absolute differences; the two right panels show percent differences. Values negative if calculated > analytical; values positive if analytical > calculated. NOTE: Axes are not to same scale. analytical data indicate that the menu analyses based on calculated values are reasonably accurate for calcium and magnesium, but overestimate iron and so- dium and underestimate the actual contents of zinc and potassium. MINERAL INTAKE OF MILITARY PERSONNEL Because food is only nutritious if consumed, it is important to consider actual nutrient intakes when assessing the adequacy of a feeding system. Table B-6 out- lines mean mineral intakes of a group of 30 Army Rangers consuming MREs during a 7-day field training exercise in 1996 (Military Nutrition Division, USARIEM, unpublished data). The last column of the table shows the approxi- mate nutrient content of the rations provided. Although mineral provision would have been adequate, intakes were relatively low for calcium, magnesium, and potassium. Energy intake during this study was 2,435 ± 547 kcal, which is ap- proximately 64 percent of the total energy content of the rations provided. In contrast, calcium intake was only about 43 percent of the ration content while magnesium, potassium, and zinc intakes were 50­60 percent of that provided. Figures B-8 through B-10 present the results of three different field and garrison studies with the data shown as proportions of the study samples with

APPENDIX B 261 TABLE B-6 Mineral Intakes of Army Rangers Consuming MREs (1996) Unit Mean ± SD DRI ~Provision Calcium mg 639 ± 212 1,000 1,500 Iron mg 15.1 ± 3.7 8 21 Magnesium mg 265 ± 61 400­420 480 Phosphorus mg 1,451 ± 387 700 2,300 Potassium mg 2,041 ± 484 4,700 3,800 Sodium mg 4,423 ± 1,301 2,300 5,000* Zinc mg 11.7 ± 2.5 11 20 *Excluding salt packet. SOURCE: Military Nutrition Division, USARIEM (unpublished data). mean dietary intakes less than the Estimated Average Requirement (EAR) and the proportions meeting the RDA or Adequate Intake (AI) level. The prevalence of inadequate dietary intakes in a population is estimated from the proportion of the population with intakes below the median requirement (i.e., EAR). RDAs and AIs are dietary intake goals for individuals that are likely to meet or exceed their requirements. Thus, individual intakes at RDA or AI levels indicate little likelihood of inadequacy (IOM, 2000b). On the other hand, usual intakes less than the RDA or AI cannot be interpreted to mean an individual's intake was inadequate. As there was insufficient scientific evidence upon which to set an EAR and, thus, a RDA for calcium, intakes can be only compared to an AI, which is a level of calcium intake associated with adequate calcium retention. Figure B-8 shows the results of a study conducted with combat support hospital staff in 1997 to evaluate the then current MRE and a test ration which was slightly higher in carbohydrate and lower in fat than the standard MRE ration and included novel ration items and packaging concepts (Baker-Fulco et al., 2002). Be aware that the concept ration was not designed to be nutritionally complete. Few men or women met their individual dietary intake goal for cal- cium; indeed, the majority of men and women reported calcium intakes less than 70 percent of the AI. Substantial proportions of both men and women had likely inadequate dietary intakes of magnesium and zinc. The low magnesium intake is of particular concern, as almost 60 percent of men and 75 percent of women had dietary intakes less than the EAR. Although the incidence of low zinc intakes was less than that for magnesium, about 20 percent of the men and more than 40 percent of the women consumed less than the EAR for zinc. Almost all subjects, including women, had apparently adequate iron intakes. Primarily because women consumed less energy than men, greater proportions of women than men (p < 0.01) failed to meet intake criteria for calcium, iron, magnesium, and zinc (Baker-Fulco et al., 2002).

262 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Calcium Iron 100% 100% 80% 80% 60% 60% 40% 40% 20% 20% 0% 0% Men Women Men Women Men Women Men Women RDA/AI Concept MRE17 Concept MRE17 <RDA/AI <EAR Magnesium Zinc 100% 100% 80% 80% 60% 60% 40% 40% 20% 20% 0% 0% Men Women Men Women Men Women Men Women Concept MRE17 Concept MRE17 KCAL: 2688 2093 2427 1834 N: 26 26 82 28 FIGURE B-8 Proportion of combat support hospital staff with mineral intakes at spe- cific Dietary Reference Intake levels while consuming MREs. Subjects consumed one of two rations, a Concept test ration or the standard MRE XVII. Mean energy intake and sample size of each ration and gender group is shown below the bottom right panel. NOTE: RDA = Recommended Dietary Allowance; AI = Adequate Intake; EAR = Estimated Average Requirement. The prevalence of inadequate dietary intakes in a population is estimated from the proportion of the population with intakes below the EAR. Individual intakes at or exceeding RDA or AI levels are likely adequate. Figure B-9 presents the results of a 1998 garrison study with 146 Army Rangers which would reflect their dietary status prior to a field deployment (Military Nutrition Division, USARIEM, unpublished data). The majority of their food intake came from outside food sources. The self-reported 3-day food record data suggest that slightly over 40 percent of these Rangers were at risk of a low magnesium intake, while only 12 percent had a zinc intake less than the EAR. More than 75 percent of the Rangers reported diets that meet the RDA (11 mg) for zinc and, in fact, half reported zinc intakes that meet or exceed the higher MDRI (15 mg). Prevalences of inadequate intakes of calcium, iron, and potassium appear low. This was the first study for which the food composition data for copper was sufficient to be able to estimate copper intakes; the data suggest the prevalence of inadequate copper intake by Rangers is low with only 5 percent reporting copper intakes less than the EAR. Ninety percent of the Rangers reported sodium intakes in excess of the AI level.

APPENDIX B 263 < EAR < RDA RDA/AI = 100% 80% 60% 40% 20% 0% Ca Fe Mg K Zn Cu n = 146 Energy Intake ~ 3090 ± 1380 kcal FIGURE B-9 Proportion of Army Rangers in garrison with mineral intakes at specific Dietary Reference Intake levels. Sample size and mean energy intake are shown below the chart. NOTE: RDA = Recommended Dietary Allowance; AI = Adequate Intake; EAR = Estimated Average Requirement. The prevalence of inadequate dietary intakes in a population is estimated from the proportion of the population with intakes below the EAR. Individual intakes at or exceeding RDA or AI levels are likely adequate. Similar results were found in a 9-day garrison study conducted with 40 Special Forces Soldiers eating predominately in a military dining facility (Figure B-10) (Tharion et al., 2004). Dietary intake data were collected by a visual estimation method for foods consumed in the dining facility; outside food intake was collected by self-reported food record. Except for magnesium, the preva- lence of inadequate intakes of the reported minerals was nil. Magnesium intakes of 16 of the 40 SF Soldiers were less than the EAR. The entire study sample achieved dietary intakes at RDA levels for iron, zinc, and copper. However, only 63 percent of the study sample achieved the higher MDRI goal for zinc. Dietary intake of all subjects exceeded the AI for sodium. CONCLUSIONS The absolute contents of minerals in operational rations, with the exception of selenium, meet ration standards. However, the concentrations (i.e., nutrient densities) of calcium, magnesium, and zinc are insufficient to ensure low preva- lences of inadequate intakes. There are inadequate data on which to evaluate the adequacy of the nutrient densities of copper and selenium. The sodium content

264 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL < EAR < RDA RDA/AI 100% 80% 60% 40% 20% 0% Ca Fe Mg K Zn Cu n = 40 Energy Intake 3221 ± 565 kcal FIGURE B-10 Proportion of Special Forces Soldiers in garrison with mineral intakes at specific Dietary Reference Intake levels. Sample size and mean energy intake are shown below the chart. NOTE: RDA = Recommended Dietary Allowance; AI = Adequate Intake; EAR = Estimated Average Requirement. The prevalence of inadequate dietary intakes in a population is estimated from the proportion of the population with intakes below the EAR. Individual intakes at or exceeding RDA or AI levels are likely adequate. of operational rations is likely higher than necessary while the content of potas- sium is less than desirable. Ongoing laboratory analyses of additional MRE items as well as compo- nents from other operational rations are needed to reliably calculate mineral composition of ration menus. Additional field studies are also needed to deter- mine energy and nutrient intakes of soldiers subsisting on operational rations so that appropriate target nutrient densities can be established. Field studies also provide food selection and consumption data to help identify the best food ve- hicles for fortification. Reliable laboratory biomarkers of mineral status are needed so that status of soldiers before deployment and changes in status during deployment can be assessed. This information is critical to the evaluation of product reformulations and revised menus. REFERENCES Baker-Fulco CJ, Kramer FM, Johnson J, Lesher LL, Merrill E, DeLany J. 2002. Dietary Intakes of Female and Male Combat Support Hospital Personnel Subsisting on Meal-Focused or Standard Versions of the Meal, Ready-to-Eat. Technical Report T-02/23. Natick, MA: USARIEM. CMNR (Committee on Military Nutrition Research). 1991. Military Nutrition Initiatives. Washing- ton, DC: Institute of Medicine Publication IOM-91-05.

APPENDIX B 265 Consolazio CF. 1976. The impact of low caloric feeding during exercise. In: Food for the Armed Forces. Technical Report NRDC TR 76-42-OTD. Natick, MA: Natick Research and Develop- ment Center. Pp. 61­94. IOM (Institute of Medicine). 1997. Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride. Washington, DC: National Academy Press. IOM. 1998. Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. Washington, DC: National Academy Press. IOM. 2000a. Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids. Wash- ington, DC: National Academy Press. IOM. 2000b. Dietary Reference Intakes for Applications in Dietary Assessment. Washington, DC: National Academy Press. IOM. 2001. Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Cop- per, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Washington, DC: National Academy Press. IOM. 2005. Dietary Reference Intakes for Water, Potassium, Sodium, Chloride, and Sulfate. Wash- ington, DC: The National Academies Press. National Research Council. 1989. Recommended Dietary Allowances. 10th Edition. Washington, DC: National Academy Press. Tharion WJ, Baker-Fulco CJ, Bovill ME, Montain SJ, Delany JP, Champagne CM, Hoyt RW, Lieberman HR. 2004. Adequacy of garrison feeding for Special Forces soldiers during training. Military Medicine 169(6):483­490. U.S. Departments of the Army, Navy, and Air Force. 2001. Nutrition Standards and Education. AR 40-25/BUMEDINST 10110.6/AFI 44-141. Washington, DC: U.S. Department of Defense Headquarters. Bioavailability of Iron, Zinc, and Copper as Influenced by Host and Dietary Factors Janet R. Hunt USDA-ARS Grand Forks Human Nutrition Research Center, Grand Forks, North Dakota INTRODUCTION To determine the mineral intake that will support or enhance performance of military personnel, the bioavailability of the minerals must be considered, in addition to the quantity of the mineral required for biological function. Bio- availability describes the biological utilization of a mineral as consumed, and is affected both by dietary and host factors. This paper briefly summarizes infor- mation on the bioavailability of iron, zinc, and copper, emphasizing topics of particular application to setting nutritional guidelines for feeding the military. IRON BIOAVAILABILITY Body iron is mainly controlled at the point of intestinal absorption rather than excretion (McCance and Widdowson, 1937), and iron bioavailability is

266 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL largely determined by the factors that affect intestinal absorption. Absorbed iron must replace approximately 1 mg of obligatory iron losses daily in men and postmenopausal women, and up to an additional 2.5 mg daily in menstruating women (Institute of Medicine, 2001). Iron absorption is substantially influenced by both host and dietary factors. Dietary factors that influence absorption include the form of dietary iron (either heme or nonheme), as well as dietary constituents consumed concurrently that help keep nonheme iron reduced and soluble. Heme iron, the protoporphyrin iron complex that enters intestinal mucosal cells intact, is absorbed more efficiently than nonheme iron. Heme iron accounts for ~40 percent of the iron in meat, poultry, and fish flesh, and constitutes 0­2.5 mg of the dietary iron consumed daily. Absorption of heme iron is enhanced by unidentified factors in meat, poultry, or fish (Layrisse et al., 1968; Martinez- Torres and Layrisse, 1971) and inhibited by calcium (Hallberg et al., 1991) when consumed in the same meal (Box B-1). Nonheme iron describes the remaining iron in foods, which has been found to form a chemically exchangeable iron pool in the intestinal lumen (Cook et al., 1972). Absorption of this nonheme iron is affected by other dietary constituents consumed in the same meal (Box B-1), that influence the solubility and reduced (ferrous) valence state. Meat, poultry, and fish (Layrisse et al., 1968; Martinez- Torres and Layrisse, 1971) and ascorbic acid (Cook and Monsen, 1977; Hallberg et al., 1986) enhance nonheme iron absorption in a dose-dependent manner, and are especially effective in the presence of inhibitors such as phytic acid and BOX B-1 Food Components that Enhance or Inhibit Iron Absorption, When Consumed Concurrently Heme Iron Absorption Enhancers Inhibitors · Meat, poultry, fish · Calcium Non-heme Iron Absorption Enhancers Inhibitors · Meat, poultry, fish · Phytic acid · Ascorbic acid · Polyphenols/tannins (tea and coffee) · Alcohol · Soy protein · Retinol? · Egg · Carotene? · Calcium · Other organic acids? · Antacids Interactions · Ascorbic acid or meat, poultry, and fish--enhancing effects are greater with phytate or polyphenols

APPENDIX B 267 polyphenols (Hallberg et al., 1989; Hallberg and Hulthen, 2000). Alcohol en- hances nonheme iron absorption, possibly by enhancing gastric acid secretion which promotes the reduced valence state (Hallberg and Hulthen, 2000). Caro- tenes been reported as enhancers of nonheme iron absorption (Garcia-Casal et al., 1998; Layrisse et al., 2000). Reports of enhancement by retinoids are incon- sistent (Garcia-Casal et al., 1998; Layrisse et al., 2000; Walczyk et al., 2003). Citric, malic, and tartaric acids enhance nonheme iron absorption at a high (100- fold) molar ratio (Gillooly et al., 1983), which may not be practically relevant. Inhibitors of nonheme iron absorption include phytic acid (inositol hexa- phosphate, the main food form of inositol phosphates) in whole grains, legumes, lentils, and nuts (Gillooly et al., 1983; Hallberg et al., 1989); iron-binding poly- phenols, such as flavonoids, phenolic acids, and their polymerization products, in tea, coffee, red wines, and a variety of other cereals, vegetables, and spices (Brune et al., 1989; Gillooly et al., 1983; Hallberg and Hulthen, 2000); soy protein (apparently independent of the phytic acid in soy), (Hurrell et al., 1992); and eggs (Callender et al., 1970; Hallberg and Hulthen, 2000; Hurrell, 2003). Calcium inhibits the absorption of both nonheme as well as heme iron (Cook et al., 1991; Hallberg et al., 1991). Zinc in a 1:5 iron:zinc molar ratio reduces iron absorption when given with water, but not with a meal (Rossander-Hulten et al., 1991). Supplemental zinc in equimolar quantities may inhibit iron absorption (Crofton et al., 1989) and impair the iron status of women with low iron reserves (Donangelo et al., 2002). Extensive research on dietary iron bioavailability has helped quantify dose effects and interactions, and algorithms have been devel- oped to calculate the iron bioavailability of diets (Hallberg and Hulthen, 2000; Reddy et al., 2000), but these need further validation. Host factors that influence iron absorption include iron stores, erythropoie- sis, hypoxia, pregnancy, and inflammation (Finch, 1994; Miret et al., 2003). Iron absorption is inversely related to iron status (Cook, 1990; Hallberg et al., 1997; Lynch et al., 1989; Roughead and Hunt, 2000; Taylor et al., 1988). With serum ferritin concentrations from 300 to ~5 µg/L, heme iron absorption can differ by nearly 3-fold, from approximately 20 to 50 percent, and the efficiency of non- heme iron absorption (from a high bioavailability diet) is influenced to a greater extent, from less than 1 to over 35 percent. Recent high iron intake reduces nonheme (Hoglund, 1969) but not heme iron absorption, independent of detect- able changes in serum ferritin (Roughead and Hunt, 2000). The genetic mutation associated with hemochromatosis in people from Northern Europe results in increased absorption of both heme and nonheme iron (Lynch et al., 1989). About 10 percent of those populations are heterozygous for the same mutation, but this does not appear to increase iron absorption (Hunt and Zeng, 2004; Roe et al., 2005). However, additional genetic as well as environmental factors may influ- ence an increasing occurrence of high serum ferritin with age, especially in adult men (IOM, 2001). Chronic inflammation is associated with anemia (e.g., anemia of chronic disease) which may partly be the result of reduced iron absorption

268 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL (Weber et al., 1988). Control of these host factors affecting iron absorption ap- pears to involve the polypeptide hepcidin, which is secreted from the liver in response to high iron stores or infectious stimuli, and both down-regulates intes- tinal iron absorption and stimulated macrophage iron uptake, reducing serum iron (Ganz, 2003). Inflammatory stress from heavy exercise and exertion may initiate a sequence of increased IL(interleukin)-6 production (Margeli et al., 2005), followed by production of hepcidin (Nemeth et al., 2004), and possibly reduced iron absorption, although the latter has not been clearly demonstrated. Gender does not directly influence iron absorption: men and women with the same iron status absorb iron similarly. However, women of child-bearing ages have considerably lower iron status than men: the 5th, 50th, and 95th per- centiles for serum ferritin are 9, 37, and 124 for U.S. women and 42, 118, and 263 for men, respectively (IOM, 2001). In the U.S., iron deficiency in women of childbearing age is more common among minorities (8­10 percent in White, non-Hispanics vs. 15­19 percent in Black, non-Hispanics and 19­22 percent in Mexican Americans) and those with low income (Looker and Cogswell, 2002; Looker et al., 1997). Because of their reduced iron status, menstruating women absorb nonheme iron about twice as efficiently as men (Hunt, 2003b). Women with low iron stores can absorb 25­30 percent of the iron from a diet with high bioavailability, or as much as 3­4 mg iron daily. However, absorption by such women can be substantially reduced by factors that decrease dietary iron bio- availability, such as low meat intake, high phytic acid, and tea consumption (Hunt, 2003b). Iron absorption can vary at least 15-fold relative to body iron stores, and 6- to-10-fold relative to dietary bioavailability, from diets with similar total iron content. However, these substantial differences in iron absorption, as measured with isotopic tracers, are slow to influence clinical indices of body iron status. In controlled trials of weeks or months duration, serum ferritin was unresponsive to differences in ascorbic acid (Cook et al., 1984; Garcia et al., 2003; Hunt et al., 1994; Malone et al., 1986; Monsen et al., 1991) calcium (Minihane and Fairweather-Tait, 1998; Sokoll and Dawson-Hughes, 1992), or several dietary factors affecting iron absorption by 4-to-6-fold (Hunt, 2003b; Hunt and Roughead, 2000; Hunt and Roughead, 1999). Dietary changes apparently require months or years to influence body iron stores, but such differences have been observed with cross-sectional studies of vegetarians, who consistently have lower iron stores than omnivores (Hunt, 2003a). Likewise, serum ferritin was posi- tively associated with ingestion of heme iron, supplemental iron, dietary vitamin C, and alcohol and negatively associated with coffee drinking, in a cross- sectional study of elderly subjects (Fleming et al., 1998). Changes in iron status are also relatively gradual with iron supplementation: serum ferritin increased by 4­5 µg/L with 20 mg iron as FeSO4 daily for 6 weeks (Hinton et al., 2000), and 10­12 µg/L with 50 mg iron as FeSO4 daily (with meals) for 12 weeks (Roughead and Hunt, 2000). Women with low iron stores

APPENDIX B 269 were unable to maintain the difference in serum ferritin achieved with 12 weeks supplementation for 12 weeks after supplements were discontinued (Roughead and Hunt, 2000). These data suggest that to improve the iron status of women with low iron stores (serum ferritin < 20 µg/L) within several weeks, supplemen- tal doses of 20­50 mg nonheme iron/day may be required on a continuing basis. Somewhat more positive results occurred with supplements containing 11 per- cent of the iron in the heme form: 9 or 27 mg daily iron increased serum ferritin by ~5 (a nonsignificant difference) or 12 µg/L (p < 0.05), respectively, and increased hemoglobin values from ~136 to 142 g/L (p < 0.05) in women with low iron stores (Fogelholm et al., 1994). These changes occurred within 1 month, with little change in 5 more months of supplementation (Fogelholm et al., 1994). A ranking of the bioavailability to humans of iron salts used for fortification is likely determined by iron valence and solubility (ferrous sulfate, ferrous succi- nate, ferrous lactate, ferrous fumarate, ferrous glycine sulphate, ferrous gluta- mate, ferrous gluconate, > ferrous citrate, ferrous tartrate, ferrous pyrophosphate > ferric sulphate, ferric citrate) (Brise and Hallberg, 1962). Chelated forms of iron such as sodium iron ethylendiaminetetraacetic acid (NaFeEDTA) or ferrous bis-glycinate are highly bioavailable and in comparison to iron salts, are less influenced by inhibitors such as phytic acid (Bovell-Benjamin et al., 2000; Hurrell, 2002). Iron fortification sources such as ferric pyrophosphate, ferric orthophosphate and elemental iron powders are relatively inert in dry foods, minimizing adverse chemical reactions that may impair food color, taste, and shelf-life, but also reducing iron absorption relative to salts such as ferrous sulfate. Some micronization and emulsification technologies appear to improve the bioavailability of ferric pyrophosphate (Fidler et al., 2004) and may be useful with other iron forms. The bioavailability of elemental iron powders, composed of relatively pure iron metal with a zero valence state, is inversely related to particle size, surface area, and solubility, and differs according to specific production processes; the bioavailability to replete anemic rats is greatest for carbonyl, followed by electrolytic, and then the several reduced iron powders (Swain et al., 2003). However, the bioavailability of elemental iron powders is difficult to determine sensitively in humans because the commercial powders cannot be isotopically labeled. ZINC BIOAVAILABILITY Both zinc absorption and excretion adaptively adjust to control body zinc in animals with zinc intakes from marginal to luxuriant (Hunt et al., 1987; Weigand and Kirchgessner, 1976a; Weigand and Kirchgessner, 1976b). Humans absorb zinc more efficiently when dietary zinc is low (Lee et al., 1993; Taylor et al., 1991; Wada et al., 1985), but this at least partly reflects the immediate effect of the amount ingested, rather than a long-term adaptation to changed zinc intake (Sandström and Cederblad, 1980; Sandström et al., 1980). As more zinc is in-

270 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL gested, absorptive efficiency decreases considerably, but the absolute amount absorbed increases. Several dietary factors may influence human zinc absorption (Lonnerdal, 2000). The zinc content and phytate content, or phytate-to-zinc molar ratio are primary factors, and these are applied in a dietary algorithm for estimating frac- tion zinc absorption from adult diets (International Zinc Nutrition Consultative Group [IZiNG], 2004). Most of the zinc in Western diets is derived from animal foods, with beef supplying about a quarter of dietary zinc (Subar et al., 1998), which is highly bioavailable. Plant sources such as legumes, whole grains, nuts and seeds are also rich in zinc, which is less bioavailable because these sources are also high in phytic acid, a zinc chelator (Harland and Oberleas, 1987). Mixed or refined diets have phytate:zinc molar ratios of 4­18, whereas unrefined cereal based diets can range from 18 to 30 (IZiNCG, 2004). Although phytic acid in unrefined foods reduces fractional zinc absorption, the higher zinc content of these foods may make these foods preferable to more refined products. For ex- ample, nearly 50 percent more zinc was absorbed from a serving of whole wheat, compared with white bread (0.22 versus 0.15 mg, respectively), because the zinc content of the whole wheat bread more than compensated for a less efficient absorption of zinc (16.6 compared to 38.2 percent, respectively) (Sandström et al., 1980). Zinc bioavailability is enhanced by dietary protein when zinc content is constant (Sandström et al., 1980), but this may differ with specific sources of protein (Davidsson et al., 1996), and the practical importance of protein may be confounded by the food zinc content, which correlates directly with protein con- tent. Women tested with diets high or low in meat (replacing refined carbohy- drates) absorbed zinc with similar efficiency, so the amount absorbed was pro- portional to the nearly 2-fold difference in dietary zinc content (Hunt et al., 1995). Calcium has been proposed to reduce zinc absorption, but tests are equivo- cal with calcium either reducing (Wood and Zheng, 1997) or not influencing human zinc absorption (Dawson-Hughes et al., 1986; Lonnerdal et al., 1984; Spencer et al., 1984). Calcium is more likely to inhibit zinc absorption in the presence of phytic acid, by forming insoluble complexes (Fordyce et al., 1987). However, this 3-way interaction has not been clearly demonstrated in humans (Lonnerdal, 2000), and was not observed when calcium was added to a soy- based infant formula (Lonnerdal et al., 1984), or when dairy products (sources of protein as well as calcium) were added to whole wheat bread, a source of phytic acid (Sandström et al., 1980). Other divalent cations could interfere with zinc absorption by competing for transport sites. Iron reduces zinc absorption when administered using supple- mental amounts of inorganic salts (iron:zinc molar ratios of 25:1), but zinc ab- sorption is unaffected in the presence of a food matrix or with more moderate ratios of iron and zinc (iron:zinc molar ratios of 2.5:1) (Lonnerdal, 2000;

APPENDIX B 271 Sandström et al., 1985; Solomons and Jacob, 1981; Whittaker, 1998). Sup- plementing diets with 2 mg copper did not affect zinc absorption by young or elderly adults (August et al., 1989). Because women generally consume less food, including less dietary zinc, the efficiency of zinc absorption from typical diets is likely to be somewhat greater for women than men. For instance, using experimental diets based on representative U.S. diet surveys, women absorbed 29 ± 8 percent, or 2.3 mg zinc from a diet containing 7.8 mg zinc and 1,570 kcal, and men absorbed 22 ± 4 percent, or 3.1 mg zinc from a diet containing 14.0 mg and 2,545 kcal (Hunt et al., 1992). It is difficult to evaluate the impact of zinc bioavailability on zinc nutrition since there are no sensitive clinical indices of marginal zinc status. Plasma zinc does not correlate with zinc absorption measurements (Hunt et al., 1995) and has been relatively insensitive to several weeks of severe dietary zinc restriction (Johnson et al., 1993; Wada et al., 1985). However, iron supplementation re- duced plasma zinc in a study of pregnant Peruvian women (O'Brien et al., 1999). Plasma zinc was also reduced in research volunteers several weeks after chang- ing to a vegetarian diet (Hunt et al., 1998; Srikumar et al., 1992), and was correlated inversely with dietary phytate:zinc molar ratios in adolescent girls consuming lacto-ovo-vegetarian diets (Donovan and Gibson, 1995), but usually has not differed between vegetarians and non-vegetarians in cross-sectional studies (Anderson et al., 1981; Donovan and Gibson, 1995; Kies et al., 1983; Krajcovicova-Kudlackova et al., 1995; Latta and Liebman, 1984). Because of lower zinc absorption, people consuming vegetarian diets, especially with phytate:zinc molar ratios exceeding 15, may require 20 to 50 percent more zinc than nonvegetarians (Hunt et al., 1998; IOM, 2001). Zinc sulfate and zinc oxide are relatively inexpensive and are the forms of zinc most commonly used for food fortification (IZiNCG, 2004). Although zinc sulfate is much more soluble in water than zinc oxide, the two forms have been found equally well absorbed when used to fortify wheat products (de Romana et al., 2003; Herman et al., 2002). In addition to these two forms, zinc chloride, zinc gluconate, and zinc stearate are generally recognized as safe by the U.S. Food and Drug Administration. COPPER BIOAVAILABILITY Much less information is available about the bioavailability of copper. Good food sources include organ meats, seafood, nuts, seeds, whole grains, and choco- late. Absorptive efficiency is inversely proportional to dietary copper content. For example, young men consuming diets containing 0.8, 1.7, or 7.5 mg/day absorbed 12, 36, and 56 percent of the dietary copper (Turnlund et al., 1989). Similarly, the greater copper content of an experimental vegetarian diet was associated with a lower fractional apparent absorption, but more total copper

272 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL absorbed, despite a 3-fold greater phytic acid content compared to a nonvege- tarian diet (Hunt and Vanderpool, 2001). Copper absorption was not reduced by supplemental ascorbic acid (Jacob et al., 1987) or by phytic acid or cellulose (Turnlund et al., 1985). Compared with men, women tended to absorb copper from similar meals slightly more efficiently, which may compensate for a typically lower dietary copper intake (Johnson et al., 1992). Copper absorption was not different be- tween young and elderly adults (Johnson et al., 1992; Turnlund et al., 1988). High zinc intakes can reduce copper absorption (IOM, 2001), and zinc supplements have been used to treat Wilson's disease, an inherited disease that results in copper toxicity (Brewer et al., 1983). CONCLUSIONS The bioavailability as well as the content of iron, zinc and copper should be considered when planning military diets. The bioavailability of these nutrients is generally high in North American diets, but bioavailability can be reduced by food choices such as the selection of a vegetarian diet. Biochemical indices are available to assess iron, but not zinc or copper nutritional status. Approximately 20 percent of menstruating women have low iron stores, and iron deficiency is more prevalent in minorities and those of low income. To address iron defi- ciency in these women, food-based approaches, including food fortification, are likely to require months or years to influence iron status, and would unnecessar- ily increase bioavailable iron for men. Iron supplementation should be evaluated for these specific women, or perhaps for all military women. REFERENCES Anderson BM, Gibson RS, Sabry JH. 1981. The iron and zinc status of long-term vegetarian women. Am J Clin Nutr 34:1042­1048. August D, Janghorbani M, Young VR. 1989. Determination of zinc and copper absorption at three dietary Zn-Cu ratios by using stable isotope methods in young adult and elderly subjects. Am J Clin Nutr 50:1457­1463. Bovell-Benjamin AC, Viteri FE, Allen LH. 2000. Iron absorption from ferrous bisglycinate and ferric trisglycinate in whole maize is regulated by iron status. Am J Clin Nutr 71(6):1563­1569. Brewer GJ, Hill GM, Prasad AS, Cossack ZT, Rabbani P. 1983. Oral zinc therapy for Wilson's disease. Ann Intern Med 99(3):314­319. Brise H, Hallberg L. 1962. Absorbability of different iron compounds. Acta Med Scand 171 (Suppl): 23­38. Brune M, Rossander L, Hallberg L. 1989. Iron absorption and phenolic compounds: Importance of different phenolic structures. Eur J Clin Nutr 43(8):547­557. Callender ST, Marney SR, Jr., Warner GT. 1970. Eggs and iron absorption. Br J Haematol 19(6): 657­665. Cook JD. 1990. Adaptation in iron metabolism. Am J Clin Nutr 51:301­308. Cook JD, Monsen ER. 1977. Vitamin C, the common cold, and iron absorption. Am J Clin Nutr 30:235­241.

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274 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Harland BF, Oberleas D. 1987. Phytate in foods. Wld Rev Nutr Diet 52:235­259. Herman S, Griffin IJ, Suwarti S, Ernawati F, Permaesih D, Pambudi D, Abrams SA. 2002. Co- fortification of iron-fortified flour with zinc sulfate, but not zinc oxide, decreases iron absorp- tion in Indonesian children. Am J Clin Nutr 76(4):813­817. Hinton PS, Giordano C, Brownlie T, Haas JD. 2000. Iron supplementation improves endurance after training in iron-depleted, nonanemic women. J Appl Physiol 88(3):1103­1111. Hoglund S. 1969. Studies in iron absorption VI. Transitory effect of oral administration of iron on iron absorption. Blood 34(4):505­510. Hunt JR. 2003a. Bioavailability of iron, zinc, and other trace minerals from vegetarian diets. Am J Clin Nutr 78(3 Suppl):633S­639S. Hunt JR. 2003b. High-, but not low-bioavailability diets enable substantial control of women's iron absorption in relation to body iron stores, with minimal adaptation within several weeks. Am J Clin Nutr 78(6):1168­1177. Hunt JR, Roughead ZK. 1999. Nonheme iron absorption, fecal ferritin excretion, and blood indexes of iron status in women consuming controlled lactoovovegetarian diets for 8 wk. Am J Clin Nutr 69:944­952. Hunt JR, Roughead ZK. 2000. Adaptation of iron absorption in men consuming diets with high or low iron bioavailability. Am J Clin Nutr 71(1):94­102. Hunt JR, Vanderpool RA. 2001. Apparent copper absorption from a vegetarian diet. Am J Clin Nutr 74(6):803­807. Hunt JR, Zeng H. 2004. Iron absorption by heterozygous carriers of the HFE C282Y mutation associated with hemochromatosis. Am J Clin Nutr 80(4):924­931. Hunt JR, Johnson PE, Swan PB. 1987. Influence of usual zinc intake and zinc in a meal on 65Zn retention and turnover in the rat. J Nutr 117:1427­1433. Hunt JR, Mullen LK, Lykken GI. 1992. Zinc retention from an experimental diet based on the U.S. FDA Total Diet Study. Nutr Res 12:1335­1344. Hunt JR, Gallagher SK, Johnson LK. 1994. Effect of ascorbic acid on apparent iron absorption by women with low iron stores. Am J Clin Nutr 59:1381­1385. Hunt JR, Gallagher SK, Johnson LK, Lykken GI. 1995. High- versus low-meat diets: Effects on zinc absorption, iron status, and calcium, copper, iron, magnesium, manganese, nitrogen, phospho- rus, and zinc balance in postmenopausal women. Am J Clin Nutr 62(3):621­632. Hunt JR, Matthys LA, Johnson LK. 1998. Zinc absorption, mineral balance, and blood lipids in women consuming controlled lactoovovegetarian and omnivorous diets for 8 wk. Am J Clin Nutr 67:421­430 Hurrell RF. 2002. Fortification: Overcoming technical and practical barriers. J Nutr 132(4 Suppl): 806S­812S. Hurrell RF. 2003. Influence of vegetable protein sources on trace element and mineral bioavailability. J Nutr 133(9):2973S­2977S. Hurrell RF, Juillerat MA. 1992. Soy protein, phytate, and iron absorption in humans. Am J Clin Nutr 56(3):573­578. International Zinc Nutrition Consultative Group (IZiNCG). 2004. Chapter 1: Overview of zinc nutri- tion. Assessment of the risk of zinc deficiency in populations and options for its control. Food and Nutrition Bulletin 25:S99­S129. IOM (Institute of Medicine). 2001. Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Washington, DC: National Academy Press. Jacob RA, Skala JH, Omaye ST, Turlund JR. 1987. Effect of varying ascorbic acid intakes on copper absorption and ceruloplasmin levels of young men. J Nutr 117(12):2109­2215. Johnson PE, Milne DB, Lykken GI. 1992. Effects of age and sex on copper absorption, biological half-life and status in humans. Am J Clin Nutr 56(5):917­925.

APPENDIX B 275 Johnson PE, Hunt CD, Milne DB, Mullen LK. 1993. Homeostatic control of zinc metabolism in men: Zinc excretion and balance in men fed diets low in zinc. Am J Clin Nutr 57(4):557­565. Kies C, Young E, McEndree L. 1983. Zinc bioavailability from vegetarian diets. Influence of dietary fiber, ascorbic acid, and past dietary practices. In: Inglett GE, ed. Nutritional Bioavailability of Zinc. Washington, DC: American Chemical Society. Pp. 115­126. Krajcovicova-Kudlackova M, Simoncic R, Babinska K, Bederova A, Brtkova A, Magalova T, Grancicova E. 1995. Selected vitamins and trace elements in blood of vegetarians. Ann Nutr Metab 39(6):334­339. Latta D, Liebman M. 1984. Iron and zinc status of vegetarian and nonvegetarian males. Nutr Rep Int 30(1):141­149. Layrisse M, Martinez-Torres C, Roche M. 1968. Effect of interaction of various foods on iron absorption. Am J Clin Nutr 21:1175­1183. Layrisse M, Garcia-Casal MN, Solano L, Baron MA, Arguello F, Llovera D, Ramirez J, Leets I, Tropper E. 2000. New property of vitamin A and beta-carotene on human iron absorption: Effect on phytate and polyphenols as inhibitors of iron absorption. Arch Latinoam Nutr 50(3): 243­248. Lee DY, Prasad AS, Hydrick-Adair C, Brewer G, Johnson PE. 1993. Homeostasis of zinc in mar- ginal human zinc deficiency--Role of absorption and endogenous excretion of zinc. J Lab Clin Med 122(5):549­556. Lonnerdal B. 2000. Dietary factors influencing zinc absorption. J Nutr 130(5):1378S­1383S. Lonnerdal B, Cederblad A, Davidsson L, Sandstrom B. 1984. The effect of individual components of soy formula and cows' milk formula on zinc bioavailability. Am J Clin Nutr 40(5):1064­1070. Looker AC, Cogswell ME. 2002. Iron deficiency--United States, 1999­2000. MMWR Weekly 51(40): 897­899. Looker AC, Dallman PR, Carrol MD, Gunter EW, Johnson CL. 1997. Prevalence of iron deficiency in the United States. JAMA 277(12):973­976. Lynch SR, Skikne BS, Cook JD. 1989. Food iron absorption in idiopathic hemochromatosis. Blood 74:2187­2193. Malone HE, Kevany JP, Scott JM, O'Broin SD, O'Connor G. 1986. Ascorbic acid supplementation: Its effects on body iron stores and white blood cells. Irish J Med Sci 155:74­79. Margeli A, Skenderi K, Tsironi M, Hantzi E, Matalas AL, Vrettou C, Kanavakis E, Chrousos G, Papassotiriou I. 2005. Dramatic elevations of interleukin-6 and acute phase reactants in athletes participating in the ultradistance foot race spartathlon: Severe systemic inflammation and lipid and lipoprotein changes in protracted exercise. J Clin Endocrinol Metab 90(7):3914­3918. Martinez-Torres C, Layrisse M. 1971. Iron absorption from veal muscle. Am J Clin Nutr 24:531­540. McCance RA, Widdowson EM. 1937. Absorption and excretion of iron. Lancet 2:680­684. Minihane AM, Fairweather-Tait SJ. 1998. Effect of calcium supplementation on daily nonheme-iron absorption and long-term iron status. Am J Clin Nutr 68:96­102. Miret S, Simpson RJ, McKie AT. 2003. Physiology and molecular biology of dietary iron absorp- tion. Annu Rev Nutr 23:283­301. Monsen, ER, Labbe RF, Lee W, Finch CA. 1991. Iron balance in healthy menstruating women: Effect of diet and ascorbate supplementation. In: Trace Elements in Man and Animals (TEMA- 7. Dubrovnic, Yugoslavia: University of Zagreb, Institute for Medical Research and Occupa- tional Health. Pp. 6.2­6.3. Nemeth E, Rivera S, Gabayan V, Keller C, Taudorf S, Pederson BK, Ganz T. 2004. IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hep- cidin. J Clin Invest 113(9):1271­1276. O'Brien KO, Zavaleta N, Caulfield LE, Yang DX, Abrams SA. 1999. Influence of prenatal iron and zinc supplements on supplemental iron absorption, red blood cell iron incorporation, and iron status in pregnant Peruvian women. Am J Clin Nutr 69(3):509­515.

276 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Reddy MB, Hurrell RF, Cook JD. 2000. Estimation of nonheme-iron bioavailability from meal composition. Am J Clin Nutr 71(4):937­943. Roe MA, Heath AL, Oyston SL, Macrow C, Hoogewerff JA, Foxall R, Dainty JR, Majsak-Newman G, Willis G, Fairweather-Tait SJ. 2005. Iron absorption in male C282Y heterozygotes. Am J Clin Nutr 81(4):814­821. Rossander-Hulten L, Brune M, Sandstrom B, Lonnerdal B, Hallberg L. 1991. Competitive inhibition of iron absorption by manganese and zinc in humans. Am J Clin Nutr 54:152­156. Roughead ZK, Hunt JR. 2000. Adaptation in iron absorption: Iron supplementation reduces nonheme- iron but not heme-iron absorption from food. Am J Clin Nutr 72(4):982­989. Sandström B, Cederblad A. 1980. Zinc absorption from composite meals II. Influence of the main protein source. Am J Clin Nutr 33:1778­1783. Sandström B, Arvidsson B, Cederblad A, Bjorn-Rasmussen E. 1980. Zinc absorption from compos- ite meals I. The significance of wheat extraction rate, zinc, calcium, and protein content in meals based on bread. Am J Clin Nutr 33:739­745. Sandström B, Davidsson L, Cederblad A, Lonnerdal B. 1985. Oral iron, dietary ligands and zinc absorption. J Nutr 115:411­414. Sokoll LJ, Dawson-Hughes B. 1992. Calcium supplementation and plasma ferritin concentrations in premenopausal women. Am J Clin Nutr 56:1045­1048. Solomons NW, Jacob RA. 1981. Studies on the bioavailability of zinc in humans: Effects of heme and nonheme iron on the absorption of zinc. Am J Clin Nutr 34:475­482. Spencer H, Kramer L, Norris C, Osis D. 1984. Effect of calcium and phosphorus on zinc metabolism in man. Am J Clin Nutr 40:1213­1218. Srikumar TS, Johansson GK, Ockerman PA, Gustafsson JA, Akesson B. 1992. Trace element status in healthy subjects switching from a mixed to a lactovegetarian diet for 12 mo. Am J Clin Nutr 55:885­890. Subar AF, Krebs-Smith SM, Cook A, Kahle LL. 1998. Dietary sources of nutrients among U.S. adults, 1989 to 1991. J Am Dietet Assoc 98:537­547. Swain JH, Newman SM, Hunt JR. 2003. Bioavailability of elemental iron powders to rats is less than bakery-grade ferrous sulfate and predicted by iron solubility and particle surface area. J Nutr 133(11):3546­3552. Taylor CM, Bacon JR, Aggett PJ, Bremner I. 1991. Homeostatic regulation of zinc absorption and endogenous losses in zinc- deprived men [published erratum appears in Am J Clin Nutr 1992 Aug;56(2):462]. Am J Clin Nutr 53(3):755­763. Taylor P, Martinez-Torres C, Leets I, Ramirez J, Garcia-Casal MN, Layrisse M. 1988. Relationships among iron absorption, percent saturation of plasma transferrin and serum ferritin concentra- tion in humans. J Nutr 118(9):1110­1115. Turnlund JR, King JC, Gong B, Keyes WR, Michel MC. 1985. A stable isotope study of copper absorption in young men: Effect of phytate and alpha-cellulose. Am J Clin Nutr 42:18­23. Turnlund JR, Reager RD, Costa, F. 1988. Iron and copper absorption in young and elderly men. Nutr Res 8:333­343. Turnlund JR, Keyes WR, Anderson HL, Acord LL. 1989. Copper absorption and retention in young men at three levels of dietary copper by use of the stable isotope 65Cu. Am J Clin Nutr 49: 870. Wada L, Turnlund JR, King JC. 1985. Zinc utilization in young men fed adequate and low zinc intakes. J Nutr 115(10):1345­1354. Walczyk T, Davidsson L, Rossander-Hulthen L, Hallberg L, Hurrell RF. 2003. No enhancing effect of vitamin A on iron absorption in humans. Am J Clin Nutr 77(1):144­149. Weber J, Were JM, Julius HW, Marx JJ. 1988. Decreased iron absorption in patients with active rheumatoid arthritis, with and without iron deficiency. Ann Rheum Dis 47(5):404­409. Weigand E, Kirchgessner M. 1976a. 65Zn-labeled tissue zinc for determination of endogenous fecal zinc excretion in growing rats. Nutr Metab 20:314­320.

APPENDIX B 277 Weigand E, Kirchgessner M. 1976b. Radioisotope dilution technique for determination of zinc ab- sorption in vivo. Nutr Metab 20:307­313. Whittaker P. 1998. Iron and zinc interactions in humans. Am J Clin Nutr 68(2 Suppl):442S­446S. Wood RJ, Zheng JJ. 1997. High dietary calcium intakes reduce zinc absorption and balance in humans. Am J Clin Nutr 65:1803­1809. Functional Metabolism of Copper, Zinc, and Iron Cathy W. Levenson Florida State University, Tallahassee INTRODUCTION The metabolism of copper, zinc, and iron occurs in every organ, tissue, and cell type of the human body. The molecular, biochemical, and physiological roles that these nutrients play are so fundamental that these metals are essential not just for optimal physiological performance, but for sustaining life itself. Any attempt to understand the effects that military service, and the inherent physical and emotional demands of training and combat, would have on metal metabo- lism must first address the actual roles of these metals and how they function in the metabolic processes that regulate physiological function. METAL TRANSPORT After intestinal absorption of dietary copper, zinc, and iron, these essential nutrients are transported through the serum to specific organs. For example, both copper and zinc appear to be transported through the serum for delivery to tis- sues via albumin as well as other small molecular weight ligands including se- rum amino acids and peptides such as glutathione (Lovstad, 2004; Stewart et al., 2003). Interestingly, the majority of serum copper, at least 65 percent in humans, is associated with the enzyme ceruloplasmin. While the enzymatic activity of this protein requires copper, and is thus frequently used as a copper status indica- tor, this protein does not appear to play a significant role in the transport and delivery of copper to tissues. Instead, the primary role of this powerful serum antioxidant appears to be its role in iron metabolism (Sharp, 2005). While the binding of copper and zinc to serum albumin and peptides is relatively non-specific, iron, in contrast, has a specialized transport system. The protein responsible for the transport and delivery of iron to tissues is transferrin. At the cellular level, target tissues take up iron via a receptor-mediated endocytotic mechanism that is specifically regulated by the availability of iron (Levenson and Tassabehji, 2004). Increases in iron availability decrease the abundance of the transferrin receptor and limit cellular iron uptake. However, in periods of low

278 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL dietary iron availability ferritin levels are decreased and translation and synthesis of the transferrin receptor is increased in an attempt to maximize cellular iron uptake (Levenson and Tassabehji, 2004). FUNCTIONAL METABOLISM OF COPPER Immune System In the military setting, it is clear that optimal immune function is essential. Exposure to a variety of novel bacterial, both intestinal and cutaneous, is an accepted part of military life, particularly in international theaters. Close living quarters increase the risk of transmission of bacterial and viral infection. Copper, zinc, and iron are all essential for immune function (reviewed in Failla, 2003). In the case of copper, a number of studies have reported neutropenia resulting from copper deficiency, the most recent of which was published just this year (Nagano et al., 2005). Other work on the functional metabolism of copper in the immune system has shown that copper deficiency impairs the activity of the cytokine interleukin-2 (IL-2) in human T-lymphocytes (Hopkins and Failla, 1997), and suppresses monocyte differentiation (Huang et al., 2001). Red Blood Cells Iron is carried to the bone marrow for incorporation into developing erythro- cytes by the serum transport protein transferrin. However, the ability of iron to bind to this protein is dependent of the ferrioxidase activity of the copper- dependent enzyme ceruloplasmin. Without copper and ceruloplasmin activity, iron is trapped in the ferrous state. Unable to bind to transferrin, iron delivery is prevented, resulting in symptoms consistent with iron deficiency including mi- crocytic, hypochromic anemia. Unfortunately, misdiagnosis of copper deficiency as iron deficiency and treatment with iron supplements could result in iron over- load. Thus, understanding the functional metabolism and interactions of these two nutrients is important as anemia would clearly impair physical performance in military personnel. While primary dietary copper deficiency is likely to be very rare, it is impor- tant to note that high levels of zinc intake inhibit copper absorption. This in turn can lead to neurotropenia, reduced ceruloplasmin activity, defective iron trans- port, and microcytic, hypochromic anemia (Willis et al., 2005). A recent report showed that supplementation with 100 mg zinc/day resulted in marked anemia and severe neutropenia associated with copper deficiency (Irving et al., 2003). Thus, while it is important that dietary zinc be adequate to meet the increased needs of military personnel, it is important that diets not be supplemented with zinc at levels that would induce copper deficiency.

APPENDIX B 279 Muscle Clearly optimal physical performance in military personnel is dependent on optimal muscle metabolism including substrate utilization, mitochondrial oxida- tion, and ATP synthesis. This is true both for both skeletal muscle and mitochondria-rich cardiac muscle. It has been known for some time that the trace metal copper is essential for the activity of cytochrome c oxidase, an essential mitochondrial enzyme used in the synthesis of ATP (Rossi et al., 1998). More recent work has suggested that copper may also regulate synthesis of other members of the mitochondrial electron transport chain such as ATP synthase (Medeiros and Wildmon, 1997). Additionally, the copper-dependent enzymes lysyl- oxidase, which is responsible for collagen cross-linking, and Cu, Zn-superoxide dismutase, a cytosolic free radical scavenger, are both needed for normal muscle function. Brain and Nervous System The role of copper in neuronal metabolism and neurological function is complex. First, the brain synthesizes ceruloplasmin. Unlike ceruloplasmin that is synthesized in the liver for export into the serum, the ceruloplasmin in the brain is anchored in cell membranes where it functions in iron metabolism. The ab- sence of ceruloplasmin activity results in brain iron toxicity (Xu et al., 2004). Other copper dependent enzymes in the brain and nervous system include dopa- mine beta-monooxygenase, that is responsible for the conversion of the neurotrans- mitter dopamine into norepinephrine. Norepinephrine is in turn converted into epinephrine, or adrenaline. Interestingly, the actual effect of copper deficiency is complex, as dietary copper deficiency in rats produced elevated dopamine beta- monooxygenase activity (Prohaska and Brokate, 2001). Regardless of the exact mechanism, these data show that copper deficiency would likely disrupt the nor- mal "fight or flight" response to stress that is associated with catecholamine pro- duction, and would come into play during both combat and training settings. Peptidylglycine alpha-amidating monooxygenase (PAM) is also copper de- pendent (Prohaska et al., 1995). This enzyme, found both intracellularly as well as in the serum, is responsible for the post-translational modification, maturation and activation of a wide variety of neurohormones including vasopressin that regulates water balance and blood pressure, gastrin and cholecystokinin (CCK) in the gastrointestinal system, calcitonin that regulates bone calcium deposition, thryotropin, substance P, and neuropeptide Y (NPY) (Eipper et al., 1993). While all of these hormones play vital roles, of particular interest to the stress of com- bat is the role that NPY may play. Our data show that NPY is regulated by copper in the central nervous system (Rutkoski et al., 1999), and that synthesis of this peptide in the adrenal gland is an important part of the physiological response and adaptation to psychological stress (Levenson and Moore, 1998).

280 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL FUNCTIONAL METABOLISM OF IRON Red Blood Cells The role of iron in hemoglobin function and oxygen transport is well known. Clearly the delivery of oxygen to muscle, particularly in times of peak demand, is essential for optimal muscle performance. Dietary iron deficiency leads to microcytic, hypochromic anemia. When dietary iron is limited, erythrocyte he- moglobin synthesis is reduced, both because iron is an essential part of the por- phyrin ring structure of hemoglobin, and because iron deficiency impairs the activity of at least one enzyme in the heme synthetic pathway. Additionally, we know that iron deficiency, and the resulting decrease in hemoglobin concentra- tion, results in increased division of pro-erythrocytes in the bone marrow, lead- ing to decreased erythrocyte diameter and volume, and impairing oxygen deliv- ery capability. Muscle For many years the fatigue and irritability associated with dietary iron defi- ciency were attributed to the anemia discussed above. While reduced oxygen delivery can certainly contribute to these symptoms, there is a growing apprecia- tion for the role of iron in skeletal and cardiac muscle. Iron is required for the electron transport chain in the mitochondrial that is responsible for ATP synthe- sis. Iron deficiency reduces mitochondrial cytochrome c, cytochrome oxidase, glycerol-3-phosphate dehydrogenase activity, and respiratory capacity (McLane et al., 1981). Furthermore, it appears that the fatigue associated with iron defi- ciency can be corrected with iron supplements, even in the absence of anemia (Brutsaert et al., 2003). An understanding of the role of iron in functional muscle metabolism is important, particularly for females who may have lower iron stores. This also illustrates the need to evaluate iron status not simply by hemo- globin or hemtocrit, but rather by other measures that may be more reflective of total body iron status such as total iron binding capacity (TIBC) and serum ferritin. Brain and Nervous System Like copper, iron is not only an essential nutrient, but, in high concentra- tions, is toxic. For many years evidence has been mounting that suggests a role for iron accumulation in the death and destruction of dopaminergic neurons of the substantia nigra. Damage to these catecholaminergic neurons results in ab- normal motor behavior, balance disturbances, tremors, and cognitive declines most frequently associated with Parkinson's disease (Zecca et al., 2004). Iron accumulation has also been associated with Fredrick's ataxia and Alzheimer's

APPENDIX B 281 disease (Zecca et al., 2004). While the long term avoidance of these disorders is important for both military personnel and the general population, the most recent epidemiological data suggest that high intakes of dietary iron alone do not cause Parkinson's disease. For example, when the diets of Parkinson's patients and age-matched controls were compared, it was found that dietary iron intakes were not different (Logroscino et al., 1998). However, it should be noted that there was an association between high dietary fat intake and Parkinson's disease that was exacerbated by high dietary iron levels, suggesting that iron-induced oxida- tive processes maybe a significant part of Parkinson's etiology (Logroscino et al., 1998). These data highlight the need to examine overall dietary intake, not just metal intake, for risk assessment. Clearly, there are many iron dependent processes in the brain. While there are data suggesting that chelation of iron may provide protection from Parkin- son's disease (Kaur et al., 2003), newly emerging data show that dietary iron deficiency may contribute to increased risk of gait and balance disorders result- ing from damage to dopaminergic neurons (Levenson et al., 2004). Mice fed an iron deficient diet for six weeks had significantly lower striatal dopamine con- centrations and poorer motor behavior scores than those fed an iron adequate control diet (Levenson et al., 2004). Other work has revealed that iron restriction impairs dopamine metabolism (Erikson et al., 2000), disrupts dopamine receptor function (Nelson et al., 1997), and induces programmed cell death mechanisms in dopaminergic neurons (Levenson et al., 2004). Future work will be needed to understand the potential risk that iron deficiency poses for normal neuronal func- tion. But for now it is clear that adequate iron status, as measured by TIBC and not just the presence of anemia, should be maintained. FUNCTIONAL METABOLISM OF ZINC Biochemical Functions The physiological functions of zinc can largely be attributed to the catalytic and structural roles of zinc bound to specific proteins. For example, zinc is known to be required for the catalytic activity of approximately 100 mammalian enzymes. Zinc dependent enzymes, such as glyceraldehyde dehydrogenase, lac- tate dehydrogenase, and DNA and RNA polymerases, are so ubiquitous that there is virtually no metabolic cycle that is not dependent on zinc. The biochemi- cal role of zinc is also illustrated by the existence of hundreds, if not thousands, of zinc-finger proteins that act as DNA-binding transcription factors to regulate gene expression. Zinc-finger proteins, that coordinate zinc in their structure through cysteines and histidines, also act as RNA binding proteins as well as participate in protein-protein interactions that regulate cellular function and metabolism.

282 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Because of the vast number of biochemical and molecular functions, it is not surprising that the functional metabolism of zinc is important. For example, cellular proliferation and differentiation that take place upon immune system activation are both dependent on zinc and appear to be disrupted in zinc defi- ciency. Secretion of a number of important cytokines such as IL-2, interferon- gamma, and tumor necrosis factor alpha are reduced by dietary zinc deficiency (Fraker et al., 2000). Furthermore, tissue growth, including the growth of new muscle tissue and wound healing, is dependent on adequate dietary zinc (Andrews and Gallagher-Allred, 1999). Brain and Nervous System Zinc is needed for many aspects of brain and nervous system function in- cluding neuronal differentiation, synaptic activity, receptor modulation and func- tion, and neuronal survival. It is well accepted that the central nervous system, particularly the hypothalamus, is responsible for regulation of appetite and feed- ing behaviors. Adequate food intake is essential not only to maintain energy balance and body weight, but also to insure adequate micronutrient intake. Our lab (Evans et al., 2004) as well as many others, has shown that when fed a zinc- restricted diet, rats develop a profound anorexia within approximately one week Given a choice between dietary carbohydrate, protein and fat, zinc deficient rats virtually eliminate carbohydrate intake (Rains and Shay, 1995). While the cellu- lar and molecular mechanisms responsible for these observations are still being investigated (Levenson, 2003), it is interesting to speculate what effect dietary zinc has on human feeding behavior. While human food intake and selection is too complex to attribute to a single factor, it has been hypothesized that zinc deficiency may play a role in the development of some cases of human anorexia nervosa (Tannhauser, 2002). There is also evidence that zinc deficiency results in decreased taste acuity in humans (Russell et al., 1983), a problem that could easily contribute to reduced food intake. Thus, given the increased energy re- quirements of combat troops and the usual decrease in apetite due to a variety of stressors during military life, maintaining adequate zinc nutriture appears to be important. However, the data regarding the involvement of zinc in food behavior of humans is not well understood. There is also a growing body of literature, spanning the last two decades, suggesting a link between clinical depression and zinc deficiency (for recent reviews see Nowak et al., 2005 and Levenson, 2006). For example, not only has it been shown that patients suffering from major depression have lower serum zinc levels, but the severity of the zinc deficiency can also be correlated with the severity of the depression (Maes et al., 1994). Patients whose symptoms are resistant to treatment have even lower serum zinc levels than those who respond to conventional pharmacological treatments (Maes et al., 1999). Zinc has also successfully been used as an adjunct to antidepressant treatment. In a double-

APPENDIX B 283 blind trial, treatment with tricyclic antidepressants or selective serotonin reuptake inhibitors was significantly enhanced when supplemented with zinc compared with a placebo (Levenson, 2006; Nowak et al., 2003). While there are many causes of depression, including genetics, there is no dispute that stressful envi- ronments and situations, both acute and chronic, are a significant factor in the development of depression and depression-like disorders (Wurtman, 2005). Given the emerging evidence that dietary zinc may play a role in the develop- ment of depression, every effort should be made to maintain the zinc status of individuals exposed to stressful living and working conditions. CONCLUSIONS An examination of the functional metabolism of the copper, zinc, and iron shows that each of these nutrients is a vital and essential regulator of the immune system, muscle metabolism and performance, red blood cell function, and the brain and nervous system. The role of trace metals in the metabolism of these different organ systems is particularly important during periods of physical and psychological stress associated with combat and training of military personnel. REFERENCES Andrews M, Gallagher-Allred C. 1999. The role of zinc in wound healing. Adv Wound Care 2: 137­138. Brutsaert TD, Hernandez-Cordero S, Rivera J, Viola T, Hughes G, Haas JD. 2003. Iron supplementa- tion improves progressive fatigue resistance during dynamic knee extensor exercise in iron- depleted, nonanemic women. Am J Clin Nutr 77:441­448. Eipper BA, Milgram SL, Husten EJ, Yun HY, Mains RE. 1993. Peptidylglycine alpha-amidating monooxygenase: A multifunctional protein with catalytic, processing, and routing domains. Protein Sci 2:489­497. Erikson KM, Jones BC, Beard JL. 2000. Iron deficiency alters dopamine transporter functioning in rat striatum. J Nutr 130:2831­2837. Evans SA, Overton JM, Alshingiti A, Levenson CW. 2004. Regulation of metabolic rate and sub- strate utilization by zinc deficiency. Metabolism 53:727­732. Failla ML. 2003. Trace elements and host defense: recent advances and continuing challenges. J Nutr 133:1443S­1447S. Hopkins RG, Failla ML. 1997. Copper deficiency reduces interleukin-2 (IL-2) production and IL-2 mRNA in human T-lymphocytes. J Nutr 127:257­262. Huang ZL, Failla ML, Reeves PG. 2001. Differentiation of human U937 promonocytic cells is impaired by moderate copper deficiency. Exp Biol Med (Maywood) 226:222­228. Irving JA, Mattman A, Lockitch G, Farrell K, Wadsworth LD. 2003. Element of caution: A case of reversible cytopenias associated with excessive zinc supplementation. CMAJ 169:129­131. Kaur D, Yantiri F, Rajagopalan S, Kumar J, Mo JQ, Boonplueang R, Viswanath V, Jacobs R, Yang L, Beal MF, DiMonte D, Volitaskis I, Ellerby L, Cherny RA, Bush AI, Andersen JK. 2003. Genetic or pharmacological iron chelation prevents MPTP-induced neurotoxicity in vivo: A novel therapy for Parkinson's disease. Neuron 37:899­909. Levenson CW. 2003. Zinc regulation of food intake: New insights on the role of neuropeptide Y. Nutr Rev 61:247­249.

284 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Levenson CW. 2006. Zinc: The new antidepressant? Nutr Rev 64(1):39­42. Levenson CW, Moore JB. 1998. Response of rat adrenal neuropeptide Y and tyrosine hydroxylase mRNA to acute stress is enhanced by long-term voluntary exercise. Neurosci Lett 242: 177­179. Levenson CW, Tassabehji NM. 2004. Iron and ageing: An introduction to iron regulatory mecha- nisms. Ageing Res Rev 3:251­263. Levenson CW, Cutler RG, Ladenheim B, Cadet JL, Hare J, Mattson MP. 2004. Role of dietary iron restriction in a mouse model of Parkinson's disease. Exp Neurol 190:506­514. Lovstad RA. 2004, A kinetic study on the distribution of Cu(II)-ions between albumin and transfer- rin. Biometals 17:111­113. Maes M, D'Haese PC, Scharpe S, D'Hondt P, Cosyns P, De Broe ME. 1994. Hypozincemia in depression. J Affect Disord 31:135­140. Maes M, De Vos N, Demedts P, Wauters A, Neels H. 1999. Lower serum zinc in major depression in relation to changes in serum acute phase proteins. J Affect Disord 56:189­194. McLane JA, Fell RD, McKay RH, Winder WW, Brown EB, Holloszy JO. 1981, Physiological and biochemical effects of iron deficiency on rat skeletal muscle. Am J Physiol 241:C47­C54. Nagano T, Toyoda T, Tanabe H, Nagato T, Tsuchida T, Kitamura A, Kasai G. 2005. Clinical features of hematological disorders caused by copper deficiency during long-term enteral nutrition. Intern Med 44:554­559. Nelson C, Erikson K, Pinero DJ, Beard JL. 1997. In vivo dopamine metabolism is altered in iron- deficient anemic rats. J Nutr 127:2282­2288. Nowak G, Siwek M, Dudek D, Zieba A, Pilc A. 2003. Effect of zinc supplementation on antidepres- sant therapy in unipolar depression: A preliminary placebo-controlled study. Pol J Pharmacol 55:1143­1147. Nowak G, Szewczyk B, Pilc A. 2005. Zinc and depression. An update. Pharmacol Rep 57:713­718. Prohaska JR, Brokate B. 2001. Dietary copper deficiency alters protein levels of rat dopamine beta- monooxygenase and tyrosine monooxygenase. Exp Biol Med (Maywood) 226:199­207. Prohaska JR, Bailey WR, Lear PM. 1995. Copper deficiency alters rat peptidylglycine alpha- amidating monooxygenase activity. J Nutr 125:1447­1454. Rains TM, Shay NF. 1995. Zinc status specifically changes preferences for carbohydrate and protein in rats selecting from separate carbohydrate-, protein-, and fat-containing diets. J Nutr 125: 2874­2879. Rossi L, Lippe G, Marchese E, De Martino A, Mavelli I, Rotilio G, Ciriolo MR. 1998. Decrease of cytochrome c oxidase protein in heart mitochondria of copper-deficient rats. Biometals 11:207­212. Russell RM, Cox ME, Solomons N. 1983. Zinc and the special senses. Ann Intern Med 99:227­239. Rutkoski NJ, Fitch CA, Yeiser EC, Dodge J, Trombley PQ, Levenson CW. 1999. Regulation of neuropeptide Y mRNA and peptide concentrations by copper in rat olfactory bulb. Brain Res Mol Brain Res 65:80­86. Sharp P. 2004. The molecular basis of copper and iron interactions. Proc Nutr Soc 63:563­569. Stewart AJ, Blindauer CA, Berezenko S, Sleep D, Sadler PJ. 2003. Interdomain zinc site on human albumin. Proc Natl Acad Sci 100:3701­3706. Tannhauser PP. 2002. Anorexia nervosa: A multifactorial disease of nutritional origin? Int J Adolesc Med Health 14:185­191. Willis MS, Monaghan SA, Miller ML, McKenna RW, Perkins WD, Levinson BS, Bhushan V, Kroft SH. 2005. Zinc-induced copper deficiency: a report of three cases initially recognized on bone marrow examination. Am J Clin Pathol 123:125­131. Xu X, Pin S, Gathinji M, Fuchs R, Harris ZL. 2004. Aceruloplasminemia: an inherited neuro- degenerative disease with impairment of iron homeostasis. Ann N Y Acad Sci 1012:299­305. Zecca L, Youdim MB, Riederer P, Connor JR, Crichton RR. 2004. Iron, brain ageing and neuro- degenerative disorders. Nat Rev Neurosci 5:863­873.

APPENDIX B 285 Absorption Mechanisms, Bioavailability, and Metabolism of Calcium and Magnesium Connie M. Weaver Purdue University, West Lafayette, Indiana INTRODUCTION The essentiality of calcium for many vital functions has led to important discoveries regarding its metabolism and bioavailability from food and supple- ment sources. Many of the changes in bone occur during early years and there- fore much of the research to answer metabolism and requirement questions has been conducted in children and young adults. The onset of osteoporosis in the elderly and adverse consequences has also spurred research on preventative mea- sures to minimize it. Among the U.S. population, calcium intake inadequacy is common, especially among women. The role of magnesium in bone health is also well recognized and deficien- cies have adverse consequences in bone formation (brittle bones), endocrine function, possibly also disrupting mood states and sleep regulation. However, magnesium deficiencies due to dietary inadequacy in the U.S. population are not common although they can occur as comorbidity to other conditions such as malabsorption or renal disfunction. Soldiers face a variety of stressors that go from intense physical activity to the psychological stressors such as anxieties or sleep disturbances. Under mili- tary situations, whether training or in combat, these stress factors may influence the metabolism of calcium or magnesium or both, resulting in daily requirements that might be different than those for the general U.S. population. In addition, typical operational rations may be inadequate regarding the levels of these two minerals or they might be designed in a way that lowers their bioavailability. This paper describes factors that influence the metabolism and absorption of magnesium or calcium and potential risks of deficiency among military popula- tion in training or combat. FUNCTION AND CONSEQUENCES OF DEFICIENCY Calcium and magnesium are the two most important minerals for bone de- velopment and maintenance that are at risk for being deficient in the diet. The proportion of the total body's calcium in bone is 99 percent for and 60 percent for magnesium. Calcium exists as hydroxyapatite in crystals and as amorphous calcium phosphate. Magnesium is important to bone quality by controlling crys- tal growth of hydroxyapatite to prevent formation of brittle bone. Accumulation of bone mass during growth and protection of loss later in life directly and linearly predicts risk of fracture (Heaney et al., 2000).

286 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Aside from the structural functions of calcium and magnesium in bone, they are also important in sustaining other living tissues. Calcium serves as a second messenger and stabilizes key proteins. Magnesium is a co-factor for numerous enzymes and is especially associated with those involved in energy metabolism. Calcium is under homeostatic regulation to maintain serum concentrations within a narrow range. Serum magnesium decreases during depletion, but not dependably. Thus, serum levels are not considered good indicators of status for either mineral. If serum levels are not maintained by adequate intakes from the diet, the skeleton serves as a large reserve. Chronic bone resorption due to inad- equate intake of calcium is associated with decreased peak bone mass and in- creased skeletal fragility depending on the lifestage (Heaney et al., 2000; Heaney and Weaver, 2003). HOMEOSTATIC REGULATION Maintenance of serum calcium at approximately 2.5 mmol/L occurs because of coordinated actions at the gut, kidney, and skeleton in response to the hor- monal regulation through the PTH-vitamin D axis (Figure B-11) (Weaver and Heaney, 2006a). When serum calcium levels fall due to dietary deficiency, cal- cium absorption increases, tubular reabsorption increases, and bone resorption is elevated. Daily calcium transfer in average young adult women is illustrated in Figure B-12. In contrast to calcium, there is no identified hormonal regulation of magne- sium (Rude and Shils, 2006). Daily magnesium transfer is illustrated in Figure B-13 (Rude and Shils, 2006). The main site of magnesium regulation is the kidney. The kidney is very efficient at conserving magnesium during periods of low intake to maintain homeostasis. During periods of dietary deficiency, uri- nary excretion can decrease to 1 mEq/day. At the other extreme, almost all of an intravenously administered magnesium load is excreted in the urine within 24 hours. ABSORPTION MECHANISMS For both calcium and magnesium, absorption occurs predominantly in the small intestine. For both ions, absorption occurs by both saturable transcellular absorption that is physiologically regulated and nonsaturable paracellular ab- sorption that is dependent on concentrations in the lumen (Weaver and Heaney, 2006a). Absorption for both calcium and magnesium increases while fractional absorption decreases with increasing dose as shown for calcium in Figure B-14 for calcium (Fine et al., 1991; Heaney et al., 1990). The range in absorption efficiency is great for both minerals, i.e., 5 to 75 percent, but average magnesium absorption is higher than calcium.

APPENDIX B 287 FIGURE B-11 Homeostatic regulation of calcium. Copyright C.M. Weaver, 2005.

288 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Bone deposition 542 mg Plasma Bone 2.5 mmol/L Net retention ~0 Bone resorption 501 mg Endogenous Absorption Fecal excretion Urine 283 mg 121 mg 203 mg Diet 1300 mg Feces 1138 mg Gut FIGURE B-12 Daily mass calcium transfer in adult women total body 1­1.2 kg. Copyright C.M. Weaver, 2005. Plasma Muscle and soft tissue Bone [180 mg] [10 g] [20 g Total] Endogenous Fecal excretion Main point of Absorption 30 mg Urine >150 mg 120 mg regulation Diet 300 mg Feces 180 mg Gut FIGURE B-13 Daily mass magnesium transfer and [body pools] total body 28­40 g. Copyright C.M. Weaver, 2005.

APPENDIX B 289 FIGURE B-14 Effect of load on calcium absorption. SOURCE: Weaver, 2001. Used with permission. BIOAVAILABILITY Most of the major sources of calcium from foods and supplements and some fortified foods have been evaluated using sensitive isotopic tracer techniques and intrinsically labeled foods and salts. Because different applications of isotopic tracer techniques produce different absolute values for calcium absorption (Wastney et al., in press), it is important to use a common referent in bio- availability studies so that relative bioavailability among various sources can be established. For foods, the referent is typically milk, and for salts, it is calcium carbonate. A comparison of bioavailability from major sources of calcium is given in Table B-7. The most potent inhibitor of calcium absorption is oxalate. However, foods that contain oxalate vary in the degree of calcium bioavailability, from the low calcium bioavailability in spinach to soy foods with calcium bio- availability comparable to milk. Phytate is a modest inhibitor of calcium absorp- tion. Enhancers of calcium absorption have received much study, but there is no known universally consistent enhancing ingredient. Some ingredients enhance calcium absorption in single studies, but the effect disappears during chronic feeding due to adaptation, i.e., lactulose and whey (Brommage et al., 1993; Zhao et al., in press). Vegetables from the Brassica family have superior bioavailability to most other sources, but the nature of this enhancement has been elusive.

290 Needed Milk Equal Cup 1.0 8.1 9.7 3.9 2.3 4.5 1.0 1.2 1.0 1.1 3.3 3.2 9.8 Servings to 1 16.3 34.9 (mg) b 9.9 5.9 2.76 9.8 96.3 11.9 24.7 42.5 21.5 97.2 77.4 94.7 85.3 29 30.1 Absorbable Ca/serving a (%) Estimated Absorption Efficiency 8.36 5.1 32.1 26.7 24.4 21.8 53.8 61.3 32.1 32.1 39.6 40.2 49.3 91.9 22.2 3 44.7 40.5 79 35 61 44 Calcium Content (mg/serving) 300 113 303 241 239 212 347 115 Calcium (g) 86 85 71 42 42 85 85 85 85 85 15 Serving Size 240 172 110 164 Absorbable for Sources Leaves Comparing Flower Green B-7 Cheese pinto red white Food Cabbage Mustard Spinach Cookies Potatoes Choy TABLE Source Foods Milk Beans, Beans, Beans, Bok Broccoli Cheddar Cheese Chinese Chinese Chinese Kale Spinach Sugar Sweet

291 food test the of 9.5 5.8 1.0 1.2 0.88 1.3 0.74 12.8 absorption alciumc of 7.54 10.1 16.6 96.3 80.0 72 82.6 93.3 81.3 109 129 102.6 113.7 ration the for adjusting then 8.54 82.0 38.0 32.1 31.0 36.3 24 43.0 41.2 34.2 37.9 37.3 27.1 load) in 20 20 0.889­0.0964 174 300 258 300 300 300 200 300 300 250 300 = absorption index. 28 28 16.8 120 240 126 240 240 (fractional absorptive absorption. milk the Malate for load, press. ×fractional in Citrate Phosphate equation same the the content Heaney, Sulfate at Calcium using and Malate Bread Set Tricalcium tested with Cereal load calcium Calcium as Foods with Salts Carbonate Citrate Citrate Glycerophosphate for milk Weaver Wheat Bran Juice to Calcium with Milk Rhubarb Whole Wheat Yogurt Fortified Tofu, Orange Soy Bread Calcium Calcium Calcium Calcium Calcium Adjusted a relative Calculated b SOURCE:

292 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL In contrast to calcium, very little is known about magnesium bioavailability. One study measured magnesium absorption from vegetables intrinsically labeled with stable isotopes of magnesium and found true absorption ranged from 52 percent to 62 percent (Schwartz et al., 1984). Absorption was lower in bran-rich sources, presumably due to the inhibition in absorption by the phytate. Several mag- nesium salts have been evaluated for magnesium absorption using a urinary elimina- tion approach. Magnesium oxide has much lower absorption than the citrate, lactate, or hydroxide salts (Bohmer et al., 1990; Lindberg et al., 1990). Bioavailability of the chloride, lactate, and aspartate salts was similar (Firoz and Graber, 2001). Enteric- coated magnesium chloride has a much lower absorption than magnesium acetate (Fine et al., 1991). In general, ensuring that magnesium and calcium are sufficient in the diets is of larger concern than the issue of bioavailability because the impact is larger given that manipulations designed to alter bioavailability are usually modest. Both nutrients are consumed in amounts below the recommended intakes in adults (IOM, 1997); the intake inadequacies are more severe for the calcium intake of women (50th percentile of 19­30 year old is 612 mg/day versus an AI of 1,000 mg). DIETARY INTERACTIONS A schematic of nutrients which affect calcium metabolism and the points of regulation is given in Figure B-15. The most important dietary interaction for calcium is with sodium. Dietary sodium increases urinary calcium loss because of the co-transport of calcium and sodium in the kidney. In adolescent girls, this effect was greater in whites than blacks (Wigertz et al., 2005). Dietary protein increases urinary calcium loss, but does not increase net calcium loss from the body and bone resorption is not increased (Kerstetter et al., 2005; Roughead et al., 2003). Dietary protein can increase bone mass which has been associated with increased serum IGF-1 levels (Dawson-Hughes, 2003). The loss in the urine with increased protein intake appears to be offset by increased calcium absorp- tion (Kerstetter et al., 2005). A diet rich in fruits and vegetables is thought to reduce urinary calcium loss, presumably through effects on acid-base balance. This effect is thought to be at least partially related to potassium content. Because active calcium absorption is vitamin D dependent, vitamin D status influences calcium absorption. It also influences bone resorption (Weaver and Heaney, 2006b). INFLUENCE OF PHYSICAL ACTIVITY There have been several studies on the interaction of calcium intake and physi- cal activity on bone health. These are reviewed in the calcium section of Chapter 3 of

APPENDIX B 293 Intestine Protein Ca Intake Protein (IGF-1) Bone Vit. D Bone formation Ca10 (PO4)6 (OH)2 Absorption Also Mg++ on Ca Pool crystal surface Bone resorption P Ca Vit. D Kidney Salt Protein Fecal Loss Vit. D Urinary Loss K+ FIGURE B-15 Summary of diet effects on calcium metabolism. Copyright C.M. Weaver, 2005. this report. There have been few studies on the impact of physical activity on calcium and magnesium requirements or metabolism (Weaver, 2000). It is plausible that weight bearing exercise could counteract a marginally deficient diet in calcium for bone strength because it can increase bone geometry (Specker and Binkley, 2003). On the other hand, physical activity could increase mineral losses from the body which could increase their requirement. The most likely route of loss is dermal, especially during situations of high heat and humidity. Our understanding of dermal losses is covered in this report (See Haymes, 2005 in this Appendix). SUMMARY Calcium and magnesium are two minerals frequently at risk for deficiency in the diet. These minerals are important for bone health as well as for many other functions. They may be at greater risk of loss during physical activity through sweat loss. The dietary component that most negatively affects calcium retention is sodium. This effect is greater in whites than blacks. Achieving ad- equate intakes are more important than concerns over bioavailability or dietary interactions because of the larger impact on nutrition for these two minerals. REFERENCES Bohmer T, Roseth A, Holm H, Weberg-Teigen S, Wahl L. 1990. Bioavailability of oral magnesium supplementation in female students evaluated from elimination of magnesium in 24-hour urine. Magnesium and Trace Elements 9:272­278.

294 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Brommage R, Binacua C, Antille S, Carrie AL. 1993. Intestinal calcium absorption in rats is stimu- lated by dietary lactulose and other resistant sugars. J Nutr 123:2186­2194. Dawson-Hughes B. 2003. Interaction of dietary calcium and protein in bone health in humans. J Nutr 133:852S­854S. Fine KD, Santa Ana CA, Porter JL, Fordtran JS. 1991. Intestinal absorption of magnesium from food and supplements. J Clin Invest 88:396­402. Firoz M, Graber M. 2001. Bioavailability of U.S. commercial magnesium preparations. Magnesium Research 14:257­262. Heaney RP, Weaver CM. 2003. Calcium and Vitamin D. In: Bilezikian JP, ed. Endocrinology and Metabolism Clinic of North America: Osteoporosis 32:181­194. Philadelphia, PA: Elsevier Science. Heaney RP, Weaver CM, and Fitzimmsons ML. 1990. The influence of calcium load on absorption fraction. Am J Clin Nutr 5(11):1135­1138. Heaney RP, Abrams S, Dawson-Hughes B, Looker A, Marcus R, Matkovic V, Weaver CM. 2000. Peak Bone Mass. Osteoporosis Intl 11:985­1009. IOM (Institute of Medicine). 1997. Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride. Washington, DC: National Academy Press. Kerstetter JE, O'Brien KO, Caseria DM, Wall DE, Insogna KL. 2004. The impact of dietary protein on calcium absorption and kinetic measures of bone turnover in women. J Clin Endoc Metab 90(1):26­31. Lindberg JS, Zobitz MM, Poindexter JR, Pak CYC. 1990. Magnesium bioavailability from magne- sium citrate and magnesium oxide. J Amer Coll Nutr 9:48­55. Roughead K, Johnson LK, Lykken GI, Hunt JR. 2003. Controlled high meat diets do not affect calcium retention or indices of bone status in healthy postmenopausal women. J Nutr 133:1020­ 1026. Rude RK. 2006. Magnesium. In: Shils ME, Shike M, Ross AC, Caballero B, Cousins RJ, eds. Modern Nutrition in Health Disease. 10th Ed. Lippincott Williams & Wilkins. Rude R, Gruber H. 2004. Magnesium deficiency and osteoporosis: Animal and human observations. J Nutr Biochem 15:710­716. Rude RKG, Wei LY, Frausto A, Mills BG. 2003. Magnesium deficiency: Effect on bone and mineral metabolism in the mouse. Calcified Tissue Int 72:32­41. Schwartz R, Spencer H, Welsh JJ. 1984. Magnesium absorption in human subjects from leafy veg- etables intrinsically labeled with stable28Mg. Amer J Clin Nutr 39:571­576. Specker B, Binkley T. 2003. Randomized trial of physical activity and calcium supplementation on bone mineral content in 3- to 5-year old children. J Bone Miner Res 18:885­892. Wastney ME, Zhao Y, Weaver CM. 2006. Kinetic Studies. In: Weaver CM, Heaney RP, eds. Cal- cium and Human Health. Humana Press. Weaver CM, Heaney RP. 2006a. Calcium. In: Shils ME, Shike M, Ross AC, Caballero B, Cousins RJ, eds. Modern Nutrition in Health Disease. 10th Ed. Lippincott Williams & Wilkins. Weaver CM, Heaney RP. 2006b. Food Sources, Supplements, and Bioavailability. In: Weaver CM, Heaney RP, eds. Calcium and Human Health. Humana Press. Wigertz K, Palacios C, Jackman LA, Martin BR, McCabe LD, McCabe GP, Peacock M, Pratt JH, Weaver CM. 2005. Racial differences in calcium retention in response to dietary salt in adoles- cent girls. Am J Clin Nutr 81:845­850. Zhao Y, Martin BR, Wastney ME, Schollum L, Weaver CM. 2005. Acute versus chronic effects of whey proteins on calcium absorption in growing rats. Exp Biol Med 230(8):536­542.

APPENDIX B 295 Drinking Water as a Source of Mineral Nutrition Gerald F. Combs, Jr. USDA-ARS Grand Forks Human Nutrition Research Center, Grand Forks, North Dakota INTRODUCTION This paper describes the content of minerals in drinkable water and explores the possibility that consumption of water during foreign deployments of the military personnel can substantially increase the daily intake of essential miner- als. The diverse sources of water consumption might result in substantial varia- tions on water mineral levels. Consumption of water could then be factored in when planning levels of these minerals in meals for individuals or populations. It is possible to use water as a vehicle to deliver minerals the intakes of which are otherwise inadequate. The applicability of this strategy to military popula- tions is explored. Also, the relevance of obtaining water from various sources is addressed; the importance of this variability in the context of provision of a wide range of mineral levels is discussed. MINERALS IN NATURAL WATER SUPPLIES The mineral contents of lakes and rivers vary with climate, local geology, the type and extent of local agriculture and extent of urbanization (Bowen, 1979) (Table B-8). In temperate areas, calcium is the dominant mineral element in fresh water, while rivers draining arid areas tend to be rich in sodium (Na) and chlorine (Cl), and tropical rivers, being more dilute in general, tend to contain greater concentrations of iron (Fe) and silicon (Si). Thus, fresh water supplies can provide nutritionally important amounts of calcium (Ca), magnesium (Mg), Fe, manganese (Mn). Industrialized countries have used municipal water as a vehicle for providing fluorine. In a few locales, surface run-off from selenium- rich soils has been found to contain biologically significant amounts of that element; but such cases are few and most water supplies are very low in sele- nium (Se). Few water supplies contain appreciable amounts of copper (Cu); however, the use of copper piping between municipal reservoirs and consumers' taps can increase the amounts of the element if the water is soft. For example, Angino (1979) found 16 percent of U.S. tap water samples to contain at least 0.2 mg Cu per L, and 6 percent to contain 0.5 mg Cu per L. Drinking water in Boston and Seattle has been estimated to provide 0.46 (Sparrow et al., 1982) and 1.3­2.2 mg Cu daily (Sharrett et al., 1982), respectively. River waters typically contain fairly large amounts of organic matter that can act as ligands for such trace elements as Cu, Fe, Mn, molybdenum (Mo), nickel (Ni), and zinc (Zn). However, the hydrolysis and formation of chelates

296 + 2 ­ ­2 3 4 I ,colloidal 4 ­2 3 + 3 + ,CrO Fe(OH) 3 ,CuCO + ,CH ,MgPO 3 ,SeO4 + ­ 3 ,MnCl ­2 4 ,NiCO ­2 4 ­2 ,ZnOH +2 +2 +2 ­ + l ,MgF g n +2 +2 ­ ,IO + a i n B(OH) Ca C Cr(OH) CuOH F Colloidal, ­ Probable Species* K M MoO N HPO SeO3 µg µg µg µgI µgM µgN µg L*] µg µg ­1 per L mg g (range) (0.2­50) (0.05­12) mg (0.03­70) (50­70) g (0.03­21) (4­10) g (0.13­43) (60­88) (0.052­0.2) µgZ mg mg 4.44 0.3 0.25 1.3 2 1.29 0.2 0.56 0.2 0.03 Seawater Mean [amount 19.35 60 10 10.77 60 412 399 Sea-Water and Fresh- in ­2 4 complexes ,CrO complexes 3 ,org. 3 + ­2 ­ Elements 4 4 ­2 ,org. +2 +2 +2 ­ n + +2 PO +2 ­ + i 2 n Probable Species B(OH) Ca Cl Cr(OH) CuOH Colloidal ­ K Mg MoO Na SeO3 Mineral Essential µgF µg µgM µg mg mg µg µg µgI mg µg mg µgN mg µgH µg µgZ L] per (range) (7­500) (2­120) (1­35) (0.1­6) (0.2­30) (50­2,700) (10­1,400) (0.5­7) (0.5­10) (0.4­6) (0.02­130) (0.03­10) (0.7­25) (0.02­27) (1­300) (0.2­1) (0.2­100) Nutritionally 5 15 7 1 3 2.2 4 8 0.5 6 0.5 0 0.2 15 (1979). Freshwater Mean [amount 100 500 B-8 *Bowen TABLE Element B1 Ca Cl Cr Cu F Fe I2 K Mg Mn Mo Na Ni P2 Se Zn SOURCE:

APPENDIX B 297 may be relatively slow processes, taking days to years to form turbid solutions; such reactions may not have come to equilibrium before river water enters the ocean so that many minerals enter ocean waters in forms that are poorly avail- able for biological utilization. The concentrations of the dominant minerals in seawater [Na, potassium (K), Mg, Ca] vary somewhat, but the mixing of the oceans results in each being in a fairly stable ratio with Cl (Bowen, 1979) (Table B-8). Concentra- tions of minor mineral constituents with relatively short residence times, how- ever, can vary according to depth or location, or both. Some [Ca, phosphorus (P), Si] are markedly depleted in the upper layers due to removal by plankton or biological precipitation; others (Cu, Fe, Mn, Se, Zn) can be relatively high in surfaces waters fed by river inputs from mineralized areas or industrialized communities. Soluble forms of minerals can exist in cationic (Ca+2, Cu+2, Fe+2, Fe+3, K+, Mg++, Mn++, Na+, Ni+, Zn++) and anionic (B4O7 , Cr2O7 , F­, I­, ­2 ­2 MoO4 , PO4 , SeO3 , SeO4 ) species; in ocean water they exist bound to ­2 ­2 ­2 ­2 various ligands (e.g., to hydroxyl or ketone ligands for B, Cr, Cu, Fe, I, Mo, P, Se, Si, Zn; to chlorine ligands for Mn; or to carbonate ligands for Cu, Ni, Zn). Insoluble forms of minerals precipitate as sediments; those near the mouths of rivers typically contain large amounts of Al and Fe as insoluble hydroxides. The main types of sediments in ocean waters include CaCO3 from the re- dissolved skeletons of marine organisms, silica from the re-dissolved skeletons of diatoms and other organisms, and Fe-containing, aluminosilicate red clays derived from continental rocks. Colloidal hydroxides of Fe, Mn and Se are known to form at the pH of seawater. Such polymers can scavenge polyvalent elements. Because their formation is dependent on redox potential, the rela- tively low concentrations of dissolved O2 at lower depths (< 1 mg L­1) results in Fe and Mn being reduced to their soluble ions (Fe+2, Mn+2) that are absorbed on colloidal hydroxides. ENTERIC ABSORPTION OF MINERALS FROM WATER The absorption and post-absorptive utilization of minerals depends on its chemical form as well as the presence or absence in the gut of factors of dietary origin that can affect those processes. For example, water-born Se (selenite, selenate) is passively absorbed at somewhat lower efficiencies (60­80 percent) than the selenoaminoacids in foods (90­95 percent) that are actively transported across the gut (Combs and Combs, 1986). The utilization of non-heme Fe can be markedly improved by including in the diet sources of ascorbic acid (e.g., or- anges) or meats both of which promote the utilization of non-heme Fe (Gordon and Godber, 1989). In similar fashion, the presence of citrate or histidine in the gut can enhance the absorption of ingested Zn, and dietary ascorbate can en- hance the antagonistic effect of Fe on Cu utilization (Salovaara et al., 2002; Swain et al., 2002).

298 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL There is no reason to think that minerals consumed in drinking water are not subject to the same determinants of bioavailability that affect their utilization from in foods. However, for the most part the bioavailability of minerals from water has not been broadly studied. Some of the determinants known to affect bioavailability of minerals from foods are constituents of the foods consumed: phytate, phosphorus and triglycerides can each reduce the lumenal solubility and, hence, the absorption of Ca; calcium absorption is also decreased by in- creasing levels of oxalic acic (see also Weaver 2005 in this Appendix). Phytate and other non-fermentable fiber components can and reduce the absorption of Zn, Mg, Fe and P. Other determinants relate to either foods or water: sulfides can react with Cu to form insoluble CuS; minerals that share transporters can be mutually inhibitory for absorption, eg., sulfite and selenite, Cd and Zn, Zn and Cu, Ca and Fe; the bioavailability of the divalent cations (Ca++, Fe++, Cu++, Zn++) can be enhanced by certain chelating substances (e.g., unidentified factors in meats, ascorbic acid in fruits) and pro-biotic factors (e.g., inulin and other fructo-oligosaccharides) (see Boeckner et al., 2001; Pallauf and Rimbach, 1997). For these reasons, the utilization of minerals from drinking water may de- pend as much on the contents of inhibitor and promoter substances also present in the gut as it does on the chemical form(s) of minerals consumed in the water. In general, the bioavailability of water-borne Fe may be improved by minimiz- ing foods containing phytates and polyphenols and by including meats and sources of ascorbic acid. Similarly, the utilization of water-born Ca should be optimized by minimizing oxalate-containing vegetables (e.g., spinach, rhubarb, beet greens, chard); and the utilization of water-born Ca, Fe, Mg, P, or Zn may be enhanced using diets low in unrefined (> 90 percent extraction), unfermented cereal grains and high-phytate products (see also Hunt 2005 in this Appendix). MINERALS IN PROCESSED WATER The processing of water, by distillation or reverse osmosis, is widely prac- ticed, including for U.S. military needs, and is rapidly growing as the principle source of new fresh water worldwide. In addition to procuring water for human consumption according to the EPA standards, these methods yield water of high purity and, thus, very low mineral content. Such water is highly corrosive which may result in minerals leaching in water such as zinc and iron but depending on the nature of the pipe. To minimize concerns about human toxicity with the potential leaching of heavy metals water processors typically add back selected mineral salts in a step referred to as "stabilization." As a whole, this purification/reconstitution process can result in mineral contents differing widely between the water product and the original source. Few commercially bottled waters not labeled as "mineral water" on the American market contain appreciable amounts of minerals. In 2003, the World Health Organization (WHO) convened an expert panel to review the evidence of health effects associated with drinking and cooking

APPENDIX B 299 water mineral content (WHO, 2004). The panel reviewed more than 80 observa- tional studies conducted in 1957­2003; most, but not all, showed inverse rela- tionships of the hardness of water supplies and the risk of cardiovascular disease (particularly, ischemic heart disease) (Calderon and Craun, in press; Craun and Calderon, in press; Monarca et al., 2003; Monarca et al., in press; Nardi et al., 2003). While the panel was unable to determine whether that relationship in- volved Ca, Mg or the combination of those elements, the primary contributors to hardness, it pointed out that low Mg intakes have been shown to increase su- praventricular beats in humans (Klevay and Milne, 2003). The panel also cited evidence that the consumption of water with high levels of Ca does not increase, but may reduce the risk of calcium oxalate urinary stones (Donato et al., 2003), unless calcium supplements were taken separate from consumption of dietary oxalate from food (Curhan et al., 1993, 1997, 2004). There are no data, however, to suggest that water-borne minerals are utilized any differently from the same minerals in foods. Available data for Ca and Mg indicate that these minerals are, in fact, widely under-consumed. The Institute of Medicine (1997) estimated that the median daily intakes of 19­30 year old Americans were: 61 percent (females) and 95 percent (males) of Adequate Intake (AI) levels for Ca, and 66 percent (females) and 82 percent (males) of Recommended Dietary Allowance (RDA) levels for Mg. Data on intake of minerals by military personnel is scant. The few studies that have collected such data reported that magnesium intakes from foods may be insufficient for much of the population and also that women, in general, may be at a higher risk of mineral deficiencies because they tend to consume less food. Therefore, water-borne minerals can contribute to the mineral nutrition of such individuals, particularly under conditions requiring high fluid intakes. WATER CONSUMPTION Water, the largest single constituent of the body, is tightly regulated and must be maintained within 1­2 percent balance to sustain thermoregulation and physical work capacity (IOM, 2004). This is accomplished by consuming water in proportion to body water losses. Because water losses are subject to environ- mental factors, water requirements can vary widely (Grandjean et al., 2003; IOM, 2004). Water is lost from the body through urine (typically, 1­2 L/day), feces (approximately 100 ml/day), insensible respiratory, trans-epidermal and evaporative losses (cumulatively, approximately 450 ml/day), and sweat. Sweat losses vary considerably (1­8 + L/day) according to environmental temperature and humidity, and to endogenous heat production during physical activity; under extreme conditions, individuals can produce as much as 3­4 L/hour. In 2003, the IOM set Adequate Intake (AI) for water (2.7 L/day for females, 19­30 years, and 3.7 L/day for males, 19­30 years, assuming that approximately 20 percent would come from foods) (IOM, 2004). Water from food can vary from 0.5­1 L/day; the

300 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL TABLE B-9 Potential Nutritional Contributions of Minerals Occurring in Freshwater Supplies Daily Need Reported Water % Daily Need (range) Met, by Rate of (19­30 y Mineral (/L), Water Consumption Element male) Mean (range)a 2 L/day 4 L/day Ca 1,000 mgb 15 3 6 (2­120) mg (0.4­24) (1­48) Cl 2,300 mgb 7 1 1 (1­35) mg (0.1­3) (0.2­6) Cr 35 µgb 1 6 12 (0.1­6) µg (1­34) (2­64) Cu 9 mgc 3 1 1 (0.2­30) µg (0.02­7) (0.1­13) F 4 mgb 100 5 10 (50­2,700) µg (3­135) (6­270) Fe 8 mgc 500 13 25 (10­1,400) µg (0.3­35) (1­70) I 150 µgc 2 3 5 (0.5­7) µg (1­9) (3­19) K 4,700 mgb 2.2 0.1 0.2 (0.5­10) mg (0.02­0.4) (0.04­1) Mg 400 mgb 4 2 4 (0.4­6) mg (0.2­3) (0.4­6) Mn 2.3 mgb 8 1 1 (0.02­130) µg (<0.01­11) (<0.01­23) Mo 45 mgc 0.5 <0.01 <0.01 (0.03­10) µg (<0.01­0.04) (<0.01­0.1) Na 1,500 mgb 6 (0.7­25) mg 1 2 (0.1­3) (0.2­7) P 700 mgc 20 6 11 (1­300) µg (0.3­86) (0.6­171) Se 55 µgc 0.2 1 1 (0.2­1) µg (1­4) (1­7) Zn 11 mgc 15 0.3 1 (0.2­100) µg (<0.01­2) (<0.01­4) SOURCE: Bowen (1979); Adequate Intake from IOM (1997, 2001, 2004); Recommended Dietary a b c Allowance from IOM (1997, 2000, 2001). balance of daily water need must be met by consuming fluids. Thus, under practical circumstances, total fluid requirements can range from 2­16 L/day (IOM, 2004; Sawka and Montain, 2003) (Figure B-16). Reports from the mili- tary point to water consumption of about 3 L/day when soldiers are under garri- son training (J. Kent and S. Corum, personal communication, U.S. Army, Au- gust 24, 2005) or 4­5 L/day when they are in sustained operations (IOM, 2005).

APPENDIX B 301 6 L/day 8 L/day 10 L/day 12 L/day 14 L/day 16 L/day 9 12 15 18 19 21 (1­72) (2­96) (2­120) (2­140) (3­168) (4­190) 2 3 4 5 5 6 (0.2­10) (0.3­13) (0.5­19) (1­22) (1­25) (1­26) 18 24 30 36 42 24 (3­98) (4­128) (5­162) (6­192) (7­226) (8­256) 2 3 3 4 5 6 (0.1­20) (0.2­27) (0.2­33) (0.2­40) (0.3­47) (0.4­54) 15 20 25 30 35 40 (8­400) (12­540) (15­670) (16­800) (20­940) (24­1,040) 38 50 63 76 88 100 (1­105) (1­140) (2­175) (2­210) (2­245) (2­280) 8 10 13 16 18 10 (4­28) (6­38) (7­47) (8­56) (10­66) (12­76) 0.3 0.4 1 1 1 1 (0.1­1) (0.1­2) (0.1­2) (0.2­2) (0.2­3) (0.2­4) 6 8 10 12 14 16 (1­9) (1­12) (1­15) (2­18) (2­21) (2­24) 2 2 3 4 4 4 (<0.01­34) (<0.01­46) (<0.01­57) (<0.01­68) (0.01­80) (0.01­96) 0.01 0.01 0.01 0.02 0.02 0.02 (0.01­0.1) (0.01­0.2) (0.01­0.2) (0.02­0.2) (0.02­0.3) (0.02­0.4) 2 3 4 4 6 6 (0.3­10) (0.4­13) (0.5­17) (0.6­20) (0.7­23) (1­26) 17 22 23 34 34 44 (1­257) (1­342) (2­428) (2­514) (3­600) (2­684) 2 3 3 4 5 6 (2­11) (2­14) (3­18) (4­22) (4­25) (4­28) 1 1 1 2 2 2 (<0.01­5) (<0.01­7) (<0.01­9) (0.02­10) (0.03­13) (0.03­14) IMPLICATIONS OF USING PROCESSED DRINKING WATER The mineral nutritional value of drinking water depends on both its mineral content and level of consumption. The use of "hard" waters can provide signifi- cant amounts of Ca, Mg, Mn, and Fe, although the latter is likely to be present in colloidal forms of limited direct nutritional value. Under circumstances of high water consumption (e.g., > 6 L/day), many natural waters can also be significant sources of Cr, F, Fe, I, and P; in fact, some water sources can provide RDA

302 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL 18 5,600 kcal/d 16 L/d 14 4,400 kcal/d 12 3,500 kcal/d 10 requirement, 3,800 kcal/d 8 6 water 1,800 kcal/d 4 daily 2 0 10 15 20 25 30 35 daily mean wet bulb temperature, degrees C FIGURE B-16 Daily water requirement as a function of environmental temperature and total energy expenditure. SOURCE: Sawka and Montain (2001). Used with permission from the International Life Sciences Institute. levels of those minerals (Table B-9). However, most processed waters are very poor sources of minerals unless they have been re-mineralized during "stabiliza- tion." In many cases, processed drinking water, including commercially bottled water may provide little, if any, essential nutrients. Variability of mineral levels among waters for consumption in military settings does not appear to be a factor to be considered when estimating amounts of minerals needed in operational rations or menus. Drinking water represents, however, a potential vehicle for delivering essen- tial minerals to troops many of whom will have high fluid intakes during hot weather duty. This presents opportunities for the military to develop mineral content standards for drinking water produced or purchased for troop consump- tion. Such standards could be useful in ensuring adequate intakes of key miner- als not easily achieved by dietary means. REFERENCES Angino, EE. 1979. Geochemistry of drinking water as affected by distribution and treatment. In: Angino EE, Sandstead HH, Comstock GW, Corbett RG, Strong JP, Voors AW, eds. Geochem- istry of Water in Relation to Cardiovascular Disease. Washington, DC: National Academy Press.

APPENDIX B 303 Boeckner LS, Schnepf MI, Tungland BC. 2001. Inulin: A review of nutritional and health implica- tions. Adv Food Nutr Res 43:1­63. Bowen HJM. 1979. Environmental Chemistry of the Elements. New York: Academic Press. Pp. 13­29. Calderon R, Craun GF. The association of cardiovascular disease risks with water hardness: A review of the epidemiological studies published from 1957­1979. In: Cotruvo J, Fawell J, eds. Nutrients in Drinking Water and the Potential Health Consequences of Long-Term Consump- tion of Demineralized and Remineralized and Altered Mineral Content Drinking Waters Wash- ington, DC: ILSI Press; In press. Combs Jr, GF, Combs SB. 1986. The Role of Selenium in Nutrition and Health. Academic Press, New York, Pp. 179­182. Craun G, Calderon R. The association of cardiovascular disease risks with water hardness: Interpret- ing the epidemiological evidence. In: Cotruvo J, Fawell J, eds. Nutrients in Drinking Water and the Potential Health Consequences of Long-Term Consumption of Demineralized and Remineralized and Altered Mineral Content Drinking Waters Washington, DC: ILSI Press; In press. Curhan GC, Willett WC, Rimm EB, Stampfer MJ. 1993. A prospective study of dietary calcium and other nutrients and the risk of symptomatic kidney stones. N Engl J Med 328(12):833­838. Curhan GC, Willett WC, Speizer FE, Spiegelman D, Stampfer MJ. 1997. Comparison of dietary calcium with supplemental calcium and other nutrients as factors affecting the risk for kidney stones in women. Ann Intern Med 126(7):497­504. Curhan GC, Willett WC, Knight EL, Stampfer MJ. 2004. Dietary factors and the risk of incident kidney stones in younger women: Nurses' Health Study II. Arch Intern Med 164(8):885­891. Donato F, Monarca S, Premi S, Gelatti U. 2003. Durezza dell'acqua potablie e mallatie cronico- degenerative. Parte III. Patologie tumorali, urolitiasi, malformazioni fetali, deterioramento delle funzioni cognitive nell'anziano, diabete mellito ed eczema atopico. Ann Ig 15:57­70. Gordon DT, Godber JS. 1989. The enhancement of nonheme iron bioavailability by beef protein in the rat. J Nutr 119:446-452. Grandjean AC, Reimers KJ, Buyckx ME. 2003. Hydration: Issues for the 21st century. Nutr Rev 61:261­271. IOM (Institute of Medicine). 1997. Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D and Fluoride. Washington, DC: National Academy Press. IOM. 2000. Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium and Carotenoids. Wash- ington, DC: National Academy Press. IOM. 2001. Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Cop- per, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium and Zinc. Washington, DC: National Academy Press. IOM. 2004. Dietary Reference Intakes for Water, Potassium, Sodium, Chloride and Sulfate. Wash- ington, DC: The National Academies Press. Klevay LM, Milne DB. 2003. Low dietary magnesium increases supraventricular ectopy. Am J Clin Nutr 57:550­554. Monarca S, Donato F, Zerbini I. In press. Drinking water hardness and cardiovascular diseases. In: Cotruvo J, Fawell J, eds. Nutrients in Drinking Water and the Potential Health Consequences of Long-Term Consumption of Demineralized and Remineralized and Altered Mineral Content Drinking Waters Washington, DC: ILSI Press. Monarca S, Zerbini I, Simonati C, Gelatti U. 2003. Durezza dell'acqua potablie e mallatie cronico- degenerative. Parte II. Malattie cardiovasculari. Ann Ig 15:41­56. Nardi G, Donato F, Monarca S, Gelatti U. 2003. Durezza dell'acqua potablie e mallatie cronico- degenerative. Parte I. Analisi dell recherché epidemiologiche. Ann Ig 15:35­40. Pallauf J, Rimbach G. 1997. Nutritional significance of phytic acid and phytase. Arch Tierernahr 50:301­319.

304 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Salovaara S, Sandberg AS, Andlid T. 2002. Organic acids influence iron uptake in the human epithe- lial cell line Caco-2. J Agric Food Chem 50:6233­6238. Sawka MN, Montain SJ. 2001. Fluid and electrolyte balance: Effects on thermoregulation and exer- cise in the heat. In: Bowman BA, Russell RM, eds. Present Knowledge in Nutrition. 8th edi- tion. Washington, DC: ILSI Press. Sharrett AR, Carter AP, Orheim RM, Feinleib M. 1982. Daily intake of lead, cadmium, copper, and zinc from drinking water: The Seattle Study of Trace Metal Exposure. Environ Res 28: 456­475. Sparrow D, Silbert JE, Weiss ST. 1982. The relationship of pulmonary function to copper concentra- tions in drinking water. Am Rev Respir Dis 126:312­315. Swain JH, Tabatabai LB, Reddy MB. 2002. Histidine content of low-molecular-weight beef proteins influences nonheme iron bioavailability in Caco-2 cells. J Nutr 132:245­251. WHO (World Health Organization). 2004. Nutrient Minerals in Drinking-Water and the Potential Health Consequences of Consumption of Demineralized and Remineralized and Altered Mineral Content Drinking-Water: Consensus of the Meeting. [Online]. Available: http://www.who.int/ water_sanitation_health/dwq/nutconsensus/en/print.html [accessed January 31, 2006]. Assessment of Zinc, Copper, and Magnesium Status: Current Approaches and Promising New Directions Carl L. Keen and Janet Y. Uriu-Adams University of California-Davis INTRODUCTION It is intuitively well understood that optimal nutritional status can favorably impact health and performance. However, the extent to which marginal deficien- cies of essential micro- and macro-nutrients might affect the performance of typically healthy individuals, including military personnel who are presumably consuming well-balanced diets is less appreciated. Even in well developed coun- tries, marginal deficiencies of several essential nutrients including zinc, copper, and magnesium are common. The frequency of these deficiencies can increase under stressful conditions. For example, military personnel undergoing energy restriction, sleep deprivation, and physical, environmental, and psychological stress during combat are at risk for suboptimal nutrition due to reduced nutrient intake, as well as stress-induced alterations in mineral metabolism and homeo- stasis. Deficiencies of these minerals can impair daily activities and functions, reduce the rate of wound healing and recovery from injuries, and increase the risk for infections due to a compromised immune function. Given the above, the assessment of an individual's nutritional status is clearly a high priority within the military. However, these assessments are often challenging as many methods currently in use lack both precision and accuracy. This paper will address the current biomarkers used to assess zinc, copper, and magnesium status, as well as the limitations of these assessments. The potential for using novel metabolic

APPENDIX B 305 profiles such as metabolomics, metallomics or breathomics as new and promis- ing methods for nutrient status assessment will also be discussed. CAUSES OF MINERAL DEFICIENCIES Mineral deficiencies can arise through multiple mechanisms (Keen et al., 2003a). Primary deficiencies are those that occur as a result of low dietary in- takes of the micronutrient. Recent reports of mineral intakes show that zinc, copper and magnesium are low in select populations even in developed countries (Briefel et al., 2000; Champagne et al., 2004; Ford and Mokdad, 2003; Olivares et al., 2004; Tarasuk et al., 2005). However, a simple inspection of an individual's dietary intake provides limited information regarding their nutritional status as multiple factors (genetic, disease, drugs, physiological, and environmental stres- sors) can cause secondary, or `conditioned' mineral deficiencies. Genetic factors including mutant genes, polymorphisms, and multiple gene defects can result in abnormal zinc and copper metabolism. For example, acrodermatitis enteropathica (characterized by zinc deficiency), is caused by mutations in the ZIP4 zinc trans- porter (Wang et al., 2002). Menke's disease (copper deficiency) and Wilson's disease (copper toxicity) are due to mutations in the copper-transporting AT- Pases, ATP7A and ATP7B, respectively (Harris, 2000). Interactions between minerals and food components including phytates, fiber, vitamins, and other minerals can reduce mineral absorption and result in a mineral deficiency in an individual despite their having a seemingly "adequate" level of intake of the mineral. A substantial body of literature has documented that drugs and certain chemicals or toxicants can produce a secondary mineral deficiency by chelating metals and decreasing their absorption, increasing their excretion, or both. Disease-associated changes in micronutrient metabolism have been noted in dia- betes. This is of particular concern given the increase in the frequency of diabe- tes that is being seen in numerous populations. Similarly, physiological stress, infection, or conditions of inflammation such as cardiovascular disease and obe- sity, can produce an acute phase response, and a subsequent re-distribution of minerals in body tissues. Lastly, excessive loss of micronutrients from sweat has been noted, a concern for individuals undergoing physical exertion, particularly under harsh environmental conditions. Urinary and fecal mineral losses during intense exercise can also be larger than during resting periods. Nutritional Biomarkers There is an essential need for an accurate evaluation of an individual's min- eral status. Ideally, the biomarker(s) used would be highly sensitive, and highly specific. These markers would be substances that reflect the activity of an en- zyme or process that is directly or indirectly impacted by a deficiency of the specific nutrient. That is, during a nutrient deficiency, the product, or precursor,

306 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL of an enzymatic reaction or process is increased, or decreased, in the blood, urine, or breath. Moreover, significant changes in the concentration of the biomarker should occur prior to extensive tissue damage. There are several issues that must be considered before choosing a nutri- tional biomarker (Box B-2). Some center around methodology such as whether the method is reliable and reproducible, whether the assay is robust enough to detect small changes over background, the length of time it takes to perform the assay (i.e., minutes, hours, or days), the risks for false positives or negatives, whether the assay can be performed in the field versus in a hospital or labora- tory, and issues regarding the stability of the biomarker prior to analysis (e.g., transport and storage issues). Other key concerns relate to the interpretation of the results. For any biomarker that is used for nutritional status assessment, it is important to establish reference ranges for various populations, as well as the within-person and between-person variance. In cases where the individual vari- BOX B-2 Questions in Choosing a Nutritional Biomarker Is the method reliable and reproducible? Do appropriate reference ranges exist for the population being studied? Is the assay robust enough (signal to noise) to be of practical value? Can the assay be done within a short time period? Is the within-person and between-person variance known? If the variance between individuals is larger than the within-person variance, do we need to maintain longitudinal records for each individual? Is there a high risk for false positives, or false negatives? Are there issues of timing relative to dietary exposure: recent versus usual intakes, acute versus chronic exposure? Is the type of measurement a direct measure (static indicator) or functional assay? Does the subject need to fast prior to the collection of a sample? What is the stability of the marker? (Can the sample be easily transported?) Does an acute change in the "marker" reflect an immediate risk, or the potential for risk?

APPENDIX B 307 ance is larger than the within-person variance, it might be necessary to establish longitudinal records for each individual. It would be important to determine whether fasting prior to the collection of a sample affects the biomarker, as well as whether the biomarker value can accurately distinguish between recent intake versus usual intakes, and between acute versus chronic exposure. Moreover, the questions of whether an acute change in the biomarker reflects an immediate risk, or the potential for risk, should be determined. Zinc Over 200 diverse metalloproteins that are involved in carbohydrate, protein, lipid, and nucleic acid metabolism require zinc as a cofactor. Zinc can act at the catalytic site as well as having stabilizing or regulatory effects. Currently, zinc status is typically assessed by measuring the zinc concentration in easily acces- sible pools such as plasma, serum, or hair, or blood cells such as erythrocytes or lymphocytes (Hambidge, 2003; IOM, 2002b). The activity of zinc-dependent enzymes such as angiotensin converting enzyme (ACE) or extracellular superox- ide dismutase (SOD) has also been used. Zinc-regulated genes such as metal- lothionein have been proposed to be useful as indices of zinc status (Cao and Cousins, 2000; Liuzzi and Cousins, 2004). The measurement of certain hor- mones and growth factors such as growth hormone and insulin-like growth fac- tor (IGF) have also been proposed to be sensitive to zinc status, however, these markers show less sensitivity. The majority of the above markers are to a large extent reflective of the zinc status of the blood pool. In this regard it is important to note that approximately 91 percent of body zinc in a normal adult is found in muscle, bone, and liver while blood represents less than 0.1 percent of total body zinc (King and Keen, 1994). Moreover, of the 0.1 percent of zinc that is in blood, only 12­22 percent of that is present in plasma. Thus, plasma represents a very small fraction of total body zinc. Numerous factors can decrease plasma or se- rum zinc concentrations including pregnancy, oral contraceptive use, and infec- tion or stress which precipitate an acute phase response, while other factors can increase plasma and serum zinc concentrations including fasting, or muscle in- jury where tissue breakdown occurs. Additionally, circadian rhythms can affect plasma and serum zinc concentrations and, thus, the time of day the sample is taken must be taken into account when evaluating the results. Given the above, it has been suggested that a suite of markers, as opposed to single markers, be used to more accurately ascertain zinc status. It is well known that zinc deficiency can result in hypogeusia (taste impairment). A report by Takeda et al. (2004) found that in patients with zinc deficiency-related hypogeusia, serum zinc was within the normal range, however, the ratio of ACE activity (apo-ACE/holo-ACE) was a more sensitive indicator of zinc status than serum zinc. The discovery of the ZnT (SLC30/CDF) and ZIP (Zrt/IRT-like proteins) families of zinc transporters involved in the regulation, export, and import of zinc in a variety of tissues has

308 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL prompted the question of whether the protein or mRNA of these proteins could be used as biomarkers for zinc status. Using quantitative real-time RT-PCR, it has been shown that modest dietary zinc supplementation in humans (15 mg Zn/ day for 10 days) increased metallothionein and ZnT1 mRNA, and decreased Zip3 mRNA in dried spots of whole blood indicating that these targets were responsive to zinc supplementation (Aydemir et al., 2006). In addition, since zinc deficiency increases oxidative stress, it has been suggested that perhaps a signature of oxidative stress markers might be used to assess zinc status (see below). Copper Copper is an essential mineral that is involved in numerous electron transfer reactions due to the metal's redox cycling capability. Copper deficiency can adversely affect energy production, glucose and cholesterol metabolism, iron metabolism, hematopoietic and immune systems, oxidative defense system, neu- ropeptide synthesis and processing, and heart and vessel integrity and function (Keen et al., 2003b; Uriu-Adams and Keen, 2005). While severe copper defi- ciency is uncommon, marginal copper deficiency may be prevalent even in de- veloped countries (IOM, 2002a; Uriu-Adams and Keen, 2005). Exercise, infec- tion, inflammation, diabetes and hypertension, and the consumption of zinc supplements can adversely affect copper metabolism (IOM, 2002a; Uriu-Adams and Keen, 2005) and precipitate a sub-clinical copper deficiency. Currently, copper status is commonly assessed by analyzing the concentra- tion of copper in plasma, serum, or blood cells such as platelets and leukocytes (IOM, 2002a). The concentration or activity of the copper-binding protein, ceru- loplasmin (Cp) in plasma, or the oxidant defense enzyme, superoxide dismutase (SOD) in erythrocytes is commonly assessed. However, given that Cp is an acute phase protein that is induced by physiological stressors such as inflammation, infection and disease, the risk for a false negative is high with respect to the identification of a "conditioned" copper deficiency. Similarly, an inflammatory response can make the interpretation of oxidative defense markers problematic. Thus, as with zinc, it has been suggested that more than one copper status index should be used to assess status. The activity of numerous copper-dependent en- zymes such as cytochrome-c oxidase, lysyl oxidase, diamine oxidase, and pep- tidylglycine a-amidating monoxygenase in blood or cells to assess copper status has been reported (Hambidge, 2003). Indices of immune status (number or bac- tericidal activity of neutrophils) have also been used. A recent report shows that the protein expression of CCS (copper chaperone for Cu/Zn superoxide dis- mutase) in erythrocytes and liver was increased in rats made mildly copper defi- cient by feeding diets that were moderately high in zinc (Iskandar et al., 2005). It will be important to test whether CCS protein expression can be used as a sensi- tive biomarker to assess copper status in humans. As copper deficiency increases

APPENDIX B 309 oxidative stress, identification of a signature oxidative stress marker for copper deficiency could be helpful (see below). Magnesium Magnesium, an abundant intracellular divalent cation, is involved in numer- ous metabolic processes and is a cofactor for over 300 enzymes. The biochemi- cal abnormalities of magnesium deficiency include hypokalemia and hypocalce- mia which can lead to clinical manifestations of muscle cramps, tetany, and tremors, arrhythmias, cardiomyopathy, convulsion, and death. A number of stud- ies suggest that magnesium intake is inadequate which could lead to compro- mised magnesium status (IOM, 1997). About 75 percent of magnesium intake is obtained from milk, meat, eggs, vegetables, fruits, grains, and nuts. However, over the past decade, magnesium intake has decreased due in part to increased consumption of refined and processed foods, which generally have low magne- sium content. In addition, food components such as phytates, phosphorus, cal- cium, protein, and fat can affect magnesium absorption. While severe magne- sium deficiency is not thought to be a common occurrence in humans, low concentrations of plasma magnesium are commonly reported. For example, low magnesium is observed in people with diabetes or asthma, in alcoholics, in pa- tients with cancer, malabsorption syndromes or renal disease, in burn patients and the elderly, as well as in individuals who exercise (Britton et al., 1994; IOM, 1997). Currently, a number of measures are commonly used to assess magnesium nutriture including magnesium concentrations in plasma or serum, or erythro- cytes or lymphocytes (IOM, 1997) although there is considerable debate about whether blood magnesium levels reflect overall magnesium status. Ionized mag- nesium in plasma or erythrocytes (measured by ion-selective electrodes) or free intracellular magnesium levels in erythrocytes (measured by nuclear magnetic resonance) have been suggested to be better indicators although these methods require rigorous validity testing and the establishment of functional cut-offs. Intravenous or parenterally administered magnesium loading (magnesium toler- ance test) have also been used to assess magnesium status in adults. The intrave- nous method is invasive, and both methods require that the subject have normal renal function. Moreover, while the magnesium tolerance test has been used to detect magnesium depletion or risk of depletion, it does not appear to be sensi- tive to magnesium supplementation conditions in normal subjects with adequate magnesium status. For example, in healthy subjects, the mean retention of an administered magnesiun load did not change significantly after three months of magnesium supplementation (350 mg/day) (IOM, 1997). Additionally, the activ- ity of magnesium-dependent enzymes (such as Na/K ATPase), or analyses of substances that have been noted to be affected by magnesium deficiency such as thromboxane B2, C-reactive protein, endothelin-1, and nitric oxide production

310 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL have been suggested as possible biomarkers for magnesium (Franz, 2004). How- ever, changes in these markers have been noted in other disease states and, thus, these are not exclusive for magnesium alone. As with other nutrients, multiple biomarker measurements may be needed for accurate assessment of status. Simi- lar to zinc and copper, magnesium deficiency can increase oxidative stress. As discussed below, the use of signature oxidative stress markers may be a potential way to assess status. FUTURE METHODS OF NUTRIENT STATUS ASSESSMENT Metabolomics Metabolomics, the measurement of comprehensive profiles of low- molecular-weight metabolites (Whitfield et al., 2004), is an exciting tool that can be employed to assess nutritional status. A recent key paper from the American Society for Nutritional Sciences Long Range Planning Committee addressed the "nutritional phenotype" in the age of metabolomics (Zeisel et al., 2005). In the future, human nutritional status may be defined and measured by integrating information obtained from genetic, transcriptomic, proteomic, and metabolomic profiles, as they respond to diet, disease, environmental, and be- havioral or lifestyle factors. We are at the beginning stages of determining what metabolites are most relevant and important to nutrition, and what metabolomic profiles constitute the normal versus pathological phenotype. The linking of proteomic and metabolomic changes has been studied with regard to the vascular system as it relates to cardiovascular disease (Mayr et al., 2004). Similarly, metabolite profiling has been used in the fields of toxicology and drug discovery, in the identification of patients with coronary heart disease, and in the metabolism of components found in the diet (Whitfield et al., 2004). An exciting prospect for the future is whether characteristic profiles for indi- vidual minerals can be identified under different degrees of deficiency condi- tions. While it is still too early to know the extent to which metabolomics will be useful in characterizing an individual's acute, versus chronic, nutritional status, it is reasonable to predict that metabolomic approaches will be invalu- able in the future tailoring of nutritional recommendations for individuals in conditions of physical stress. In our opinion, a high research priority should be the identification of metabolomic signatures for acute and chronic deficien- cies, of the essential micro- and macro-molecules. It is important to note that the metabolomic signatures associated with both chronic and acute deficien- cies are likely to be different for individuals in diverse geographical regions, particularly when considering the profiles of individuals at extreme tempera- tures or altitudes. Thus, results obtained from subjects in mild temperate zones, such as those in the typical research university, may not be appropriate for military personnel engaged in activities in extreme environments.

APPENDIX B 311 Metallomics Recently, bioinorganic speciation analysis of metal and metalloid species within a cell or tissue type (metallomics) has been described as a new method for examining the role of metals in health and disease (Szpunar, 2004; Szpunar, 2005). As minerals are essential for biochemical function, the determination of not only the concentration of an individual metal species but also its distribution among cellular compartments of different cell types and the identification of the cellular bio-ligand to which it is complexed could shed light on what happens when cells are exposed to external stimuli, disease, physiological stress or nutri- ent deprivation, or toxicity. In the future, once this method is validated, body fluids or tissue biopsies may undergo metallomics as a way to assess nutrient status. Similar to metabolomics, research in this area should be considered a high priority for the military. Breathomics The odors found in breath have been used as a diagnostic tool for centuries. For example, the smell of fruity, rotten apples in the breath of diabetics has been used for centuries to diagnose diabetic ketoacidosis. Similarly, renal failure is associated with "urine-like" breath, while liver failure has been associated with fetor hepaticus. People with selenium deficiency have been noted to have breath that smells of garlic or alcohol. Dr. Michael Phillips and his team at New York Medical College have argued that breathomics may represent a rapid, non- invasive diagnostic tool for disease and wellness. This technique entails the trap- ping of metabolites (volatile organic compounds, VOCs) produced in the body either at basal levels or after a load test, from breath samples. After breath collection, the VOCs are released from the trap using automated thermal de- sorbers, separated by gas chromatography, and analyzed using mass spectrom- etry. The data collected are further analyzed by computer taking into account VOCs present in room air. There are several advantages of breath testing includ- ing that the breath collection apparatus is portable, and it is user-friendly and easy to operate. Importantly, there is minimal to no discomfort to patients. Fi- nally, breath VOCs can be identified and quantified with picomolar sensitivity (10-12 mol/L). However, as with other "omic" technologies (e.g., metabolomics), the total number of different VOCs is greater than 3,000 making data interpreta- tion somewhat problematic. With the above said, breathomics has been used to characterize different profiles of oxidative stress in patients with certain cancers, cardiovascular disease, diabetes, and preeclampsia (Moretti et al., 2004; Phillips et al., 2003; Phillips et al., 2004a; Phillips et al., 2004b). Profiles of oxidative stress or damage markers in breath samples may also prove useful in assessing mineral deficiency-induced alterations to the oxidant defense system. An imbalance in the production and elimination of reactive

312 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL oxygen species (ROS) by the oxidant defense system can result in oxidative stress and damage to macromolecules. For example, copper deficiency decreases the activity of CuZnSOD which results in increased superoxide anions. One consequence of the increased superoxide anion concentration is an increase in the formation of peroxynitrite leading to protein nitration (Beckers-Trapp et al., 2006). In zinc deficiency, oxidative stress results in lipid peroxidation and DNA damage (Olin et al., 1993; Oteiza et al., 1995). Similar findings have been noted in magnesium deficiency (Rayssiguier et al., 1993; Stafford et al., 1993). The peroxidation of lipids results in the production of lipid free radicals including non-volatile products such as conjugated dienes, lipid hydroperoxides, malon- dialdehyde and 4-hydroxynonenal, and volatile products such as alkanes and methylated alkanes. The volatile products can then be examined in breath sam- ples. This technique has been used to detect lipid peroxidation in copper defi- cient rats as an index of whole body oxidative stress (Saari et al., 1990). It remains to be seen whether individual nutrient deficiencies can be precisely identified by specific profiles of markers of oxidative stress or damage. We would suggest that the characterization of breath "oxidative stress profiles" asso- ciated with acute and chronic nutritional deficiencies should be a high priority research area for the military. However, perhaps a more critical question is whether breath testing can also be a potential method for the rapid evaluation of an individual's response to food. For example, if one were interested in identify- ing antioxidant effects of certain foods, one could use an experimental design similar to a drug intervention study. Normal controls and disease group subjects, e.g., cardiovascular disease, would be dosed with the candidate food. Serial breath tests would be obtained and oxidative stress profiles could be assessed. With regard to health and disease, in theory, with relatively small advancements in technology, one could envision an initial breath test in the field, followed by an onsite analysis of the sample at a more comprehensive hospital. An indi- vidual's response to nutritional therapy could then be followed with respect to markers of oxidative stress and tissue damage and repair. The expedient assess- ment of oxidative damage and response of the individual to different conditions could represent a breakthrough in patient care even if the method ultimately is not sensitive enough to determine specific nutrient (e.g., zinc, copper, or magne- sium) deficiencies. CONCLUSIONS Most of the biomarkers commonly used for assessing zinc, copper, and magnesium status, particularly with respect to the identification of functional deficiencies, have low sensitivity and specificity. As a consequence, there can be a high risk for false negatives, as well as false positives. The use of multiple biomarkers for a given mineral may in part compensate for the above, however, problems still exist. Lab-on-a-chip technologies may soon allow for the rapid

APPENDIX B 313 evaluation of multiple biomarkers that collectively provide information on a set of key nutrients. Evolving "omic" technologies will provide new assessment approaches that should allow for considerable improvements in our ability to correctly identify functional mineral deficiencies in short periods of time. With the identification of polymorphisms that increase an individual's risk for the development of, or susceptibility to, certain mineral deficiencies, consideration should be given to the development of individualized "omic" profiles that reflect their "optimal" status for nutrients of concern. ACKNOWLEDGEMENTS This work was supported by National Institutes of Health grants HD-26777, HD01743, and AT-00652 and a gift from the International Copper Association. REFERENCES Aydemir TB, Blanchard RB, Cousins FJ. 2006. Zinc supplementation of young men alters metal- lothionein, zinc transporter, and cytokine gene expression in leukocyte populations. Proc Natl Acad Sci USA 103:1699­1704. Beckers-Trapp ME, Lanoue L, Keen CL, Rucker RB, Uriu-Adams JY. 2005. Abnormal development and increased 3-nitrotyrosine in copper-deficient mouse embryos. Free Radical Biology and Medicine 40(1):35­44. Briefel RR, Bialostosky K, Kennedy-Stephenson J, McDowell MA, Ervin RB, Wright JD. 2000. Zinc intake of the U.S. population: Findings from the third National Health and Nutrition Examination Survey, 1988­1994. J Nutr 130:1367S­1373S. Britton J, Pavord I, Richards K, Wisniewski A, Knox A, Lewis S, Tattersfield A, Weiss S.1994. Dietary magnesium, lung function, wheezing, and airway hyperreactivity in a random adult population sample. Lancet 344:357­362. Cao J, Cousins RJ. 2000. Metallothionein mRNA in monocytes and peripheral blood mononuclear cells and in cells from dried blood spots increases after zinc supplementation of men. J Nutr 130:2180­2187. Champagne CM, Bogle ML, McGee BB, Yadrick K, Allen HR, Kramer TR, Simpson P, Gossett J, Weber J. 2004. Dietary intake in the lower Mississippi delta region: Results from the Foods of our Delta Study. J Am Diet Assoc 104:199­207. Ford ES, Mokdad AH. 2003. Dietary magnesium intake in a national sample of U.S. adults. J Nutr 133:2879­2882. Franz KB. 2004. A functional biological marker is needed for diagnosing magnesium deficiency. J Am Coll Nutr 23:738S­741S. Hambidge M. 2003. Biomarkers of trace mineral intake and status. J Nutr 133 (Suppl 3):948S­ 955S. Harris ED. 2000. Cellular copper transport and metabolism. Annu Rev Nutr 20:291­310. IOM (Institute of Medicine). 1997. Magnesium. In: Dietary Reference Intakes for Calcium, Phos- phorus, Magnesium, Vitamin D, and Fluoride. Washington, DC: National Academy Press. Pp. 190­249. IOM. 2002a. Copper. In: Dietary Reference Intakes: Vitamin A, Vitamin K, Arsenic, Boron, Chro- mium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Washington, DC: National Academy Press. Pp. 224­257.

314 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL IOM. 2002b. Zinc. In: Dietary Reference Intakes: Vitamin A, Vvitamin K, Arsenic, Boron, Chro- mium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Washington, DC: National Academy Press. Pp. 442­501. Iskandar M, Swist E, Trick KD, Wang B, L'Abbe MR, Bertinato J. 2005. Copper chaperone for Cu/ Zn superoxide dismutase is a sensitive biomarker of mild copper deficiency induced by moder- ately high intakes of zinc. Nutr J 4:35­44. Keen CL, Clegg MS, Hanna LA, Lanoue L, Rogers JM, Daston GP, Oteiza P, Uriu-Adams JY. 2003a. The plausibility of micronutrient deficiencies being a significant contributing factor to the occurrence of pregnancy complications. J Nutr 133:1597S­1605S. Keen CL, Hanna LA, Lanoue L, Uriu-Adams JY, Rucker RB, Clegg MS. 2003b. Developmental consequences of trace mineral deficiencies in rodents: Acute and long-term effects. J Nutr 133:1477S­1480S. King JC, Keen CL. 1994. Zinc. In: Shils ME, Olson JA, Shike M, eds. Modern nutrition in health and disease. Philadelphia, PA: Lea & Febiger. Pp. 214­230. Liuzzi JP, Cousins RJ. 2004. Mammalian zinc transporters. Annu Rev Nutr 24:151­172. Mayr M, Mayr U, Chung YL, Yin X, Griffiths JR, Xu Q. 2004. Vascular proteomics: Linking proteomic and metabolomic changes. Proteomics 4:3751­3761. Moretti M, Phillips M, Abouzeid A, Cataneo RN, Greenberg J. 2004. Increased breath markers of oxidative stress in normal pregnancy and in preeclampsia. Am J Obstet Gynecol 190:1184­ 1190. Olin KL, Shigenaga MK, Ames BN, Golub MS, Gershwin ME, Hendrickx AG, Keen CL. 1993. Maternal dietary zinc influences DNA strand break and 8-hydroxy-2'-deoxyguanosine levels in infant rhesus monkey liver. Proc Soc Exp Biol Med 203:461­466. Olivares M, Pizarro F, de Pablo S, Araya M, Uauy R. 2004. Iron, zinc, and copper: Contents in common Chilean foods and daily intakes in Santiago, Chile. Nutrition 20:205­212. Oteiza PI, Olin KL, Fraga CG, Keen CL. 1995. Zinc deficiency causes oxidative damage to proteins, lipids and DNA in rat testes. J Nutr 125:823­829. Phillips M, Cataneo RN, Ditkoff BA, Fisher P, Greenberg J, Gunawardena R, Kwon CS, Rahbari- Oskoui F, Wong C. 2003. Volatile markers of breast cancer in the breath. Breast J 9:184­191. Phillips M, Boehmer JP, Cataneo RN, Cheema T, Eisen HJ, Fallon JT, Fisher PE, Gass A, Greenberg J, Kobashigawa J, Mancini D, Rayburn B, Zucker MJ. 2004a. Heart allograft rejection: Detec- tion with breath alkanes in low levels (the HARDBALL study). J Heart Lung Transplant 23:701­708. Phillips M, Cataneo RN, Cheema T, Greenberg J. 2004b. Increased breath biomarkers of oxidative stress in diabetes mellitus. Clin Chim Acta 344:189­194. Rayssiguier Y, Gueux E, Bussiere L, Durlach J, Mazur A. 1993. Dietary magnesium affects suscep- tibility of lipoproteins and tissues to peroxidation in rats. J Am Coll Nutr 12:133­137. Saari JT, Dickerson FD, Habib MP. 1990. Ethane production in copper-deficient rats. Proc Soc Exp Biol Med 195:30­33. Stafford RE, Mak IT, Kramer JH, Weglicki WB. 1993. Protein oxidation in magnesium deficient rat brains and kidneys. Biochem Biophys Res Commun 196:596­600. Szpunar J. 2004. Metallomics: A new frontier in analytical chemistry. Anal Bioanal Chem 378: 54­56. Szpunar J. 2005. Advances in analytical methodology for bioinorganic speciation analysis: Metallo- mics, metalloproteomics and heteroatom-tagged proteomics and metabolomics. Analyst 130: 442­465. Takeda N, Takaoka T, Ueda C, Toda N, Kalubi B, Yamamoto S. 2004. Zinc deficiency in patients with idiopathic taste impairment with regard to angiotensin converting enzyme activity. Auris Nasus Larynx 31:425­428. Tarasuk V, Dachner N, Li J. 2005. Homeless youth in Toronto are nutritionally vulnerable. J Nutr 135:1926­1933.

APPENDIX B 315 Uriu-Adams JY, Keen CL. 2005. Copper, oxidative stress, and human health. Mol Aspects Med 26:268­298. Wang K, Zhou B, Kuo YM, Zemansky J, Gitschier J. 2002. A novel member of a zinc transporter family is defective in acrodermatitis enteropathica. Am J Hum Genet 71:66­73. Whitfield PD, German AJ, Noble PJ. 2004. Metabolomics: An emerging post-genomic tool for nutrition. Br J Nutr 92:549­555. Zeisel SH, Freake HC, Bauman DE, Bier DM, Burrin DG, German JB, Klein S, Marquis GS, Milner JA, Pelto GH, Rasmussen KM. 2005. The nutritional phenotype in the age of metabolomics. J Nutr 135:1613­1616. Environmental Stressors During Military Operations Robert Carter III, Samuel N. Cheuvront, Andrew J. Young, and Michael N. Sawka U.S. Army Research Institute of Environmental Medicine, Natick, Massachusetts INTRODUCTION U.S. fighting doctrine states that "U.S. Army forces must be prepared to fight and win on short notice anywhere in the world, from blistering deserts to frigid wastelands, in rain forests and mountains--and all types of terrain" and that soldiers are the most important and most vulnerable part of the war fighting system (Department of the Army, 2003). Military operations require soldiers to perform strenuous exercise for long hours and will push them to their physi- ologic limits, often with minimal logistical support so troops may find them- selves under-equipped for the hostile environmental conditions. Harsh environ- ments limit use of air support and crew-served vehicles, thereby placing a greater combat burden on dismounted soldiers who must sustain high metabolic rates to traverse rugged terrain and carry heavy loads. These environmental and work load conditions can impose significant adverse consequences on soldier perfor- mance and health. HEAT STRESS Soldiers encounter heat stress from environmental conditions, body heat production and the clothing or equipment they wear. Heat stress increases sweat rate and circulatory responses to dissipate body heat (Mack and Nadel, 1996). When the climatic condition is warmer than skin, it also causes the body to gain heat from the climate, and, thus, increases the amount of heat the body must dissipate (Sawka et al., 1996). In addition, exercise increases metabolic rate above resting levels, and, thus, increase the rate at which heat must be dissipated to keep core temperature from increasing to dangerous levels. Climatic heat stress and physical exercise interact synergistically, and may push physiological systems to their limits (Sawka and Young, 2000).

316 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL If the body stores heat, skin and core temperature will increase. In response, the body initiates heat loss responses (sweating and increased skin blood flow). Unless heat stress exceeds the thermoregulatory system's capacity to dissipate heat, the heat loss responses will increase until they restore heat balance and core temperature stops increasing. However, if climate or clothing limits heat loss below the rate of heat production, then increases in sweating and skin blood flow will not restore heat balance but will only increase physiological strain. Heat stress increases skin blood flow that elevates skin temperature (Rowell, 1986). Skin temperature generally increases with ambient temperature but re- mains below core temperature. When sweating does not occur, increasing skin blood flow will elevate skin temperature, and decreasing skin blood flow will lower skin temperature nearer to ambient temperature. Thus, heat loss by con- duction, convection and radiation is controlled by varying skin blood flow, and thereby skin temperature. Maintaining a high skin blood flow helps dissipate heat but strains the car- diovascular system during physical work in the heat. High skin blood flow is associated with pooling of blood in compliant skin and subcutaneous vascular beds. This pooling reduces cardiac filling and stroke volume, thus requiring a higher heart rate to maintain cardiac output (Rowell, 1986). For these conditions, the primary cardiovascular challenge is to have sufficient cardiac output to si- multaneously support high skin blood flow for heat dissipation and high muscle blood flow for metabolism. To help compensate for reduced cardiac filling, sym- pathetic activity is increased to elevate myocardial contractility and to divert blood flow from the viscera to skin and muscle. Changes in Metabolism Acute heat stress increases the metabolic rate to perform submaximal exer- cise, possibly because the rate of ATP utilization to develop a given muscle tension is increased as muscle temperature increases. Aerobic metabolism and muscle total adenine pool may decrease, while oxygen debt, blood and muscle lactate accumulation, skeletal muscle glycogen utilization and inosine 5-mono- phosphate concentration may all increase during exercise with higher muscle temperatures (Young, 1990). The increased glycogen utilization is probably me- diated by elevated epinephrine and muscle hyperthermia. In addition, lactate uptake and oxidation by the liver (and probably non-exercising muscle) are im- paired during exercise-heat stress. Elevated muscle temperature does not appear to alter oxidative adaptations or mitochondria biogenesis (Young, 1990). Heat acclimatization usually lowers total metabolic rate during exercise due to reductions in aerobic and anaerobic components, but this effect is probably too small to reduce heat storage (Sawka et al., 2000). On the other hand, changes in substrate metabolism induced by heat acclimatization may help to improve

APPENDIX B 317 endurance. Blood and muscle lactate accumulation and muscle glycogen deple- tion during exercise are often reduced following heat acclimatization. Changes in Body Fluids and Electrolytes Sweating rate is dependent upon the environmental conditions, clothing worn, exercise intensity, and heat acclimatization state. Soldiers working in hot weather often have sweating rates of 0.3 to 1.2 L/hour (Sawka and Young, 2000). Persons performing more intense activity while wearing more clothing or equip- ment often have sweating rates of 1 to 2 L/hour (IOM, 2004). In comparison, athletes performing high intensity exercise in the heat commonly have sweating rates of 1.0 to 2.5 L/hour (Mack and Nadel, 1996). Fluid requirements will vary in relation to ambient temperature, clothing worn, acclimatization state, and physical activity levels (IOM, 2004). Daily fluid requirements might range (for sedentary to very active persons) from 2­4 L/day in temperate environments and from 4­12 L/day in hot environments (IOM, 2004). Figure B-17 demonstrates the distribution of daily sweating rates for soldiers performing military activities in desert and tropic climates. 40 35 30 Tropics 25 INCIDENCE 20 15 Desert PERCENT 10 5 0 0 2 4 6 8 10 12 SWEAT LOSS, L/24 hr. FIGURE B-17 Distribution of daily sweating rates for active soldiers in desert and tropical climates. Percent incidence refers to the percentage of the subject population achieving the given daily sweat loss. SOURCE: Molnar (1947).

318 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Sweat is hypotonic to extracellular fluid and contains electrolytes, primarily sodium chloride and to a lesser extent potassium, which are lost in sweat. Sweat sodium concentration averages ~40 mEq/L (range 10­100 mEq /L) and varies depending upon diet, sweating rate, hydration status, and heat acclimatization state. Heat acclimatized persons have relatively low sodium concentrations (> 50 percent reduction) in sweat. Sweat potassium concentration averages 5 mEq/L (range 3­15 mEq/L), calcium averages 1 mEq/L (range 0.3­2 mEq/L) and mag- nesium averages 0.8 mEq/L (range 0.2­1.5 mEq/L). Electrolyte supplementation is not necessary, except for their first several days of heat acclimatization, as normal dietary sodium intake will cover the sweat losses as heat acclimatization occurs (IOM, 2004). It is important that unacclimatizated soldiers replace their sweat and electro- lyte losses while performing exercise in the heat. If sodium losses are not re- placed, the extracellular fluid volume will also decrease in volume and, conse- quently, dehydration will occur. If sweat losses are not replaced then a body water deficit, or dehydration will occur. Both types of dehydration (hypertonic hypovolemia or isomotic hypovolemia) reduce physical exercise and cognitive performance and increases the potential for heat strain. The greater the water deficit the greater the adverse consequences mediated by dehydration. COLD STRESS Human thermoregulatory adaptations to cold stress are modest and less un- derstood than adaptations to chronic heat (Young, 1996). Cold stress environ- ments include not only exposure to extremely low temperatures (for example, Arctic regions), but also cold-wet exposures (for example, rain, immersion) in warmer ambient temperatures (Toner and McArdle, 1996). In the cold, heat balance and requirements for shivering are dependent upon the severity of cli- matic cold stress, effectiveness of vasoconstriction as well as the intensity and mode of exercise (Sawka and Young, 2000). Cold-induced vasoconstriction decreases blood flow to peripheral tissues and makes them susceptible to cold injury (O'Brien et al., 2005). Reduced muscle temperature degrades finger dexterity and muscular strength while reduced core temperature can degrade the ability to achieve maximal metabolic rates and sub- maximal endurance performance. Body composition is the most important physi- ological determinant of thermoregulatory tolerance to cold exposure. The clothing insulation required for warmth and comfort is much higher during rest and light activity than during strenuous activity and over insulation can cause heat stress that elicits sweating, wet clothing and dehydration. Each of those factors can have undesirable affects on soldier performance and cold injury susceptibility. Cold exposure elicits a peripheral vasoconstriction resulting in a decrease in peripheral blood flow which reduces convective heat transfer between the body's core and shell (skin, subcutaneous fat, and skeletal muscle). During whole-body

APPENDIX B 319 cold exposure, the vasoconstrictor response spreads throughout the body's pe- ripheral shell. Vasoconstriction begins when skin temperature falls below about 35°C, and becomes maximal when skin temperature is about 31°C or less. The vasoconstrictor response to cold exposure retards heat loss and help to defend core temperature, but at the expense of a decline in temperature of peripheral tissue which also makes them susceptible to cold injury (O'Brien et al., 2005). In effect, insulation is increased effectively but skin temperature declines. Changes in Metabolism In addition to vasoconstriction, another major mechanism elicited to defend body temperature during cold exposure is an increased metabolic heat produc- tion, from shivering, which helps offset heat losses. Muscle is generally the source of the increased metabolic heat production in humans and this heat pro- duction can be increased even further with exercise. Cold exposure can increase muscle energy metabolism during exercise and reduce exercise performance (Young et al., 1996). Blood lactate concentrations during exercise in cold may be higher than in temperate conditions depending on whether experimental con- ditions allow shivering to occur during exercise. Changes in Body Fluids and Electrolytes Another physiological response sometimes elicited by cold exposure is diuresis. Termed cold-induced diuresis (CID), this response is actually second- ary to the cold-induced vasoconstriction and resulting redistribution of body fluids from the peripheral to central circulation. Exercise minimizes cold- induced vasoconstriction and the reduction in peripheral blood flow suppresses or blunts CID. For this reason, and because the effect is self-limiting (i.e., CID diminishes as body water content falls), this response to cold is not of major physiological significance. CID elicits isoosmotic dehydration. Clothing insulation needed for warmth and comfort in cold environments is much higher during rest and light activity than during strenuous activity. Therefore, if one begins exercising vigorously while wearing clothing selected for sedentary activities in the cold, sweating and the resultant drinking fluid requirements can increase substantially. Further, sweat can accumulate in clothing, compromising its insulative properties which will again be necessary when exercise stops. Adequate fluids must be ingested to replace these losses or dehydration will ensue. As ob- served by LeBlanc, man in the cold is not necessarily a cold man (Young, 1996). ALTITUDE STRESS When soldiers ascend to higher altitudes, atmospheric oxygen pressure de- clines and reduced O2 diffusion from the alveolus to blood causes a fall in arte-

320 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL rial O2 pressure (PaO2), O2 saturation of hemoglobin (SaO2), and arterial O2 content (CaO2). Because of the relationship between PaO2 and hemoglobin, sig- nificant decrements in resting SaO2 do not emerge until the altitude exceeds ~2,400 m. Although the resting SaO2 is well preserved up to ~2,400 m, the drop in PaO2 decreases the diffusion of oxygen from the lungs to the blood and then from the blood to the cells. This decrease in oxygen diffusion rate becomes apparent during aerobic exercise as an arterial oxygen desaturation occurs at altitudes as low as 1,000 m. Thus, exercise performance deteriorates at altitudes slightly greater than 1,000 m, even though resting SaO2 is near sea-level values. With altitudes higher than 1,000 m, the decrements in aerobic exercise perfor- mance are even more noticeable (Fulco et al., 1998). Changes in Body Fluids and Electrolytes Soldiers ascending to high altitude normally experience diuresis and natri- uresis that mediate a reduced total body water at a new equilibrium level. These responses are initiated within several hours of hypoxic exposure and occur con- tinuously during altitude acclimatization (Sawka et al., 2000). As persons ascend to higher altitudes there are additional fluid and electrolyte losses. Total body water losses of ~1­7 percent have been reported. Renal function is well pre- served during rest and exercise at high altitude. A hypoxia mediated stimulation of arterial chemoreceptor is believed to mediate the renal excretory responses by increasing overall flow and filtration fraction. High altitude exposure has pro- found affects on fluid regulatory hormones that help mediate dehydration. Atrial natriuretic peptide (a hormone released by walls of the cardiac atrium in re- sponse to high sodium concentration or stretching of the atria and acts to excrete sodium and water, and to cause vasodilation in the circulatory system) as well as glucocorticoid responses are elevated while aldosterone responses are blunted by high altitude exposure. In addition, high altitude exposure lessens vasopressin (an anti-diuretic hormone) responses at rest, by increasing the osmotic threshold for vasopressin release so that free water excretion is increased; high altitude also reduces thirst. During exercise at high altitude, vasopressin responses are decreased relative to the exercise intensity; however, the osmotic threshold is not changed. Other water losses, such as respiratory water loss at altitude is higher than respiratory water loss at sea level (Young et al., 1996). Persons ascending to high altitude will have plasma volume reductions that are proportional to the ascended altitude and exposure duration (Sawka et al., 2000). Plasma volume will be decreased ~10 percent at an altitude of 3,000 m. An inappropriate thirst response coupled with increased water loss and a transient diuresis, can result in rapid dehydration when military operations are conducted at high altitude; this higher risk of dehydrations along with low oxy- gen pressure, may result in substantial decrements in military performance.

APPENDIX B 321 ENERGY EXPENDITURE AND HEAT STRAIN Daily energy expenditure is a function of the terrain, load carried, duration, and intensity of the work task. Table B-10 provides daily energy expenditures (measured by doubly labeled water) for military activities. Clearly, soldiers are active and average daily energy expenditures of > 4,400 kcal. Extreme military operational scenarios can demand daily energy expenditures > 6,000 kcal/day. Such energy expenditures would only be for "very elite" soldiers with an excep- tionally demanding mission. Combat foot soldiers carry their own supplies at high energy expenditure costs. The loads they carry can be very heavy depend- ing on what phase of a mission they are performing. A recent field study by Dean and Dupont (2003) in which soldier loads were measured during actual operations in Afghanistan revealed that soldiers in the traveling phase of a mis- sion carried an average of 59.3 kg (131 lb). A by product of muscular contraction is metabolic heat production that is transferred from the active muscle to blood and the body core. Since skeletal muscle contraction is ~20 percent efficient, and then ~80 percent of expended energy is released as heat that needs to be dissipated from the body to avoid heat TABLE B-10 Daily Energy Expenditures (Measured by Double-Labeled Water) of Military Activities Group Activity Kcal/day Army Special Forces Combat exercise, temperate 3,400 Army Engineers Build road and airstrip at altitude 3,549 Army Transportation Company Garrison 3,568 Marine Combat Engineers Construction 3,668 Israeli Infantry Combat exercise, summer 3,937 Army Support hospital 3,960 Army Ranger Training course 4,010 Army Ranger Training course 4,090 Marine Artillery exercise, desert 4,115 Marine Combat exercise, winter 4,198 Army Artillery exercise, winter 4,253 Israeli Infantry Combat exercise, winter 4,281 Army Special Forces Combat exercise, winter 4,558 Marine Crucible, women 4,679 Australian Infantry Jungle training 4,750 Army Special Forces Assessment school 5,183 Army Ranger Combat exercise 5,185 Norwegian Ranger Training course 6,250 Marine Crucible, men 6,067 Average 4,405 SOURCE: Departments of Army and Air Force (2003).

322 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL storage and increasing body temperature. Heat exchange between body and the environment is governed by biophysical properties dictated by surrounding air or water temperature; air humidity; air or water motion; solar, sky, and ground radiation; and clothing. These biophysical properties combined with the meta- bolic rate can result in either heat or cold strain. SUMMARY Military operations can occur in harsh climates and extremely hostile environ- ments with minimal logistical support. Environmental stresses can induce physi- ologic strain and reduce military performance. The alterations in metabolism, body fluids and electrolyte concentrations due to environmental exposures to heat, cold, or altitude can alter the mineral levels in body compartments due to alterations in sweat and urine output. The role of that these losses contribute to performance degradation is unclear but, if they are substantial and may affect performance or health, they need to be considered when recommending mineral intake for military operations or training under extreme environmental conditions. DISCLAIMER The views, opinions and findings contained in this report are those of the authors and should not be construed as an official Department of Army position or decision, unless so designated by other official documentation. Approved for public release; distribution is unlimited. REFERENCES Dean CE, Dupont F. 2003. Modern Warrior's Combat Load. Dismounted Operations in Afghanistan. April­May 2003. Letter Report. Ft. Leavenworth, KS: U.S. Army Center for Army Lessons Learned. Departments of Army and Air Force. 2003. Heat Stress Control and Heat Casualty Management. Technical Bulletin Med 506. Washington, DC: Headquarters, Departments of Army and Air Force. Fulco CS, Rock PB, Cymerman A. 1998. Maximal and submaximal exercise performance at altitude. Aviat Space Environ Med 69:793­801. Institute of Medicine. 2004. Water. In: Dietary Reference Intakes: Water, Potassium, Sodium, Chlo- ride, and Sulfate. Washington, DC: The National Academies Press. Mack GW, Nadel ER. 1996. Body fluid balance during heat stress in humans. In: Fregly MJ, Blatteis CM, eds. Handbook of Physiology, Section 4, Environmental Physiology. New York: Oxford University Press. Pp. 187­214. O'Brien C. 2005. Reproducibility of the cold-induced vasdilation response in the human finger. J Appl Physiol 98(4):1334­1340. Rowell LB. 1986. Human Circulation: Regulation during Physical Stress. New York: Oxford Uni- versity Press. Sawka MN, Young AJ. 2000. Physical exercise in hot and cold climates. In: Garrett WE, Kirkendall DT, eds. Exercise and Sport Science. Philadelphia, PA: Lippincott, Williams and Wilkins. Pp. 385­400.

APPENDIX B 323 Sawka MN, Wenger CB, Pandolf KB. 1996. Thermoregulatory responses to acute exercise-heat stress and heat acclimation. In: Fregly MJ, Blatteis CM, eds. Handbook of Physiology, Section 4, Environmental Physiology. New York: Oxford University Press. Pp. 157­185. Sawka MN, Convertino VA, Eichner ER, Schnieder SM, Young AJ. 2000. Blood volume: Impor- tance and adaptations to exercise training, environmental stresses, and trauma/sickness. Med. Sci Sports Exerc 32:332­348. Toner MM, McArdle WD. 1996. Human thermoregulatory responses to acute cold stress with spe- cial reference to water immersion. In: Fregly MJ, Blatteis CM, eds. Handbook of Physiology, Section 4, Environmental Physiology. New York, NY: Oxford University Press. Pp. 379­418. U.S. Army Training and Doctrine Command. 2003. Operations. FM 100-5. Washington, DC: Head- quarters, Department of the Army. Young AJ. 1990. Energy substrate utilization during exercise in extreme environments. In: Pandolf KB and Hollozsy JO, eds. Exercise and Sport Sciences Reviews. Baltimore, MD: Williams and Wilkins. Pp. 65­117. Young AJ. 1996. Homeostatic responses to prolonged cold exposure: Human cold acclimatization. In: Fregly MJ, Blatteis CM, eds. Handbook of Physiology, Section 4, Environmental Physiol- ogy. New York, NY: Oxford University Press. Pp. 419­438. Young AJ, Sawka MN, Pandolf KB. Physiology of cold exposure. 1996. In: Marriott BM, Carlson SJ, eds. Nutritional Needs in Cold and in High-Altitude Environments. Washington, DC: Na- tional Academy Press. Pp. 127­147. Mineral Sweat Losses During Exercise Emily M. Haymes Florida State University, Tallahassee The extreme environmental conditions during military operations or training may affect sweat losses of minerals and, therefore, those losses constitute a risk factor for mineral inadequacy and potential performance decrements and need to be considered when evaluating mineral requirements for military personnel. Al- though other physical or psychological stressors might also alter mineral me- tabolism, sweat due to exercise and high temperatures appears to be one major mechanism of substantial mineral losses. Although less frequently studied, fecal and urinary losses might also be of importance with intense physical activity and they are also reported in this paper when known. Several studies have measured mineral concentration in sweat during and following exercise. The concentrations of calcium, magnesium, iron, and zinc have been found to vary widely in the sweat during exercise. Several factors appear to influence sweat mineral concentrations including the methods used to collect the sweat and site of sweat collection, the environmental conditions in which the sweat is collected, and duration of the exercise sweat collection. Much less is known about sweat losses of copper or other minerals. Because many studies have been conducted under different environmental situations or using various collection methods, the results of the studies presented show apparent discrepancies; in addition, because of the special extreme environments endured

324 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL by military personnel, care should be taken when extrapolating these results to mineral losses of military personnel. Only the studies of prolonged exercise in the heat by Consolazio and colleagues (Consolazio et al., 1962, 1964) have measured sweat mineral losses in conditions that simulate those experienced by military personnel. FACTORS AFFECTING SWEAT MINERAL CONCENTRATIONS The site of sweat collection influences the sweat concentration and can lead to inconsistent results if this factor is overlooked. Most of the studies that mea- sured sweat mineral concentrations during exercise have used regional sites (e.g., back, arm) for sweat collection. Two studies that directly compared whole body sweat mineral content with regional site sweat collections using patches and arm bags found higher mineral concentrations in the sweat from the regional sites (Jacob et al., 1981; Palacios et al., 2003). The conclusion from these studies is that the use of regional sites to project whole body mineral loss led to overesti- mation of total body sweat mineral losses. The environment, and particularly environment temperature and humidity, influences sweat losses of iron and zinc, being lower during exercise in warm environments compared to cooler temperatures (Tipton et al., 1993; Waller and Haymes, 1996). Because sweat rates are higher in warm environments, the de- crease in sweat mineral concentration appears to be due to sweat dilution. The total amount of sweat iron and zinc lost was the same in the two environments. Another factor that cannot be overlooked when considering sweat losses is acclimatization, that is, the physiological adaptation to the environment that occurs over days or weeks. For instance, sweat calcium loss was observed to decrease after one week of exercise in a hot environment (Conolazio et al., 1962). Another physiological adaptation observed is a decrease in mineral con- centration over time. Serial sweat collections made during prolonged exercise have found that sweat zinc and iron concentrations decrease over time (DeRuisseau et al., 2002; Paulev et al, 1983; Tipton et al., 1993; Waller and Haymes, 1996). A similar decrease in sweat iron concentration over time was found when subjects sat in a sauna (Brune et al., 1986). Although sweat rates increase over time during exercise and sauna exposure, Brune et al. (1986) sug- gested the first sweat secreted by sweat glands may contain cellular debris and external contaminants. DeRuisseau et al. (2002) recently reported lower sweat iron and zinc concentrations during the second hour of exercise than the first hour. Sweat rates remained constant during the final 90 minutes of exercise. All these research data point to apparent inconsistencies in sweat losses of minerals that may be due not only to differences in methods or laboratories but also to critical factors such as collection methods, environmental conditions, and acclimatization.

APPENDIX B 325 SWEAT MINERAL CONCENTRATIONS DURING EXERCISE Calcium Whole body sweat calcium concentrations were measured during exercise in two studies. Mean sweat calcium concentration was 52 ± 36 mg/L during exer- cise in a warm (34.5°C) humid environment (Shirreffs and Maughan, 1997). Costa et al. (1969) found exercise sweat calcium concentrations of 72 ± 10 mg/L and 74 ± 17 mg/L on two different diets that did not differ in calcium intake in a cooler (24.5°C) environment. Several studies have used pads or sponges to collect sweat from various sites during exercise. Sweat calcium concentrations of 30 ± 5 mg/L and 44 ± 12 mg/L were found during exercise by runners and firefighters, respectively (Bullen et al., 1999; O'Toole et al., 2000). Verde et al. (1982) found lower sweat calcium concentrations during indoor (40 ± 20 mg/L) and outdoor (54 ± 46 mg/L) exer- cise than during a sauna (94 ± 46 mg/L). Klesges et al. (1996) collected sweat from the trunk of basketball players using t-shirts and found mean sweat calcium concentration was 65 ± 31 mg/L. Palacios et al. (2003) found patches placed on eight body sites for 24 hours overestimated total body dermal calcium loss by more than threefold. Whole body dermal calcium loss collected using cotton underwear, shirt, pants, and socks in these same subjects averaged 103 ± 22 mg/ day (Palacios et al., 2003). Magnesium Sweat magnesium concentrations during exercise measured using whole body techniques vary from 12 ± 12 mg/L (Shirreffs and Maughan, 1997) and 15 ± 3 mg/L (Costa et al., 1969) to 55 ± 2 mg/L (Costill et al., 1976). Regional sweat collections with sponges yielded mean magnesium concentrations of 7 ± 2 mg/L and 10 ± 2 mg/L during indoor and outdoor exercise, respectively (Verde et al., 1982). Verde and colleagues observed a decrease in sweat magnesium concentration as the sweat rate increased. Although dermal magnesium loss in young women was 35 ± 13 mg/day using a whole body technique, use of sweat patches to measure sweat magnesium also have been found to overestimate daily dermal magnesium loss (Palacios et al., 2003). Copper Only one study was found that measured sweat copper concentrations dur- ing exercise. Aruoma et al. (1988) collected sweat samples from the back, chest, abdomen and arm by scraping the skin with plastic tube following heavy exer- cise. Sweat copper concentrations were 0.89 mg/L (abdomen), 0.73 mg/L (chest), 0.56 mg/L (back), and 0.52 mg/L (arm). Whole body dermal copper loss has

326 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL been found to average 0.34 mg/day (Jacob et al., 1981). Mean sweat copper concentration collected in arm bags in the same study was 0.11 mg/L. Turnlund et al. (1990) also used arm bags to estimate dermal copper loss. Mean copper loss from the arm ranged from 0.5 to 5.7 µg/day, but this study was conducting with subjects at rest. Other studies that measure copper losses in the sweat have been conducted but they were using either questionable methods of copper quan- tification, sweat induction, or collection. Iron Many studies have examined the concentration of iron in the sweat. Green et al. (1968) estimated dermal uptake and loss of iron using 59 Fe. Mean dermal iron loss was 0.24 mg/day in sedentary men and women. Slightly higher dermal iron loss (0.33 ± 0.15 mg/day) was found using whole body dermal collection method by Jacob et al. (1983). Mean sweat iron concentration measured using an arm bag was 0.076 mg/L in the same subjects. Wheeler et al. (1973) measured dermal iron loss using a whole body tech- nique during habitual daily activity and with the addition of two hours of exer- cise with two different levels of dietary iron. Dietary iron intake and iron loss through the gastrointestinal and urinary tracts were simultaneously measured. Mean iron intake and losses are presented in Table B-11. The subjects were in negative iron balance during the lower dietary iron intake phase of the study. Sweat iron concentrations were lower during the two exercise phases (0.13 mg/L and 0.15 mg/L) compared to the habitual physical activity phase (0.20 mg/L). However, subjects had higher sweat rates during the exercise phases. Mean sweat iron concentrations during exercise also decrease over time. Paulev et al. (1983) observed sweat iron on the back decreased from 0.20 mg/L to 0.13 mg/L over 30 minutes of exercise. Waller and Haymes (1996) found arm bag sweat iron concentrations decreased from 30 to 60 minutes of exercise in TABLE B-11 Iron Intake and Losses During Habitual Activity With and Without 2 Hours of Exercise Iron Intake Urinary Iron Loss Dermal Iron Loss Fecal Iron Loss Trial (mg/day) (mg/day) (mg/day) (mg/day) HA 34.8 0.22 0.38 22.0 HA/Ex1 36.8 0.23 0.32 26.0 HA/Ex2 17.5 0.19 0.34 25.2 NOTE: HA = habitual daily exercise; HA/Ex = habitual daily exercise with additional hours of exercise. SOURCE: Wheeler et al. (1973).

APPENDIX B 327 warm (0.21 mg/L to 0.08 mg/L) and neutral environments (0.31 mg/L to 0.14 mg/L). Significant decreases in sweat iron concentration were also found be- tween 30 minutes (0.19 mg/L) and 120 minutes (0.11 mg/L) by DeRuisseau et al. (2002). Total sweat iron loss was significantly lower during the second hour of exercise. Regional sweat iron concentrations vary with higher concentrations found in sweat from the chest (0.50 mg/L) and abdomen (0.49 mg/L) than from the arm (0.28 mg/L) and back (0.20 mg/L) (Aruoma et al., 1988). Zinc Dermal zinc loss measured using whole body techniques has been found to average 0.50 mg/day in men (Jacob et al., 1981) and 0.67 mg/day in women (Hess et al., 1977). Consumption of a low zinc diet by men reduced dermal zinc loss to 0.29 mg/day and zinc repletion increased dermal zinc loss to 0.62 mg/day (Milne et al., 1983). Sweat zinc concentrations decrease over time during exercise. Tipton et al. (1993) found sweat zinc was lower after 60 minutes of exercise (0.41 mg/L) than at 30 minutes (0.97 mg/L). Similar findings were reported by DeRuisseau et al. (2002) with higher zinc concentrations during the first hour (0.90 mg/L) than the second hour (0.56 mg/L) of exercise. Lower sweat zinc concentrations were also observed during one hour of exercise in a warm environment (0.52 ± 0.41 mg/L) than in a neutral environment (0.87 ± 0.87 mg/L) (Tipton et al., 1993) but the total amount of zinc lost were not different in the two environments. Just like with the other minerals, Aruoma et al. (1988) found higher sweat concentrations on the abdomen (0.83 mg/L) than the back (0.48 mg/L), arm (0.44 mg/L) and chest (0.42 mg/L). Cordova and Navas (1998) observed significantly higher fa- cial sweat zinc concentrations in athletes during the competitive season (0.83 mg/L) than during training (0.28 mg/L). Significantly higher serum cortisol lev- els were also found in the athletes during the competitive season. CONCLUSION Mineral sweat concentrations measured during exercise are quite variable and it is obvious from the results presented in this paper that the methods of sweat collection need to be similar in order to compare results, preferably whole body collection methods. Sweat iron and zinc concentrations decrease over time during prolonged exercise and are lower in warm environments when the sweat rate is higher. Differences in sweat iron, zinc, and copper concentrations have been found from different regions of the body. Calcium and magnesium concen- trations in exercise sweat samples also vary. Use of patches over small areas of skin surface leads to overestimation of dermal calcium and magnesium losses compared to whole body dermal loss techniques. There is not much data on sweat losses with exercise for selenium or other trace minerals.

328 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL For more accurate and comparable results, whole body collection as op- posed to the use of patches or small skin areas should be the collection method of sweat and sweat should be collected over a period of time and under the specific environmental conditions of interest. For example, for military situations envi- ronmental conditions could be extreme and, therefore, temperature and humidity should mimic those extreme conditions while the study is being conducted. In addition, with acclimatization to the environment the amount of minerals in the sweat decreases. It would then be advisable to conduct studies that last for at least five days so that the effect of acclimatization on sweat mineral losses can be determined. REFERENCES Aruoma OI, Reilly T, MacLane D, Halliwell B. 1988. Iron, copper and zinc concentrations in human sweat and plasma: The effect of exercise. Clin Chim Acta 177:81­88. Brune MD, Magnusson B, Persson H, Hallberg L. 1986. Iron losses in sweat. Am J Clin Nutr 43:438­443. Bullen DB, O'Toole ML, Johnson KC. 1999. Calcium losses resulting from an acute bout of moderate-intensity exercise. Int J Sport Nutr 9:275­284. Consolazio, CF, Matoush, LO, Nelson, RA, Hackler, LR, Preston, EE. 1962. Relationship between calcium in sweat, calcium balance, and calcium requirements. J Nutr 78:78­88. Consolazio, CF, Nelson, RA, Matoush, LO, Hughes, RC. 1964. The Trace Mineral Losses in Sweat. Denver, CO: U.S. Army Medical Research and Nutrition Laboratory, Report No. 284. Cordova A, Navas FJ. 1998. Effect of training on zinc metabolism: Changes in serum and sweat zinc concentrations in sportsmen. Ann Nutr Metab 42:274­282. Costa F, Calloway DH, Margen S. 1969. Regional and total body sweat composition of men fed controlled diets. Am J Clin Nutr 22:52­58. Costill DL, Cote R, Fink W. 1976. Muscle water and electrolytes following varied levels of dehydra- tion in man. J Appl Physiol 40:6­11. DeRuisseau KC, Cheuvront SN, Haymes EM, Sharp RG. 2002. Sweat iron and zinc losses during prolonged exercise. Int J Sport Nutr Exer Metab 12:428­437. Green R, Charlton R, Seftel H, Bothwell T, Mayet F, Finch C, Layrisse M. 1968. Body iron excretion in man. Am J Med 45:336­353. Hess FM, King JC, Margen S. 1977. Zinc excretion in young women on low zinc intakes and oral contraceptive agents. J Nutr 107:1610­1620. Jacob RA, Sandstead HH, Munoz JM, Klevay LM, Milne DB. 1981. Whole body surface loss of trace metals in normal males. Am J Clin Nutr 34:1379­1382. Klesges RC, Ward KD, Shelton ML, Applegate WB, Cantler ED, Palmieri GMA, Harmon K, Davis JP. 1996. Changes in bone mineral content in male athletes: Mechanisms of action and inter- vention effects. JAMA 276:226­230. Milne DB, Canfield WK, Mahalko JR, Sandstead HH. 1983. Effect of dietary zinc on whole body surface loss of zinc:impact on estimation of zinc retention by balance method. Am J Clin Nutr 38:181­186. O'Toole ML, Johnson KL, Satterfield S, Bush AJ, Koo WW, Klesges RC, Applegate WB. 2000. Do sweat calcium losses affect bone mass during firefighter training? J Occup Environ Med 42:1054­1059. Palacios C, Wigertz K, Weaver CM. 2003. Comparison of 24-hour whole body versus patch tests for estimating body surface electrolyte losses. Int J Sport Nutr Exer Metab 13:479­488.

APPENDIX B 329 Paulev PE, Jordal R, Pedersen NS. 1983. Dermal excretion of iron in intensely training athletes. Clin Chim Acta 127:19­27. Shirreffs SM, Maughan RJ. 1997. Whole body sweat collection in humans:an improved method with preliminary data on electrolyte content. J Appl Physiol 82:336­341. Tipton K, Green NR, Haymes EM, Waller M. 1993. Zinc loss in sweat of athletes exercising in hot and neutral temperatures. Int J Sport Nutr 3:261­271. Turnlund JR, Keen CL, Smith RG. 1990. Copper status and urinary and salivary copper in young men and three levels of dietary copper. Am J Clin Nutr 51:658­664. Verde T, Shephard RJ, Corey P, Moore R. 1982. Sweat composition in exercise and in heat. J Appl Physiol 53:1540­1545. Waller MF, Haymes EM. 1996. The effects of heat and exercise on sweat iron loss. Med Sci Exer Sport 28:197­203. Wheeler EF, El-Neil H, Willson JOC, Weiner JS. 1973. The effect of work level and dietary intake on water balance and the excretion of sodium, potassium and iron in a hot climate. Br J Nutr 30:127­137. Stress Factors Affecting Homeostasis: Weight Loss and Mineral Status Steven B. Heymsfield Merck and Co. Inc., Rahway, New Jersey INTRODUCTION Weight loss in adults follows when energy losses as heat and solid matter exceed energy provided by foods. Negative energy balance is accompanied by corresponding losses of all major body compartments, including body cell mass (BCM), extracellular fluid, bone mineral, and fat. Minerals, with their respective multiple corresponding functions, are associated with each of these compart- ments and are hence lost from tissues with weight loss. Minerals are ingested with foods, and are distributed to the tissue functional sites. Losses then follow through urine, stool, skin, and other portals of exit (Figure B-18). Under conditions of controlled weight loss on nutritionally ad- equate diets, the ensuing resorption of BCM and other lean compartments re- leases minerals into the available body pool. Environmental, physical, and psychological demands during military train- ing and operations may alter metabolism in ways that exacerbate the mineral losses already occurring during weight loss diets. Other stressful factors during military lifestyle that accompany negative energy balance may be heat or cold temperatures, increased physical activity, psychological stressors, and undesir- able medical conditions such as diarrhea, vomiting, and fever. When subjects experience these conditions while ingesting diets that may be inadequate with respect to mineral and vitamins, disproportionate tissue mineral depletion with adverse functional consequences for health or performance might occur. Al-

330 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Function Cell Mass Bone Skin Other Body Intake Extracellular Output Fluid Urine Stool Absorption FIGURE B-18 Intake, distribution, and losses of a representative mineral. though an evaluation of all potential interventions would not be feasible, this paper reviews the potential adverse effects resulting from mineral inadequacies due to a number of different weight loss interventions, including surgeries or weight loss diets. REVIEW OF WEIGHT LOSS LITERATURE Selective mineral depletion with related functional consequences develops with weight loss programs that provide poor quality diets or weight-loss proce- dures that interfere with normal absorptive processes. Moreover, selective min- eral losses compounded on adverse environmental or medical conditions can facilitate depletion of mineral reserves, as for example that might follow with extreme heat and sodium loss. Intestinal Malabsorption Depending on the intervention used to attain weight loss, intestinal malab- sorption of nutrients may accompany weight loss; malabsorption can lead to a net disproportionate mineral loss. Two interventions can serve as models for the typical malabsorption response that may occur with weight loss. The first intervention is that which is based on pharmaceutical that specifi- cally decrease absorption of some nutrients. For example, a mild malabsorption that occurs with the use of the weight loss medication Xenical (orlistat) (Davidson et al., 1999). Orlistat is a gastrointestinal lipase inhibitor that limits absorption of dietary fat by about 30 percent. The result is a small, but clinically

APPENDIX B 331 important, increase in fecal fat excretion. Chelation results in binding of divalent cations such as calcium and there are also increased losses of fat soluble vita- mins. Long-term studies of up to four years (Davidson et al., 1999; Torgerson et al., 2004) show some reduction in selected fat soluble vitamins, with occasional values below the normal range. Minimal changes are observed in serum calcium levels and there are as of yet no reports of excessive osteoporosis risk, possibly because mineral and multivitamin supplementation is recommended with use of orlistat. Military personnel may use Xenical for weight loss along with popular diets, as discussed below, potentially compounding depletion of fat soluble vita- mins and minerals such as calcium and magnesium. A second more potent malabsorptive intervention involves surgical alter- ations of gastrointestinal anatomy to promote weight loss. The rate of bariatric surgery (surgery performed to reduce the size of the stomach or to bypass so that fewer calories are absorbed or both) use as a means of managing severe obesity is increasing rapidly. Bariatric surgical procedures provide a good model of malabsorptive weight loss effects on minerals. Various alterations in gastrointestinal anatomy are accompanied by surgical procedures such as the jejunoileal bypass, roux-en-y gastric bypass, and bilio- pancreatic diversion (Bloomberg et al., 2005). Mineral and vitamin absorption can be compromised, depending on the specific procedure. The modern practice is to routinely provide oral multivitamin and mineral supplementation and parenteral vitamin B12 as needed following the surgical procedure, although de- ficiencies are occasionally recognized. Iron, absorbed in the duodenum and proxi- mal jejunum, is deficient in 6­36 percent of evaluated subjects (Alvarez-Leite, 2004; Bloomberg et al., 2005; Ortega et al., 2004). Calcium and vitamin D, with duodenal and proximal jejunal absorption, are reported deficient in 10­63 per- cent of patients evaluated following surgery, depending on the specific operation (Alvarez-Leite, 2004; Bloomberg et al., 2005; Ortega et al., 2004). Zinc defi- ciency is reported in 10­50 percent of patients and may be accompanied by alopecia (Alvarez-Leite, 2004; Bloomberg et al., 2005; Ortega et al., 2004). Data on magnesium and selenium is limited. Most of the available studies are based on non-randomly selected subject groups and the actual prevalence of mineral deficiency is unknown, although clearly a mechanistic basis and the accompany- ing population specific observations support disturbances in mineral pools with surgically-induced weight loss. Low-Quality Very Low Calorie Diets Diets intended for weight loss vary in caloric content and can be generally divided into categories including: fasting; very low calorie diets (VLCDs) that provide less than 800 kcal/day; and low calorie diets that provide greater than 800 kcal/day but less than the caloric requirement for weight maintenance. Low calorie diets include a vast array of recommended intakes including balanced or

332 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL even supplemented meals and popular diets such as Atkins, South Beach, The Zone, Weight Watchers, and SlimFast, or meal replacements. Fasting as a means of weight control was first popularized by Bloom begin- ning in 1959 (Bloom, 1959). The treatment was usually carried out under medi- cal supervision and water replacement prevented dehydration. Ketosis and meta- bolic acidosis were predictable components of the total fast, and popularity waned when reports emerged of total body potassium depletion, cardiac arrhythmias, and sudden death (Schucker and Gunn, 1978). Although the mechanism of dis- turbed cardiac function was never fully established, hypotheses included essen- tial nutrient depletion and cardiac protein depletion with myofiber atrophy. Fasting was replaced, in the late nineteen sixties, with the protein-sparing modified fast or VLCD. These diets usually provided ~300 kcal/day, mainly protein, along with some other essential nutrients including minerals and vita- mins (Lockwood and Amatruda, 1984; Vertes et al., 1977). Weight loss on the early VLCDs was rapid and required medical supervision with frequent electro- lyte and mineral evaluations. These VLCDs were often ketogenic and, as with total fasting, induced a metabolic acidosis. In the mid nineteen seventies the closely monitored VLCD-type of program gave way to the widely used "liquid protein" diet popularized in diet books such as the "Last Chance Diet" (Linn and Stuart, 1976). These over-the-counter liquid diet formulas often included a poor-quality protein source and were lacking in minerals, vitamins, and other essential nutrients (Lantigua et al., 1980; Licata et al., 1981). Mineral and electrolyte deficiencies, accompanied by muscle weak- ness and cardiac arrhythmias, were reported in the medical literature (Isner et al., 1979; Michiel et al., 1978). Even after excluding patients with obvious deficien- cies, there existed a sudden and near sudden-death group with a characteristic "torsade de pointes" form of ventricular fibrillation (Sours et al., 1981). The specific mechanism leading to sudden death was never fully elucidated, although Lantigua et al. reproduced cardiac rhythm disturbances in carefully monitored obese patients using a typical liquid protein formula (1980) along with negative balances of nitrogen, phosphorus, potassium, calcium, and magnesium (Figure B-19). Sodium balance remained near zero while early potassium loss was par- ticularly rapid. The group later showed adequate diet mineral, vitamin, and trace element supplementation restored zero or near zero balances of nitrogen, phos- phorus, potassium, calcium, and magnesium (Figure B-20) and also abolished the adverse cardiac effects (Amatruda et al., 1983, 1988). These and related observations led the modern "supplemented" VLCD that remains in use today. Although the specific mechanism of fatal outcomes with VLCD dieting may never be known, there is uniform agreement that providing subjects with a low energy diet that lacks adequate minerals and vitamins can lead to serious adverse consequences. The specific required amount of minerals and electrolytes has not been rigorously established, but rather accomplished on an empirical basis using recommended daily allowance and other guideline values.

APPENDIX B 333 N K Na Mg Ca P FIGURE B-19 Mean nitrogen and mineral balances in obese subjects using a control days (­9 to 0) or a typical liquid protein formula (days 0 to 40). Subjects with arrhythmias are denoted by solid lines and subjects without them by dashed lines. SOURCE: Lantigua et al. (1980). Refeeding as an Experimental Model Adequacy of mineral and electrolyte requirements was engendered not only by events highlighted with nutritionally inadequate VLCDs, but also during the refeeding interventions of malnourished adults and children. Information regard- ing needs for minerals can also be gain from refeeding interventions. The classic study of Rudman et al. (1975) was carried out in the era following introduction of parenteral feeding solutions. The authors prepared various intravenous feed- ing solutions ranging from nutritionally complete to devoid of nitrogen, phos- phorus, potassium, or sodium. These formulas were then fed to malnourished patients at a calorie level producing weight gain. The complete intravenous formula led to anabolism with corresponding positive balances of nitrogen, phos- phorus, potassium, sodium, and calcium in proportions typical of "normal" pro- toplasm, extracellular fluid, adipose tissue, and bone. Elimination of formula nitrogen led to cessation of increments in protoplasm and extracellular fluid;

334 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL (meq) NA Mg SODIUM -8 0 10 20 30 40 (mg) K MAGNESIUM (meq) -8 0 10 20 30 40 POTASSIUM -8 0 10 20 30 40 P (mg) (mg) Ca PHOSPHORUS CALCIUM -8 0 10 20 30 40 -8 0 10 20 30 40 FIGURE B-20 Mean mineral balances per four-day period in six obese subjects using a control (days ­8 to 0) or typical liquid protein formula (days 0 to 40). The data represent the mean + / ­ SEM. SOURCE: Amatruda et al. (1983). bone mineral and adipose tissue growth continued. Likewise, feeding subjects with formulas lacking in phosphorus, potassium, or sodium led to corresponding failures to produce incremental gains in combinations of protoplasm, extracellu- lar fluid, and bone. Withdrawal of phosphorus or sodium interrupted protoplasm, extracellular fluid, and bone repletion. Repletion of protoplasm was limited when the intravenous feeding solution was potassium-free. Refeeding from a malnour- ished state, whether from disease as in Rudman's patients or through a period of starvation-induced weight loss in a military setting, clearly benefits from an adequate intake of all essential minerals, electrolytes, and trace elements. Malnutrition is common in children from developing nations and these ex-

APPENDIX B 335 periences can also serve to inform about mineral needs. When these children are renourished, optimum growth may not be achieved if the administered diets are inadequate in zinc. Simmer et al. (1988) investigated whether zinc becomes deficient during malnutrition and, thus, limits the rate of weight recovery of malnourished children. The mean zinc intake was 3.7 mg/day and one group of children was supplemented with 50 mg for two weeks. The rate of early weight gain was similar in the un-supplemented and supplemented groups, but by the second refeeding week the supplemented group had a weight gain rate 73 per- cent greater than the un-supplemented group. The zinc content of polymorpho- nuclear cells increased in the zinc supplemented group (p < 0.001) but not in the un-supplemented group. Khanum et al. (1988) reported a similar observation in a larger group of malnourished children. Popular Diets As with the general population, excess weight is a concern to military per- sonnel and regulations regarding military weight standards may encourage those afflicted to attempt weight loss on a popular diet. Programs such as Weight Watchers are highly developed and the prescribed food intake is adequate in minerals and other essential nutrients. Similarly, other commercially available products, such as the liquid meal replacement SlimFast, are well studied and amply supplemented with minerals and vitamins. Of greater concern is the regu- lar appearance of popular diets that are not rigorously developed with respect to the underlying weight loss nutritional theory or to the nutrient content of sug- gested foods. This section describes some potential health risks if military per- sonnel would adhere to some of these diets. The widely used Atkins diet promotes ketosis, early diuresis, and associated metabolic acidosis if rigorously adhered to. Some concern has been expressed for the potential renal effects of high protein intake and the potential for low vitamin D and calcium intake. Metabolic acidosis over the long term can lead to osteoporosis. Freedman et al. (2001) examined the three Atkin's Diet New Diet Revolution phases, induction, ongoing, and maintenance in relation to the Food Guide Pyramid and the dietary reference intakes. Calcium, magnesium, iron, and potassium intakes would be low across the three phases while phosphorus, sodium, and zinc intakes would be within an acceptable intake range. Freedman et al. (2001) also provide an extensive tabulation of potential nutri- tional inadequacies, including low mineral content, for other popular plans includ- ing Carbohydrate Addict's diet, Sugar Busters!, and Ornish. As with Atkin's, these diet plans are low in calcium, magnesium, iron, and potassium. Vasilaris and colleagues (2004) examined micronutrient intake in overweight subjects on an ad libitum fat-reduced, high simple-carbohydrate diet. The au- thors observed a lower zinc intake in men and lower vitamin B12 intake in men

336 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL and women ingesting a fat-reduced simple carbohydrate-rich diet compared to a habitual, normal-fat diet, but not below recommended levels. More studies are needed of the mineral adequacy of popular diets potentially ingested by military personnel before recommending their use. The intake of essential micronutrients by subjects in these diets has been reported in a few studies. As expected, a consequence of the diets being low in minerals is that most subjects embarking on a low calorie diet without profes- sional guidance will often ingest inadequate minerals and vitamins (Cifuentes et al., 2004). Of particular note is the low calcium intake observed in overweight and obese women ingesting low calorie diets for weight loss. Riedt et al. (2005) examined the influence of energy restriction and calcium intake on bone mineral density in overweight post-menopausal women. Weight loss resulted in loss of bone at several anatomic sites (e.g., trochanter and spine) in women consuming 1 g Ca/day and was abolished at calcium intakes of ~1.7 g/day. A reduction in circulating estradiol or a rise in parathyroid hormone (PTH) and cortisol may explain bone mobilization, possibly because of Ca-PTH axis suppression. SUMMARY These collective observations highlight the critical importance of mineral content of weight loss diets and the functional and pathological consequences that ensue when mineral supplementation is inadequate. Not only the weight loss diets, but surgical interventions that aim at decreasing absorption of energy sources, may have severe consequences in the mineral balance and therefore performance of soldiers. Of special concern is calcium, because studies have shown that even when meeting the adequate intake for calcium, weight loss diets can result in excessive bone loss, potentially increasing stress fractures. Most of these studies have been carried out in otherwise healthy subjects living and working in stable developed environments. The additional burdens imposed by some military conditions, heat, intense physical activity, and other potential mineral pool stresses, may lead to serious deficiencies and related func- tional consequences when accompanying weight loss. Therefore, when adhering to interventions to attain a specific weight within the military, individuals should seek dietary guidance from appropriate experts so that micronutrient intake and, especially, mineral intake is adequate. REFERENCES Alvarez-Leite JI. 2004. Nutrient deficiencies secondary to bariatric surgery. Curr Opin Clin Nutr Metab Care 7:569­575. Amatruda JM, Biddle TL, Patton ML, Lockwood DH. 1983. Vigorous supplementation of a hypo- caloric diet prevents cardiac arrhythmias and mineral depletion. Amer J Med 74:1016­1022.

APPENDIX B 337 Amatruda JM, Richeson JF, Welle SL, Brodows RG, Lockwood DH. 1988. The safety and efficacy of a controlled low-energy ("very-low-calorie") diet in the treatment of non-insulin-dependent diabetes and obesity. Arch Inter Med 148:873­877. Bloom WL. 1959. Fasting as an introduction to the treatment of obesity. Metabolism 8:214­220. Bloomberg RD, Fleishman A, Nalle JE, Herron DM, Kini S. 2005. Nutritional Deficiencies follow- ing Bariatric Surgery: What Have We Learned? Obes Surg 15:145­154. Cifuentes M, Riedt CS, Brolin RE, Field MP, Sherrell RM, Shapses SA. 2004. Weight loss and calcium intake influence calcium absorption in overweight postmenopausal women. Am J Clin Nutr 80:123­130. Davidson MH, Hauptman J, DiGirolamo M, Foreyt JP, Halsted CH, Heber D, Heimburger DC, Lucas CP, Robbins DC, Chung J, Heymsfield SB. 1999. Weight control and risk factor reduc- tion in obese subjects treated for 2 years with Orlistat: A randomized controlled trial. JAMA 281:235­242. Freedman M, King J, Kennedy E. 2001. Popular diets: A scientific review. Obesity Research Suppl 1:1S­40S. Isner JM, Sours HE, Paris AL, Ferrans VJ, Roberts WC. 1979. Sudden, unexpected death in avid dieters using the liquid-protein-modified-fast diet. Circulation 60:1401­1412. Khanum S, Alam AN, Anwar I, Akbar Ali M, Mujibur Rahaman M. 1988. Effect of zinc supplemen- tation on the dietary intake and weight gain of Bangladeshi children recovering from protein- energy malnutrition. Eur J Clin Nutr 42:709­714. Lantigua RA, Amatruda JM, Biddle TL, Forbes GB. 1980. Cardiac arrhythmias associated with a liquid protein diet for the treatment of obesity. Med Intell 303:735­738. Licata AA, Lantigua R, Amatruda J, Lockwood D. 1981. Adverse effects of liquid protein fast on the handling of magnesium, calcium and phosphorus. Amer J Med 71:767­772. Linn R., Stuart SL. 1976. The Last Chance Diet. Secaucus, NJ: Lyle Stuart. Lockwood DH, Amatruda JM. 1984. Very low calorie diets in the management of obesity. Ann Rev Med 35:373­381. Michael RR, Sneider JS, Dickstein RA, Hagman HH, Eich RH. 1978. Sudden death in a patient on a liquid protein diet. N Engl J Med 298:1005­1007. Ortega J, Sala C, Flor B, Jimenez E, Escudero D, Martinez-Valls J, Lledo S. 2004. Vertical banded gastroplasty converted to Roux-en-Y gastric bypass: Little impact on nutritional status after 5-year follow-up. Obes Surg 638­643. Riedt CS, Cifuentes M, Stahl T, Chowdhury HA, Schlussel Y, Shapses SA. 2005. Overweight post- menopausal women lose Bone with moderate weight reduction and 1g/day calcium intake. J Bone Miner Res 20:455­463. Rudman D, Millikan WJ, Richardson TJ, Bixler TJ, Stackhouse J, McGarrity WC. 1975. Elemental balances during intravenous hyperalimentation of underweight adult subjects. J Clin Invest 55: 94­104. Schucker RE, Gunn WJ. 1978. A National Survey of the Use of Protein Products in Conjunction with Weight Reduction Diets Among American Women. Atlanta, GA: Centers for Disease Control and Prevention. Simmer K, Khanum S, Carlsson L, Thompson RP. 1988. Nutritional rehabilitation in Bangladesh-- the importance of zinc. Am J Clin Nutr 47:1036­1040. Sours HE, Frattali VP, Brand CD, Feldman RA, Forbes AL, Swanson RC, Paris AL. 1981. Sudden death associated with very low calorie weight reduction regimens. Am J Clin Nutr 34:453­461. Torgerson JS, Hauptman J, Boldrin MN, Sjostrom L. 2004. XENical in the prevention of diabe- tes in obese subjects (XENDOS) study: A randomized study of orlistat as an adjunct to lifestyle changes for the prevention of type 2 diabetes in obese patients. Diabetes Care 27:155­161. Vasilaras TH, Astrup A, Raben A. 2004. Micronutrient intake in overweight subjects is not deficient on an ad libitum fat-reduced, high-simple carbohydrate diet. Euro J Clin Nutr 58:326­336.

338 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Vertes V, Genuth SM, Hazelton IM. 1977. Supplemented fasting as a large scale outpatient program. JAMA 238:2151­2153. Protein Turnover and Mineral Metabolism Henry C. Lukaski USDA-ARS Grand Forks Human Nutrition Research Center, Grand Forks, North Dakota INTRODUCTION Military personnel are regularly exposed to multiple stressors during opera- tional training and combat. Factors including food restriction, sleep deprivation, increased physical activity, psychological stressors, harsh environments, and in- fection promote weight loss and body composition changes, as well as impair- ments in some biological functions and altered nutritional status (Baker-Fulco, 1995; Shippee, 1993). Decreases in lean body mass (Friedl et al., 2000) that parallel increases in the circulating concentrations of minerals, specifically intra- cellular cations, such as magnesium, zinc, and copper (Shippee, 1993), suggest an important link between protein turnover and mineral excretion. This review examines the effects of stressors on indirect measures of protein catabolism and biochemical indices of mineral nutritional status. It describes findings in injured patients and the limited observations in healthy adults partici- pating in controlled exercise and dietary interventions. A summary of the find- ings of intensive, military training under diverse conditions is provided. This information is assembled into a model that integrates experimental findings that link protein catabolism and mineral excretion. PROTEIN AND MINERAL METABOLISM AFTER INJURY Following moderate to severe injury, there is a period of excess protein breakdown as measured by increased urinary excretion of nitrogen, creatinine potassium, phosphorus, sulfur, magnesium, and zinc (Cuthbertson et al., 1972). Urinary nitrogen was significantly correlated with urinary zinc, potassium, and creatinine (r = 0.46­0.66) in patients with different types of trauma. These find- ings suggest a general association between a marker of protein breakdown, nitro- gen, and intramuscular cations and metabolites. Supportive information comes from data in which patients were labeled with radioactive zinc (65Zn) that was incorporated into skeletal muscle, a major reservoir of zinc, before elective surgery (Fell et al., 1973). Urinary nitrogen and zinc excretions increased 80 and 100 percent, respectively, after surgery; these outputs were significantly correlated (r = 0.84­0.98). Compared to pre-surgical

APPENDIX B 339 values, fractional 65Zn excretion increased significantly from 10 percent to 20 percent after surgery. The authors concluded that the zincuria indicated loss of muscle. Additional evidence of accelerated muscle breakdown and zinc losses is available. Significant increases in urinary excretion of 3-methylhistidine, an in- dex of myofibrillar protein breakdown (Munro and Young, 1978), creatinine and zinc were observed in patients either undergoing orthopedic surgery or in re- sponse to trauma (Threlfall et al., 1981). The outputs were related to the severity of injury and reached peak values seven days after injury or surgery. Other investigators (Askari et al., 1982; Berger et al., 1996) have confirmed these findings and emphasized the need for adequate zinc intake to accommodate the increased zinc losses. OTHER STRESSORS AFFECTING PROTEIN AND MINERAL METABOLISM Whereas the effects of severe injury on increased protein breakdown and mineral excretion is well established, there is a paucity of information describing metabolic perturbations with stressors that impose mild and moderate injury. An area of interest is the effect of increased physical activity on protein and mineral metabolism. Endurance Exercise Studies have examined the effects of intense, endurance exercise on zinc homeostasis. Compared to pre-exercise values, plasma zinc concentrations in- creased (5­10 percent) in men immediately after a bout of prolonged, endurance running (Anderson et al., 1984; Cordova and Alvarez-Mon, 1995; Van Rij et al., 1986). However, 2 hours after completing the exercise, plasma zinc concentra- tions decreased significantly. Exercise, however, was associated with significant increases in urinary zinc ranging from 20 to 40 percent compared to values determined on days without exercise. While it is speculated that the decrease in plasma zinc concentration may be related to zinc sequestration in liver and other soft tissues (Oh et al., 1978; Shinogi et al., 1999), the increase in urinary zinc is the result of muscle breakdown and mobilization from other stores. The impact of differences in dietary zinc, however, should not be dismissed because zinc intake was not assessed. Stressors such as prolonged endurance exercise increase amino acid oxi- dation and urinary nitrogen excretion (Lemon, 1998). Unfortunately, studies that have investigated metabolic responses to endurance exercise have not concurrently determined indices of muscle protein breakdown and mineral excretion.

340 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Effects of Energy Deficit and Exercise An experimental model that may provide insight into the effects of multiple stressors on protein and mineral metabolism is reduced energy intake and in- creased physical activity. Briefly, obese women were studied on a metabolic unit for five months (Lukaski, unpublished results). During the first 28 day period, they received a nutritionally balanced diet with energy sufficient to maintain admission body weight. Energy intake was reduced by 25 percent during the next 28 days, and then further reduced to 50 percent of maintenance levels for the following two 28 day periods. Aerobic physical activity increased progres- sively with the start of the energy restriction. Body weight decreased modestly (3 percent) with a 25 percent reduction in energy intake; it decreased (8 percent and 14 percent) significantly with an energy restriction of 50 percent of weight maintenance levels. Urinary nitrogen excretion increased significantly at the end of each period of 50 percent energy reduction (1.5 to 1.7 g/day). Urinary zinc output increased significantly (0.1 mg/day) in parallel with the restricted energy intake. These findings indicate a concomitant loss of muscle protein and zinc with a 50 percent reduction of energy intake required to maintain body weight and increased physical activity. SEMISTARVATION IN A MULTISTRESSOR ENVIRONMENT Training of candidates for Special Forces such as Rangers exposes military personnel to semi-starvation, heavy energy expenditures and other psychological and physical challenges (Moore et al., 1992). A number of metabolic perturba- tions have been observed (Friedl et al., 2000; Shippee, 1993). Hormonal re- sponses include a significant decrease in testosterone, thyroid hormones and insulin-like growth factor 1 (IGF-1), and a significant increase in cortisol con- centrations in blood. Metabolic parameters also were impacted. Blood urea ni- trogen, -hydroxybutyrate and lactate increased significantly during training. Magnesium, copper, zinc, and ferritin increased significantly (12, 30, 25, and 70 percent, respectively) whereas iron decreased significantly (30 percent). Taken together, these findings indicate that the conditions of Ranger training promoted catabolism of body protein and mobilization of minerals as a result of many factors including food deprivation, injury, and inflammatory processes. LINK BETWEEN PROTEIN CATABOLISM AND MINERAL OUTPUT Situations and conditions that expose humans to multiple stressors elicit hor- monal and immune responses that adversely impact protein and mineral metabo- lism (Figure B-21). Hypocaloric intake increases catabolic hormones (glucagon, catecholamines, and cortisol) and decreases anabolic hormone (insulin and IGF-1) concentrations in the circulation (Keys et al., 1950). The magnitude of the caloric deficit, by restriction and increased energy expenditure, directly influences these

APPENDIX B 341 Stressors Metallothionen Cortisol, catecholamines, Ketosis cytokines, glucagon, IGF-1 Sequestration "Make or break" muscle of minerals Decreased Protein catabolism circulating (breakdown > synthesis) minerals AA Efflux Mineral loss in urine Mineral Loss FIGURE B-21 Effects of stress on protein turnover and mineral metabolism. NOTE: AA = amino acids; IGF-1 = insulin-like growth factor-1. hormonal responses. Similarly, inadequate energy intake and increased exercise, particularly in conjunction with injury, affect circulating cytokines. Tumor necro- sis factor (TNF-´) and interleukin-1 (IL-1) and other immuno-regulatory proteins are involved in the acute phase response. Interestingly, IL-6 which is increased in response to exercise, is anti-inflammatory (Petersen and Pedersen, 2005). Because the circulating concentrations of these cytokines increase in response to energy stress, heavy exercise and injury, they, together with the catabolic hormones, up- regulate intracellular signal transduction pathways to increase protein catabolism and promote amino acid efflux (Glass, 2003; Tisdale, 2002). Caloric deprivation also increases fatty acid mobilization and promotes ketosis. Both amino acid efflux and ketosis increase mineral excretion in the urine. Increases in circulating mineral concentrations stimulate metallothionein synthesis (Oh et al., 1978). Metallothionein removes minerals from the circula- tion and sequesters them in tissues such as the liver and kidney; it scavenges ionized minerals and reduces the potential for oxidative damage. The increase in synthesis of thionein requires about 2 hours. Thus, there is a period during which minerals are lost into the urine. However, with a half-life of 5­6 hours, metal- lothionein serves to attenuate loss of minerals acutely. SUMMARY AND CONCLUSIONS Evidence from diverse sources indicates a clear link between muscle protein breakdown and losses of minerals. Studies in patients with acute phase response

342 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL and healthy individuals participating in endurance exercise reveal increased losses of nitrogen and zinc in the urine. Obese adults participating in a program of energy restriction and increased aerobic physical activity also experience a similar pattern of increased urinary nitrogen and zinc loss. Studies of soldiers exposed to semi-starvation and other stressors show a hormonal pattern of in- creased catabolism and release of minerals into the circulation that suggests an increased loss of muscle and minerals, specifically zinc and magnesium, as part of an acute phase response. There is a need to concurrently determine nutrient and mineral intakes, mea- sures of mineral nutritional status, and losses together with hormonal and cyto- kine measurements in soldiers under conditions simulating strenuous training and operations. This information is needed to critically evaluate adequacy of minerals, particularly zinc and magnesium, in rations to compensate for losses during active training. DISCLAIMER Mention of a trademark or proprietary product does not constitute a guaran- tee of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. U.S. Department of Agriculture, Agricultural Research, Northern Plains Area is an equal opportunity/affirmative action employer and all agency services are available without discrimination. REFERENCES Anderson RA, Polansky MM, Bryden NA. 1984. Strenuous running: Acute effects of chromium, copper, zinc, and selected clinical variables in urine and serum of male runners. Biol Trace Elem Res 6:327­336. Askari A, Long CL, Blakemore WS. 1982. Net metabolic changes of zinc, copper, nitrogen, and potassium balances in skeletal trauma patients. Metabolism 31(12):1185­1193. Baker-Fulco CJ. 1995. Overview of dietary intakes during military exercises. In: Not Eating Enough. Washington, DC: National Academy Press. Berger MM, Cavadini C, Chiolero R, Dirren H. 1996. Copper, selenium and zinc status and balances after major trauma. J Trauma 40(1):103­109. Cordova A, Alvarez-Mon M. 1995. Behavior of zinc in physical exercise: A special reference to immunity and fatigue. Neurosci Biobehav Rev 19(3):439­445. Cuthbertson DP, Fell GS, Smith CM, Tilstone WJ. 1972. Metabolism after injury. I. Effects of severity, nutrition, and environmental temperature on protein, potassium, zinc, and creatine. Br J Surg 59(12):925­931. Fell GS, Fleck A, Cuthbertson DP, Queen K, Morrison C, Bessent RG, Husain SL. 1973. Urinary zinc levels as an indication of muscle catabolism. Lancet Feb. 10, 1(7798):280­282. Friedl KE, Moore RJ, Hoyt RW, Marchitelli LJ, Martinez-Lopez LE, Askew EW. 2000. Endocrine markers of semistarvation in healthy lean men in a multistressor environment. J Appl Physiol 88:1820­1830. Glass DJ. 2003. Molecular mechanisms modulating muscle mass. Trends Mol Med 9:344­350.

APPENDIX B 343 Keys A, Brozek J, Henschel A, Mickelsen O, Taylor HL.1950. The Biology of Human Starvation. Minneapolis, MN: University of Minnesota Press, vols. 1 and 2. Lemon PWR. 1998. Effects of exercise on dietary protein requirements. Int J Sport Nutr 8(4): 426­447. Moore RJ, Friedl KE, Tulley RT, Askew EW. 1993. Maintenance of iron status in healthy men during an extended period of stress and physical activity. Am J Clin Nutr 58:923­927. Munro HN, Young VR. 1978. Urinary excretion of N gamma-methylhistidine (3-methylhistidine): a tool to study metabolic responses in relation to nutrient and hormonal status in health and disease of man. Am J Clin Nutr 31(9):1608­1614. Oh SH, Deagen JT, Whanger PD, Weswig PH. 1978. Biological function of metallothionein. V. Its induction in rats by various stressors. Am J Physiol 234(3):E282­E285. Petersen AMW, Pedersen BK. 2005. The anti-inflammatory effect of exercise. J Appl Physiol 98(4):1154­1162. Shinogi M, Sakaridani M, Yokoyama I. 1999. Metallothionein induction in rat liver by dietary restriction or exercise and reduction of exercise-induced hepatic lipid peroxidation. Biol Pharm Bull 22(2):132­136. Shippee RL. 1993. Nutritional status and immune function of Ranger trainees given increased caloric intake. Briefing for the National Academy of Sciences. In: Marriott BM, ed. Review of the Results of Nutritional Intervention, US Army Ranger Training Class 11/92 (Ranger II). Wash- ington, DC: National Academy Press. Pp. 86­104. Threlfall CJ, Stoner HB, Galasko CS. 1981. Patterns in the excretion of muscle markers after trauma and orthopedic surgery. J Trauma 21(2):140­147. Tisdale MJ. 2002. Biochemical mechanisms of cellular catabolism. Curr Opin Clin Nutr Metabol 5(4):401­405. Van Rij AM, Hall MT, Dohm GL, Bray JT, Pories WJ. 1986. Changes in zinc metabolism following exercise in human subjects. Biol Trace Elem Res 10:99­101. Physical Activity and Tyrosine Supplementation: Two Effective Interventions Against Stress-Induced Immunosuppression Monika Fleshner University of Colorado, Boulder INTRODUCTION Excessive Sympathetic Nervous System Output Is Detrimental to Health Stimulation of the sympathetic nervous system (SNS) is a hallmark of the acute stress response (Goldstein, 1987). SNS activation has many physiological consequences such as increased heart rate, respiration and blood flow to muscles, that work in concert to promote the "fight or flight" response (Goldstein, 1996; Jansen et al., 1995). SNS activation is a powerful feature of the acute stress response that is adaptive when the response is acute and constrained. If, how- ever, SNS activation is frequent or excessive, it can produce negative health consequences (Seals and Dinenno, 2004). For example, chronically elevated SNS

344 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL responses are believed to mechanistically contribute to the etiology of "meta- bolic syndrome," a key antecedent to clinical atherosclerotic diseases that in- clude visceral adiposity, glucose intolerance, insulin resistance, dyslipidemia and hypertension (Baron, 1990; Julius et al., 1992; Lind and Lithell, 1993). In addi- tion, it has been reported in both the human and animal literatures that chronic or excessive SNS activation can lead to arterial wall thickening (Chen et al., 1995; Pauletto et al., 1991; Xin et al., 1997), hypertension (Lind and Lithell, 1993), - and - adrenergic receptor desensitization (Abrass, 1986; Dinenno et al., 2002; Xiao and Lakatta, 1992) and immunosuppression (Irwin, 1993; Kennedy et al., 2005a). The negative consequences of frequent or excessive SNS activity have been convincingly demonstrated in transgenic mice lacking functional 2A adrenergic receptor (ADR) autoinhibition in the midbrain. Due to the lack of normal 2AADR central nervous constraint on SNS drive, these mice have chronically activated peripheral SNS responses and rapidly develop cardiac dys- function (Baum et al., 2002). Stress Modulates Immune Function Exposure to physical or psychological stress modulates the immune response (Adell et al., 1988; Laudenslager, 1994; Maier et al., 1994; Plotnikoff, 1991). Stress is neither globally immunosuppressive nor immunopotentiating, but rather immunomodulatory. Factors that impact the effect of stress on the immune re- sponse include the following: the duration and intensity of stressor exposure (Monjan, 1976); the perceived controllability of the stressor (Laudenslager, 1983); the timing and measure of the immune response (e.g., days versus hours, acquired versus innate (Deak et al., 1999; Fleshner et al., 1998); and the physi- ological state of the organism (e.g., young versus old, anxious versus calm, healthy versus ill, and physically active versus sedentary (Ader et al., 1991; Bonneau 1997; Brown, 1988; Dishman, 1995; Fleshner et al., 2002; Moraska and Fleshner, 2001). Animal Model of Acute Stress My laboratory has been studying the behavioral and physiological conse- quences of exposure to a well-characterized animal model of stress. This model of stress involves exposing rats to random, intermittent (average intertrial inter- val of 60 seconds), inescapable tailshocks (100 shocks of 1.6mA for a duration of 5 seconds), administered when the rats are lightly restrained in Plexiglas tubes. The use of this stressor is important for several reasons. First, a great deal is known about the behavioral, neural, endocrine, and immunological conse- quences of exposure to this acute stressor (Brennan, 1995, 1996; Campisi and Fleshner, 2003; Campisi et al., 2002, 2003; Day et al., 2004; Deak, 1997, 1999; Deak et al., 1997; Fleshner et al., 1993, 1995a,b,c,d, 1998, 2002; Gazda et al.,

APPENDIX B 345 2003; Greenwood et al., 2003a,b; Laudenslager, 1994; Maier, 1998; Maier et al., 1994; Milligan et al., 1998; Moraska and Fleshner, 2001; Moraska et al., 2002; Nguyen, 1998; Nguyen et al., 1998, 2000; O'Conner, 1999; Watkins, 1990). Second, the effects of acute stressor exposure on immune function are stressor dependent (Ader et al., 1991; Plotnikoff, 1991), therefore the use of a consistent stressor is necessary to advance our understanding of the mechanism responsible for stress-induced immunomodulation. Third, tail-shock stress allows the admin- istration of a discrete, consistent, and quantifiable stressor that does not produce physical injury. SUPRESSION OF ACQUIRED IMMUNITY BY STRESS In Vivo Generation of Antibody Against Keyhole Limpet Hemocyanin as a Measure of Acquired Immunity Acquired immunity is characterized by two primary features, exquisite anti- gen specificity and immunological memory. The effector cells of the acquired immune response include T cells and B cells. Our assessment of acquired im- mune function has been the generation of an immunoglobulin response to key- hole limpet hemocyanin (KLH Ig). This measure of immune function has both experimental advantages, as well as clinical relevance that include the following: (1) the cells involved with the generation of this response remain in the hor- monal milieu of the organism; (2) the kinetics of the developing response can be easily monitored; (3) use of a benign protein does not produce the behavioral confounds associated with the generation of sickness; (4) antibody reflects a functionally important end product of the immune system; (5) measurement of the antigen specific antibody response more accurately reflects the function of acquired immunity; (6) measurement of KLH Ig is quantifiable making the results directly comparable across studies; (7) the cells involved with this response are T cells and B cells, two primary players in acquired immune re- sponses; (8) the antibody response generated against KLH is similar to the im- munological response generated after vaccination to tetanus toxoid; (9) a re- duction in specific antibodies to bacteria, virus, or soluble toxin could render the organism more susceptible to disease caused by these pathogens; (10) KLH is clinically relevant because it is used as a immunotherapeutic in the treatment of cancer (Gilewski, 1996; Jurincic-Winkler, 1995, 1996; Lamm, 1993; Livingston, 1995), and stress-induced modulation of the antibody response to KLH could affect the efficacy of this type of vaccination and immunotherapy. Finally, re- sults from measuring responses to KLH in animals can be easily tested in hu- mans (Smith et al., 2004).

346 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Spleen Is Site for Stress-Induced KLH Antibody Suppression Rats that are immunized with KLH and exposed to a single session of ines- capable tailshock have a long-term (+ 21 days) reduction in serum levels of KLH IgM, IgG and IgG2a (Fleshner et al., 1995d, 1998; Gazda et al., 2003; Laudenslager et al., 1988). We know that the final site of stress-induced immu- nomodulation is the spleen because if we remove the spleen from adult male rats prior to intraperitoneal immunization with KLH and stressor exposure, we elimi- nate the stress-induced reduction in KLH Ig (Fleshner, 2005). Importantly, the stress-associated suppressive effect is specific to the generation of antibody to the antigen. Total serum IgM and IgG is not reduced (Fleshner et al., 1992; Smith et al., 2004). Cellular Mechanisms of Stress-Induced KLH Antibody Suppression The generation of an antibody response to a T cell dependent soluble pro- tein, such as KLH, involves the interaction of antigen presenting cells (APC; B cells or dendritic cells), T helper cells (Th) and B cells. Following intraperitoneal injection of KLH, antigen is transported to the draining lymph nodes and spleen. B cells expressing the B cell receptor that bind KLH must receive T cell help from the KLH-specific T helper cells in the form of co-stimulation and cytokines. The Th "help" facilitates B cell proliferation, B cell differentiation into antibody secreting cells (Clark and Ledbetter, 1994; Foy et al., 1996), and Ig isotype switching (IgM to IgG or IgG2a, [Stevens et al., 1988]). The proliferation of KLH-specific Th and B cells is greatest in the draining lymph nodes and spleen 4­7 days after KLH injection (Fleshner et al., 1995d, 1998; Gazda et al., 2003). Using flow cytometric analysis (Fleshner et al., 1995a; Fleshner et al., 1998), ELISPOT (Laudenslager, 1994), and antigen-specific proliferative assays (Gazda et al., 2003), we have determined that the suppression in KLH Ig is likely due to a failure of the stressed rats to increase KLH-specific T helper cell numbers (Fleshner et al., 1995a, 1998). With fewer KLH T helper cells, there is less T cell help, and fewer KLH-specific B in the spleen (Laudenslager, 1994). Fewer KLH-specific B cells lead to a reduction in serum KLH Ig. Thus, tailshock- induced suppression of KLH Ig is a well-characterized animal model of stress- induced immunosuppression. Excessive Sympathetic Nervous System Response Suppresses Acquired Immunity Although the specific mechanism responsible for stress-induced suppression of KLH Ig remains under investigation, excessive SNS output likely plays a role. Most primary and secondary lymphoid tissues (including the spleen) re- ceive dense SNS innervation (Felten, 1987; Meltzer, 1997) and Th cells (Kohm

APPENDIX B 347 and Sanders, 2000, 2001; Sanders, 1997; Swanson et al., 2001), B cells (Kasprowicz et al., 2000; Kohm et al., 2002; Podojil and Sanders, 2003; Podojil et al., 2004) and monocytes-macrophages-dendritic cells (Takahashi et al., 2004) express adrenergic receptors 2ADR. If we focus on the role of the SNS in stress-induced immunomodulation, there is evidence that SNS contributes to stress-induced suppression of specifically the KLH Ig response (Irwin, 1993). Although earlier work suggested that high concentrations of norepinephrine (NE) could suppress various aspects of immunity, more recent data support the hypothesis that splenic NE depletion, not circulating or splenic NE elevation, may be responsible for stress-induced suppression of in vivo KLH Ig responses. There are several lines of evidence to support this shift in dogma from "too much NE" to "too little NE." First, if one examines the past literature demon- strating that high levels of NE are immunosuppressive, many studies were done in vitro, examined mitogen-stimulated proliferative or cytokine responses, and tested pharmacological concentrations of NE (Malarkey et al., 2002; Ramer- Quinn, 1997). Under these circumstances, NE suppresses immune function and can be fatal to immune cells (Del Rey et al., 2003). Second, activation status of the immune cells was rarely considered in these earlier studies. For example, it was recently reported that modulation of dendritic cell function following NE exposure occurred only in the early phases of dendritic cell activation (Maestroni, 2002), and 2ADR are differentially expressed on naïve versus stimulated B cells (Sanders et al., 2003). Thus, past research supporting a simple view that too much NE is responsible for stress-induced suppression of in vivo immune re- sponses has limitations. Recent evidence is consistent with the dogmatic shift that too little NE may be responsible for stress-induced suppression of in vivo antibody responses and that dynamic interactions between SNS and immune cells occur to produce opti- mal Ig responses. For example, during the generation of an in vivo antibody response to KLH, NE is released from peripheral nerves innervating the spleen (Kohm et al., 2000). NE binding to the B cell 2ADR stimulates the expression of costimulatory molecules (Kohm et al., 2002), Ig production (Kasprowicz et al., 2000), and splenic germinal center formation (Kohm, 1999). In addition, splenic NE depletion produced by surgical denervation (Fleshner, 2006), phar- macological lesion [6-OHDA,] (Kohm and Sanders, 1999) or pharmacological competition (-methyl-p-tyrosine (Kennedy et al., 2005b) prior to immunization with KLH reduces the antibody response. Thus, splenic NE depletion in the absence of stress is sufficient to supress KLH Ig. Furthermore, central activa- tion of the SNS in the absence of stressor exposure with an 2AADR antagonist (Mirtazapine, Mirt) that acts in the brain to release the SNS from 2AADR- mediated inhibition (Dazzi et al., 2002), elevates blood NE for longer duration and to a higher level than that produced by stress. Yet, in spite of high blood concentrations of NE at the time of immunization, Mirt produces neither splenic NE depletion nor KLH Ig suppression (Kennedy et al., 2005b). Blood NE is

348 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL derived from spillover of NE released by nerve terminals in sympathetically innervated tissues. We speculate that the lack of splenic NE depletion in spite of equal or greater blood concentration on NE after Mirt injection may be due to a more global, whole body activation of the SNS; whereas tailshock stress may activate more selective central SNS circuits (Greenwood et al., 2003b) perhaps excessively driving SNS output to select tissues such as the spleen. BEHAVIORAL AND PHARMACOLOGICAL INTERVENTIONS TO PREVENT STRESS-RELATED ADVERSE EFFECTS ON IMMUNE SYSTEM Introduction Based on our results it follows that to prevent the negative consequences of activation of the acute stress response one would need interventions that pre- vent splenic NE depletion by either (1) constraining excessive SNS output or (2) providing additional substrate to prevent splenic NE depletion in the face of intense SNS drive. Such approaches are optimal because they would not elimi- nate SNS responses. In that way, the stressed organism could reap the positive physiological effects of SNS activation but avoid the negative immunological consequences of excessive SNS activation and splenic NE depletion. We have evidence that exercise and tyrosine supplementation are both possible interven- tions that satisfy this goal. Physical Activity Constrains SNS Activation We have conducted a series of studies investigating the impact of tailshock on various aspects of the stress response including SNS activation, splenic NE depletion and KLH Ig suppression. Physical active status was varied in these studies by housing animals with either a mobile or locked running wheels. In these conditions, male F344 rats will run an average distance of 15 km/week (Campisi et al., 2003; Greenwood et al., 2003a,b). Nearly 100 percent of their running occurs during the dark part of their circadian cycle (Solberg, 1999). This level of activity produces physiological changes that are indicative of "metabolic fitness". In some rat strains, wheel running reduces body weight gain (Noble et al., 1999), body fatness (Podolin, 1999), triglycerides concentrations (Suzuki, 1995), and increases lipid metabolism (Podolin, 1999), HDL/LDL ratio (Kennedy et al., 2005a), muscular hypertrophy (triceps and plantaris (Ishihara et al., 1998), red blood cell hemoglobin content (Kennedy et al., 2005a), and endurance. Animals that lived sedentary life styles with locked running wheels, and were exposed to tailshock stress, had excessive SNS responses leading to splenic NE depletion and KLH Ig suppression (Kennedy et al., 2005b). In contrast, rats that were physically active for 6 weeks prior to exposure to tailshock stress,

APPENDIX B 349 Run Sedentary l)µ l)µ ng/ ng/ (NE, (NE, Norepinephrine Norepinedrine Stressor onset FIGURE B-22 Effect of Physical Activity (freewheel running) on supression of stress- induced (10, 50, or 100 5-second, 1.6mA tailshocks) splenic NE depletion in adult male F344 rats. (*P < 0.05). SOURCE: Greenwood et al. (2003b). had constrained SNS responses such that tailshock elevated blood levels of NE but did not drive the response excessively, did not lead to splenic NE depletion (Figure B-22) and did not produce KLH Ig suppression (Figure B-23) (Green- wood et al., 2003b; Moraska and Fleshner, 2001). Thus, physical activity prevented the negative effects of acute stress on acquired immunity by constrain- ing SNS drive (Fleshner, 2005). Tyrosine Supplementation Prevents Stress-Induced Splenic NE Depletion Tyrosine is a precursor for the synthesis of NE (and dopamine, DA), and during times of intense SNS drive can be rate limiting (Acworth et al., 1988; Gibson and Wurtman, 1977; Milner and Wurtman, 1987). It has been previously reported in rats that tyrosine administration can prevent stress-induced brain NE depletion (Lehnert et al., 1984) and stress-induced behavioral deficits (Brady et al., 1980; Reinstein et al., 1984). In addition, tyrosine supplementation has been used to reduce headaches, tension and fatigue in men exposed to cold stress (Banderet and Lieberman, 1989) and more recently tyrosine was used to reduce elevated blood pressure associated with combat training (Deijen and Orlebeke, 1994; Deijen et al., 1999). The possible effect that tyrosine could have in hu- mans on stress-induced immunosupression has yet to be tested.

350 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL (OD) (OD) IgG2a IgG2a anti-KLH anti-KLH FIGURE B-23 Prevention of stress-induced (tailshocks) supression of anti-keyhole lim- pet hemocyanin (KLH) IgG2a by physical exercise on running wheels in rats (P < 0.01). Physically active rats exposed to stress were protected against stress-induced immuno- suppression. SOURCE: Moraska and Fleshner (2001). Using our animal model of stress-induced immunosuppression, we have re- cently reported (Kennedy et al., 2005b) that rats treated with tyrosine (400 mg/kg) 30 minutes prior to stressor exposure are protected from both stress-induced splenic NE depletion (Figure B-24) and KLH Ig suppression (Figure B-25). In this study we also replicated the previous findings that tyrosine presents stress-induced brain NE depletion. Importantly, blood concentrations of NE in the tyrosine treated stressed rats were equal to saline treated stressed rats, yet tyrosine completely prevented the suppression in KLH Ig. These data further support our hypothesis that stress-induced suppression of KLH Ig requires splenic NE depletion and not circulating NE elevation (Kennedy et al., 2005b). CONCLUSION The data presented here support the hypothesis that stress-induced immuno- suppression is due to excessive activation of the sympathetic nervous system (SNS). Future work should strive to further develop interventions, such as exer- cise and tyrosine supplementation, that allow us to reap the positive physiologi- cal effects, while minimizing the maladaptive consequences, of activation of the acute SNS stress response.

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APPENDIX B 357 Mineral Intake Needs and Infectious Diseases Davidson H. Hamer Boston University and Tufts University, Boston, Massachusetts INTRODUCTION Several minerals play central roles in the immune response and thus may be integral to protective responses to pathogens causing human infections. The inflammatory response associated with acute infections, especially diarrheal dis- ease, may result in enhanced excretion of certain essential minerals including copper, iron, and zinc, and thus contribute to deficiencies of these minerals. During the last decade, there has been extensive research in children in resource- poor areas of the world that has demonstrated the beneficial effects of zinc for the prevention and adjunctive treatment of common infectious diseases. In con- trast, despite its important role in immune function, iron supplementation has been occasionally associated with a small increased risk of infection. Unfortu- nately, there is little data available regarding the role of mineral supplements in adults for prevention or treatment of infection with the exception of zinc for the common cold--these studies have shown variable results. Although military per- sonnel are at increased risk for enhanced losses of minerals due to physical stress as well as exposure to infectious pathogens, there is a dearth of data regarding the potential role of mineral supplements in this population. IMPACT OF STRESS ON INFECTION Numerous psychological and physical stressors exert immunomodulatory effects, which may increase the risk of infection acquisition or result in greater severity of infections (Peterson et al., 1991). Several stress-responsive neu- rotransmitters and neuropeptides have been shown to interact with cells of the immune system in vitro, resulting in either enhancement (e.g., -endorphin) (Maestroni and Conti, 1989) or suppression (e.g., glucocorticoids) (Cupps and Fauci, 1982) of the immune response. Animal models of stress and viral infec- tion have mostly shown increased mortality (reviewed in Peterson et al., 1991) or other complications such as paralysis in a poliomyelitis model in mice and monkeys. Similar results have been found in animal models of bacterial infec- tion and stress (i.e., forced exercise), with the majority demonstrating increased mortality associated with stress (Peterson et al., 1991). Relatively little pathogen-specific data exist on the association between stress and infections in humans. Most studies evaluated viral pathogens (e.g., poliomyelitis, hepatitis A, and herpes simplex virus) or upper respiratory infec- tions, which are predominantly also due to viruses (see review by Peterson et al., 1991). With a few exceptions, the results of most studies suggested an associa-

358 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL tion between physical or psychosocial stress and increased frequency, duration, or severity of symptoms. Two studies involved military trainees. A prospective study of marine recruits in North Carolina found, in addition to an association with winter, higher rates of colds in those who were white, well educated, and had slower promotion (Voors et al., 1968). A four-year prospective seroepi- demiological study of infectious mononucleosis in West Point Military Acad- emy cadets demonstrated an association between increased risk of clinical illness and three psychosocial factors: having fathers who were overachievers, being highly motivated, and struggling academically. In summary, while animal models have consistently demonstrated an asso- ciation between stress and increased mortality from infection, the limited human studies suggest an association between increased physical or psychological stress and enhanced morbidity from viral infections. Relatively little is known about the impact of stress on bacterial, fungal, or parasitic infections in humans. Al- though the evidence is limited, it is likely that the physical and severe psycho- logical stressors to which the military are exposed increase the likelihood of acquisition while also potentially worsening the severity of infectious diseases. EFFECTS OF STRESS AND INFECTION ON MINERAL EXCRETION Systemic infections produce an acute phase response that results in alter- ations of mineral status indicators including elevation of serum ferritin (Elin et al., 1977) and erythrocyte protophoryin (Stoltzfus et al., 2000), and reduction of plasma zinc concentrations (Duggan et al., 2005; Strand et al., 2004). In addition to their impact on measures of iron and zinc status, acute infections may also lead to accelerated losses of essential minerals. Fecal Mineral Losses There are a number of different potential causes of mineral loss from the gastrointestinal tract (Box B-3). A consequence of mineral loss, whether due to infectious or malabsorptive processes, is a net negative balance. For example, acute and persistent diarrhea in children has been associated with low serum or plasma zinc concentrations (Castillo-Duran et al., 1988; Chaudhary et al., 1996; Naveh et al., 1982). Although the acute phase response during infectious diar- rhea contributes to depression of plasma zinc, at least one study found low rectal mucosal zinc concentrations in children with chronic diarrhea (Sachdev et al., 1990), thus suggesting that the infectious process can lead to a net negative balance. Two studies have rigorously evaluated the impact of acute diarrhea on trace mineral balance (Table B-12). Castillo-Duran and colleagues (1988) evaluated the magnitude of zinc and copper losses in young children with acute diarrhea requiring hospitalization. Fourteen infants, aged 3 to 14 months, with acute diar-

APPENDIX B 359 BOX B-3 Causes of Fecal Mineral Loss 1. Infectious Etiologies · Tropical sprue · Acute diarrhea · Persistent diarrhea 2. Non-Infectious Causes of Malabsorption · High phytate diets · High concentrations of competing divalent cations · Crohn's disease · Celiac disease · Short-bowel syndrome · Intestinal bypass · Pancreatic insufficiency rhea were compared to a control group of 15 infants of similar age, birth weight, and nutritional status. Mean fecal losses of copper and zinc were higher in the diarrhea group during the initial 48 hours. When repeat metabolic balance stud- ies were performed on days 6 and 7 of admission, fecal zinc losses were similar for the two groups whereas copper balance remained negative only for the diar- rhea group. There was a strong correlation between fecal weight and fecal losses for both minerals, and a negative correlation between fecal and plasma zinc concentrations. A study of 24 male Guatemalan children, aged 7 to 23 months, with acute, dehydrating diarrhea found increased fecal excretion of copper, iron, and zinc during oral rehydration therapy (Ruz and Solomons, 1990). Although this study lacked a control group and did not carry out a follow-up evaluation after resolution of diarrhea, it nevertheless also demonstrated significant linear correlations between mineral excretion and fecal volume. These studies both demonstrate that fecal losses of copper, iron, and zinc during acute diarrhea are likely to induce a negative balance of these minerals. Although limited data exist for adults, one study that evaluated zinc losses in patients with a variety of gastrointestinal disorders (primarily Crohn's disease and ischemic bowel) found substantial small intestinal zinc losses that persisted over time (Table B-12) (Wolman et al., 1979). Similar to the pediatric studies of acute diarrhea, this adult study found a significant correlation between intestinal zinc losses and the weight of contents lost or excreted. Positive zinc balance was nearly reached in patients receiving 6 mg of zinc per day intravenously and was easily achieved if 12 mg/day was administered. In contrast to the patients with diarrhea, positive zinc balance could be attained in those without diarrhea with only 3 mg of zinc per day. Although this study involved hospitalized patients

360 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL TABLE B-12 Gastrointestinal Mineral Losses in Diarrhea Author Study population Fecal copper Fecal iron Fecal zinc Castillo-Duran Infants with 55.7 ± 21.2 µg/ Not evaluated 159.4 ± 59.9 et al., 1988 acute diarrhea kg body µg/kg body weight/day weight/day Healthy infants 28.8 ± 6.7 µg/kg Not evaluated 47.4 ± 6.4 body weight/ µg/kg body day weight/day Ruz and Solo- Young children 1.61 µg/kg/hour 6.33 µg/kg/ 6.08 µg/kg/ mons, 1990 with acute (38.6 µg/kg/day)a hour hour (145.9 diarrhea (151.9 µg/kg/ µg/kg/day)a day)a Wolman et al., Adults with Not evaluated Not evaluated 15.15 µg/g of 1979 gastrointestinal intestinal disease contentb aEstimated 24 hour output based on hourly measurement. bPatients with intact small intestine. with serious intestinal pathology, it nevertheless demonstrates the important con- tribution of diarrhea to fecal zinc losses and its negative impact on zinc balance. Urinary and Sweat Mineral Losses There are a number of different factors that influence urinary mineral excre- tion including age, gender, physical exercise, urine pH, high protein food, high fiber food, coffee, tobacco, and alcohol. A study of healthy volunteers from the Canary Islands found urinary zinc levels that were 19-fold and nearly 8-fold greater than copper and iron, respectively (Rodriguez and Diaz, 1995). Men had significantly greater daily excretion of zinc than women although these differ- ences were no longer significant when urinary excretion was controlled for crea- tinine, an indicator of glomerular filtration rate and overall renal function. In terms of copper and iron, women had greater daily urinary excretion of both these minerals than men. Routine exercise was associated with reduced urinary excretion of copper, iron, and zinc. Patients with increased urinary nitrogen ex- cretion, due to probable hypercatabolism, had higher urinary zinc losses than those with lower urinary nitrogen excretion (Wilman et al., 1979). Moderate amounts of iron and zinc can also be lost in sweat. Studies of young, physically fit adults have demonstrated that sweat losses of both iron and zinc are greatest in the first 30 minutes of exercise and thereafter diminish (DeRuisseau et al., 2002; Waller and Haymes, 1996). Estimated whole body iron

APPENDIX B 361 loss is greater during exercise than at rest and is greater overall in men than women (Waller and Haymes, 1996). During two hours of exercise, sweat losses were 3 percent and 1 percent of the Recommended Daily Allowance (RDA) of iron RDA and 9 percent and 8 percent of the zinc RDA for men and women, respectively (DeRuisseau et al., 2002). Of the different factors influencing the excretion of essential minerals, there are two that have implications for the mineral needs of the military. First, inten- sive sweating associated with physical exercise contributes to enhanced losses of iron and zinc; however, it appears that homeostatic mechanisms serve to reduce sweat losses of iron and zinc during prolonged exertion. Although data are lim- ited regarding the impact of fever on sweat losses, it is likely that prolonged febrile illnesses will contribute to total body losses of iron and zinc. The second and potentially more significant factor is the enhanced fecal excretion of several minerals during acute and persistent diarrhea. Infectious diarrhea, especially if prolonged, is likely to result in a negative balance for copper, iron, magnesium, and zinc. Consequently, supplemental minerals, especially zinc, need to be con- sidered for military personnel with diarrhea. ROLE OF ZINC IN THE PREVENTION OF INFECTION Subclinical Zinc Deficiency and Risk of Infection Cross-sectional studies of children in Papua New Guinea and pregnant women in Malawi have shown associations between suboptimal zinc status, based on hair zinc levels, and falciparum malaria (Gibson and Huddle, 1998; Gibson et al., 1991). A prospective study of children aged 12 to 59 months who had recovered from a recent episode of acute non-dysenteric diarrhea was car- ried out in an urban slum in New Delhi (Bahl et al., 1998). Thirty two percent of children had low plasma zinc concentrations ( 8.4 µmol/L). Children with low baseline plasma zinc levels had a 47 percent higher risk of diarrhea during the three month observation period than those with normal zinc. Although the over- all risk of acute lower respiratory infection (ALRI) was not significantly higher in the low plasma zinc group, boys as opposed to girls with low plasma zinc had a four-fold higher risk of developing an episode of ALRI during the 90 day observation period. The prevalence of ALRI was about three-fold higher in zinc- deficient children, possibly as a result of longer duration episodes of ALRI in this group. In summary, while the cross-sectional studies suggested an association be- tween malaria and zinc deficiency, there were multiple other factors including poor zinc intake and high phytate intake that contributed to the poor zinc status. In contrast, the longitudinal design of the Indian study provides stronger evi- dence of an association between subclinical zinc deficiency and the subsequent risk of infection.

362 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Prevention of Childhood Diarrhea and Pneumonia with Zinc Supplements Similarly to the studies on zinc deficiency above, there are, there are virtually no studies of the effect of zinc for the prevention of infections in adults. Therefore, this section will review the results of seminal studies in children, even though the results cannot be extrapolated to adults with any level of confidence. Several stud- ies in recent years have shown that zinc supplementation in children normalizes immune function and dramatically reduce infectious disease morbidity and mortal- ity (Sazawal et al., 1997, 1998; Sempertegui et al., 1996). A pooled analysis of studies of zinc supplementation for the prevention of diarrhea and pneumonia in children in developing countries found that, in trials that provided 1­2 times the RDA of elemental zinc 5 to 7 times per week, the pooled odds ratios (OR) for diarrheal incidence and prevalence were 0.82 (95 percent confidence interval, CI, ranging from 0.72 to 0.93) and 0.75 (95 percent CI ranging from 0.63 to 0.88), respectively (ZICG, 1999). The OR for pneumonia was 0.59 (95 percent CI rang- ing from 0.41 to 0.83) for zinc-supplemented children. This pooled analysis found a 33 percent reduction in the incidence of persistent diarrhea but this effect only trended towards significance (OR 0.67, 95 percent CI, ranging from 0.42 to 1.06). Similarly, zinc supplementation was associated with a non-significant reduction of dysentery of 13 percent. A more recent study of zinc supplementation given for a period of 14 days each time a child had an episode of diarrhea demonstrated reductions in the duration and incidence of diarrhea and a reduced incidence of ARI (acute respiratory infection) (Baqui et al., 2002). The non-injury death rate was also 51 percent lower in the zinc intervention clusters, suggesting that zinc supplementation reduced mortality. Prevention of Malaria with Zinc In contrast to the extensive evidence base for the efficacy of zinc in the prevention of diarrheal disease and ALRI in children in resource-poor settings, there is relatively limited data regarding zinc and malaria prevention. A trial in The Gambia found that zinc supplementation was associated with a 32 percent reduction in health centre visits for slide-confirmed malaria, though this differ- ence did not attain statistical significance (Bates et al., 1993). While this finding was provocative, the study was not optimally designed for this outcome, and had several important limitations including the lack of a precise definition of malarial illness and a small sample size. Subsequent work from Papua New Guinea pro- vided more convincing evidence of a protective effect of zinc. In a community- based study, a 46-week period of zinc supplementation in preschool children significantly reduced Plasmodium falciparum-attributable health centre atten- dance by 38 percent (p = 0.037) (Shankar et al., 2000). Episodes accompanied by any level of parasitemia were also reduced by 38 percent (p = 0.028) and

APPENDIX B 363 episodes with parasitemia 100,000 per µL were reduced by 69 percent (p = 0.009). A community-based trial of zinc supplementation in Burkina Faso on the incidence of febrile episodes of falciparum malaria, the severity of malaria epi- sodes, or anemia in children aged 6 to 31 months demonstrated that the cross- sectional prevalence of falciparum malaria and of P. falciparum, P. malariae, and P. ovale parasitemia were all significantly lower in children supplemented with zinc (p = 0.001) for all comparisons to placebo (Muller et al., 2001). In addition, the mean density of P. falciparum increased significantly (p = 0.001) during the study in the placebo group relative to the zinc group. Thus, zinc supplementation appeared to provide benefits in terms of several key malario- metric measures. Other beneficial effects of zinc supplementation, such as a significant reduction of the number of days with diarrhea (p = 0.002) and a trend towards reduced mortality (relative risk, RR 0.41, 95 percent CI 0.15­1.19, p = 0.1) were also noted. However, this study failed to find any benefit of zinc on the incidence of clinical malaria episodes (defined as temperature 37.5°C and parasites/µL). There are several potential explanations for the lack of a protec- tive effect of zinc for malaria in this study. First, the sample size was too small to measure this effect, since the proportion of febrile malaria episodes of all chil- dren with positive blood smears was quite small. Second, the prevalence of clinical zinc deficiency in the population under study was low. Using a cut-off point for zinc deficiency of 60 µg/dL (ZICG, 1999), only a small proportion of these children were zinc deficient at baseline, as the mean zinc concentration was 76.5 mg/dL. Theoretically, zinc might have a greater effect on clinical ma- laria if used in a population where zinc deficiency was widespread. Thus, in summary there is ample evidence that zinc supplementation serves to protect children against diarrheal disease, ALRI, and possibly malaria. In addition, there is growing evidence that zinc supplementation may reduce mor- tality in young children (Baqui et al., 2002; Muller et al., 2001; Sazawal et al., 2001). At present there is no data available regarding the potential benefits of zinc supplementation for prevention of malaria in healthy adults. TREATMENT OF INFECTIONS WITH ZINC Treatment of Diarrheal Disease with Zinc There are several potential mechanisms by which zinc might have a benefi- cial effect on the duration of diarrhea. These include improved absorption capac- ity (Golden and Golden, 1985), increased brush border disaccharidase activity (Gebhard et al., 1983), faster regeneration of intestinal epithelium (Bettger and O'Dell, 1981), a reduction of gut permeability, and an enhanced immune re- sponse (Shankar and Prasad, 1998), which may result in more rapid clearance of

364 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL enteropathogens. Based on these potential mechanisms, a large number of stud- ies have evaluated the role of zinc in the treatment of acute and persistent diar- rhea in children. Zinc supplementation, as an adjunct to oral rehydration therapy, reduced the duration and severity of acute and persistent diarrhea in several randomized controlled trials (Bhutta et al., 1999; Roy et al., 1997; Sachdev et al., 1988, 1990; Sazawal et al., 1995, 1997). A pooled analysis of randomized, controlled trials of zinc for acute diarrhea found that zinc reduced the mean duration of diarrhea by 16 percent (95 percent CI: 7 percent, 26 percent) (ZICG, 2000). Zinc- supplemented children also had a 15 percent lower probability of continuing diarrhea on a given day in the acute diarrhea studies and a 24 percent lower probability of continuing diarrhea in the persistent diarrhea trials. There was also a 42 percent lower rate of death or treatment failure in the persistent diarrhea studies. An analysis of the cost-effectiveness of zinc as adjunct therapy to stan- dard management of acute childhood diarrhea, including dysentery, found that the use of zinc significantly improved the cost-effectiveness of standard treat- ment for both dysenteric and non-dysenteric diarrhea (Sommerfelt et al., 2004). An alternative approach to the management of acute diarrhea that has been recently evaluated is the addition of zinc to oral rehydration solution (ORS). In a zinc-deficient rat model of diarrhea, treatment with ORS plus zinc resulted in a recovery of normal plasma zinc levels and improved histology of the intesti- nal villi relative to rats treated with ORS minus zinc, suggesting that the supple- mental zinc helped to improve intestinal epithelial integrity (Altaf et al., 2002). Two studies in children have evaluated ORS-zinc in children. One of these, a small study of young children in Cuba, found no advantage of ORS-zinc over ORS alone (Bahl et al., 2001). In contrast, a study of young children with acute diarrhea in North India found that ORS-zinc was more efficacious than ORS alone for reducing the duration of an episode of diarrhea (Bahl et al., 2001). However, ORS-zinc was less efficacious than zinc supplements given sepa- rately from ORS. Although the addition of zinc to ORS was not associated with tolerability problems in these two studies, this approach does not appear to be as efficacious as oral zinc supplementation, perhaps because the total amount of zinc administered on a daily basis is greater when oral supplements are administered. To date there have been no evaluations of zinc for the man- agement of diarrhea in adults. Treatment of Pneumonia, Measles, and Malaria with Zinc Two studies evaluated the efficacy of zinc as an adjunct to antimicrobial therapy for children with severe ALRI (Brooks et al., 2004; Mahalanabis et al., 2004). In the first study, children aged 2­23 months with severe pneumonia received 20 mg of zinc per day plus standard antibiotics until hospital discharge (Brooks et al., 2004). Children who received zinc had a reduced duration of

APPENDIX B 365 severe pneumonia including shorter duration of tachypnea, hypoxia, and chest in-drawing. The overall duration of pneumonia was 4 days in children treated with zinc versus 5 days in those who received placebo. The second study in- volved the administration of 10 mg of zinc twice daily for 5 days to children aged 2­24 months with severe ALRI (Mahalanabis et al., 2004). Zinc treatment significantly reduced the duration of fever and very ill clinical status as judged by the study pediatrician in boys but not girls. Since this finding arose from post hoc subgroup analysis, it needs to be validated in a gender-stratified, randomized controlled trial. These two studies provide early suggestions of a potential thera- peutic effect of zinc for severe pneumonia in very young children. Whether zinc will prove to be a useful therapeutic adjunct for the treatment of pneumonia in older children or for selected respiratory pathogens remain open questions. The utility of zinc supplementation for the treatment of measles has been evaluated in only one study (Mahalabis et al., 2002). Children, aged 9 months to 15 years, hospitalized in India for measles were randomized to zinc or placebo in addition to routine supportive care. Treatment with zinc had no impact on the time to recovery or the proportion of children who were judged to be cured by day six. In order to evaluate the potential role of zinc as an adjunct in the treatment of acute, uncomplicated falciparum malaria, a randomized, placebo-controlled, multi-centre trial was undertaken (ZAP Study Group, 2002). Children (n = 1,087) between the ages of 6 months and 5 years with fever and 2,000/µL asexual forms of P. falciparum in a thick blood smear were enrolled at sites in Ecuador, Ghana, Tanzania, Uganda, and Zambia. Children were randomized to receive zinc (20 mg/day for infants, 40 mg/day for older children) or placebo for four days as well as chloroquine, the standard first line treatment for malaria in all sites at the time of study initiation. There was no effect of zinc on the median time to reduction of fever (zinc = 24.2 hr versus placebo = 24.0 hr, p = 0.37), reduction of parasitemia in the first 72 hr (zinc group = 73.4 percent; placebo group = 77.6 percent, p = 0.11), or hemoglobin concentration during the three day period of hospitalization or four week follow-up period. This carefully de- signed study thus failed to demonstrate any benefits of zinc as an adjunct to the treatment of malaria. As with prevention, there is no data available on the ben- efits of zinc supplementation for treatment of malaria in adults. Treatment of the Common Cold with Zinc Viral upper respiratory tract infections are a major cause of physician visits and time lost from work or education in the United States. Because of its im- munomodulatory activities (Shankar and Prasad, 1998) and in vitro inhibitory activity against rhinoviruses (Korant et al., 1974), zinc lozenges have been ex- tensively evaluated as a therapeutic strategy for the common cold. Randomized controlled trials of zinc salt lozenges have yielded mixed results. A meta-

366 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL analysis by Jackson and colleagues (1997), which included 6 trials, found a summary odds ratio of 0.5 (95 percent CI, 0.19­1.29) for the presence of any cold symptoms at 7 days. A subsequent study in adults with common cold symp- toms for less than 24 hours found a significant reduction in the mean overall duration of cough, nasal discharge, and cold symptoms (4.5 versus 8.1 days) (Prasad et al., 2000). Although this carefully designed study showed a reduction of more than 3 days of cold symptoms in adults taking zinc acetate lozenges, a repeat meta-analysis failed to find evidence of the effectiveness of zinc for re- ducing the duration of the common cold (Jackson et al., 2000). Even though the revised meta-analysis did not include the study by Prasad and colleagues (2000), there nevertheless is little evidence to support the routine use of zinc for the treatment of the common cold. A recent IOM report also concluded that there is conflicting evidence arising from studies in the elderly population and that data on the effects of non-pharmacological levels of zinc on immunity in young healthy adults are not available (IOM 2005). IRON AND INFECTION There are a number of harmful effects of iron deficiency on cellular immu- nity that are reversible with iron supplementation (reviewed in Oppenheimer, 2001). These include reduced neutrophil function, decreased numbers of T- lymphocytes associated with thymic atrophy, defective T-lymphocyte-induced proliferative responses, impaired natural killer cell activity, decreased production of macrophage migration inhibition factor, and impaired delayed-type hyper- sensitivity. On the other hand, there is little indication of major deficiencies of humoral immunity in iron-deficient humans. Despite the solid evidence of abnor- malities of cellular immunity associate with iron deficiency, there has been a long standing controversy regarding the relationship between iron status and susceptibility to infection. Iron is required for both the human host and pathogens for survival and replication. In the setting of acute infection, there is a rise in iron-binding pro- teins such as serum ferritin, which has been proposed as a defensive maneuver by the host that limits the amount of iron available to pathogens (Weinberg, 1978). Findings from studies in the 1970s suggested that iron treatment resulted in aggravation of pre-existing or latent bacterial or parasitic infections. Neonates with iron deficiency in New Zealand who were treated with parenteral iron dex- tran at birth had a 6-fold increase in gram-negative sepsis (Barry and Reeve, 1977). Similarly, adult Somali nomads with iron deficiency treated with oral iron supplementation had significantly increased clinical malaria attacks relative to a placebo control group (Murray et al., 1978). Subsequent studies also suggested that iron therapy increased a child's risk of developing malaria or aggravated the clinical severity of an episode (Oppenheimer et al., 1986; Smith et al., 1989). In

APPENDIX B 367 contrast, other investigators did not find a negative effect of iron supplementa- tion on malaria (Bates et al., 1987; Harvey et al., 1989). During the last two decades, there have been a large number of randomized controlled trials of the effect of iron supplementation on the risk of infections in children. A systematic review of many of these studies found that the pooled estimate of the incidence rate ratio of infection episodes for iron versus placebo was 1.02 (95 percent CI 0.96­1.08) (Gera and Sachdev, 2002). Although there was a slight increased incidence of several infection types in iron supplemented children, the only forms of infection that was significantly increased was diar- rheal disease (11 percent increased risk) and malaria parasitemia (43 percent increase). A meta-analysis that focused only on malaria found a 17 percent in- creased risk of P. falciparum parasitemia in randomized, controlled trials of iron supplementation (Shankar et al., 1998). There was also a non-significant 9 per- cent increase in the risk of clinical malaria. However, iron supplementation was associated with substantial benefits in terms of hemoglobin improvement and a reduced risk of severe anemia. In summary there is limited evidence of a harmful effect of iron supplemen- tation on risk of infection. Although there may be a small increased risk of malarial parasitemia, it appears that the risk of clinical malaria attacks is not significantly increased and that the benefits of iron in terms of improvement of hematological status far outweigh the risks of supplementation. IMPLICATIONS FOR MILITARY PERSONNEL MINERAL REQUIREMENTS Physical and psychological stresses contribute to an enhanced risk of infec- tion and accelerated mineral losses. Infections, especially diarrhea, may also con- tribute to increased losses of essential minerals. There has been a vast amount of research done in recent years on the importance of maintaining adequate zinc status for the prevention of infection. Although much of this work has been performed in young children in resource-poor countries, these results neverthe- less have implications for military personnel. In circumstances where there is enhanced excretion of zinc, especially in the setting of acute or prolonged diar- rhea, zinc supplementation plays an adjunctive role in the management of diar- rhea. Based on the limited evidence available, zinc is most efficacious when provided as oral supplements rather than being mixed with ORS. Maintenance of adequate zinc status should be a priority as this should serve to reduce the risk of infection in military personnel. In contrast to the ample evidence of the benefits of zinc supplementation or maintenance of zinc status for the treatment and prevention of infections at least in children, the role of iron in preventing or resisting infections is less clear. In the setting of anemia due to iron deficiency, there is a need for iron replacement.

368 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL However, in most other circumstances, there is insufficient evidence of a mean- ingful benefit of iron supplementation to decrease the incidence or severity of infections. In addition, there may be a slight risk of harm from this intervention since iron is also an essential element for many pathogens. Taking into consider- ation the potential risks and benefits that iron supplementation might impart to the immune system, it seems prudent to continue research efforts in this area before iron supplementation to improve immune responses is recommended. REFERENCES Altaf W, Perveen S, Rehman KU. 2002. Zinc supplementation in oral rehydration solutions: Experi- mental assessment and mechanisms of action. J Am Coll Nutr 21:26­32. Anonymous. 2001. Effect of zinc supplementation on clinical course of acute diarrhea. Report of a meeting, New Delhi, 7­8 May 2001. J Health Popul Nutr 19:338­346. Bahl R, Bhandari N, Hambidge KM, Bhan MK. 1998. Plasma zinc as a predictor of diarrheal and respiratory morbidity in children in an urban slum setting. Am J Clin Nutr 68 (Suppl):414S­ 417S. Baqui AH, Black RE, El Arifeen S. 2002. Effect of zinc supplementation started during diarrhoea on morbidity and mortality in Bangladeshi children: Community randomized trial. Br Med J 325:1­7. Barry DMJ, Reeve AW. 1977. Increased incidence of gram-negative neonatal sepsis with intramus- cular iron administration. Pediatrics 60:908­912. Bates CJ, Powers HJ, Lamb WH, Gelman W, Webb E. 1987. Effect of supplementary vitamins and iron on malaria indices in rural Gambian children. Trans R Soc Trop Med Hyg 81:286­291. Bates CJ, Evans PH, Dardenne M. 1993. A trial of zinc supplementation in young rural Gambian children. Br J Nutr 69:243­255. Bettger WJ, O'Dell BL. 1981. A critical physiological role of zinc in the structure and function of biomembranes. Life Sc 28:1425­1438. Bhutta ZA, Nizami SQ, Isani Z. 1999. Zinc supplementation in malnourished children with persistent diarrhea in Pakistan. Pediatrics 103:1­9. Brooks WA, Yunus M, Santosham M. 2004. Zinc for severe pneumonia in very young children: Double-blind placebo-controlled trial. Lancet 363:1683­1688. Castillo-Duran C, Vial P, Uauy R. 1988. Trace mineral balance during acute diarrhea in infants. J Pediatr 113:452­457. Chaudhary S, Verma M, Dhawan V. 1996. Plasma vitamin A, zinc and selenium concentrations in children with acute and persistent diarrhoea. J Diarrhoeal Dis Res 14:190­193. Cupps TR, Fauci AS. 1982. Corticosteroid-mediated immunoregulation in man. Immunol Rev 65: 133­155. DeRuisseau KC, Cheuvront SN, Haymes EM, Sharp RG. 2002. Sweat iron and zinc losses during prolonged exercise. International J Sport Nutr Exerc Met 12:428­437. Duggan C, MacLeod W, Krebs NF. 2005. Plasma zinc concentrations are depressed during the acute phase response in children with falciparum malaria. J Nutr 135:802­807. Elin RJ, Wolf SM, Finch C. 1977. Effect of induced fever on serum iron and ferritin concentrations in man. Blood 49:147­153. Gebhard RL, Karouani R, Prigge WF, McClain CJ. 1983. Effect of severe zinc deficiency on activity of intestinal disaccharidases and 3-hydroxy-3-methyl-glutaryl coenzyme A reductase in the rat. J Nutr 113:855­859. Gera T, Sachdev HP. 2002. Effect of iron supplementation on incidence of infectious illness in children: systematic review. Br Med J 325:1142.

APPENDIX B 369 Gibson RS, Huddle JM. 1998. Suboptimal zinc status in pregnant Malawian women: Its association with low intakes of poorly available zinc, frequent reproductive cycling, and malaria. Am J Clin Nutr 67:702­709. Gibson RS, Heywood A, Yaman C.1991. Growth in children from the Wosera subdistrict, Papua New Guinea, in relation to energy and protein intakes and zinc status. Am J Clin Nutr 53: 782­789. Golden BE, Golden MHN. 1985. Zinc, sodium and potassium losses in the diarrhoeas of malnutrition and zinc deficiency. In: Mills CF, Bremner I, Chesters JK, eds. Trace Elements in Man and Animals TEMA 5. Aberdeen, UK: Rowett Research Institute. Harvey PW, Heywood PF, Nesheim MC.1989. The effect of iron therapy on malarial infection in Papua New Guinean schoolchildren. Am J Trop Med Hyg 40:12­18. Jackson JL, Peterson C, Lesho E. 1997. A meta-analysis of zinc salts lozenges and the common cold. Arch Intern Med 157:2373­2376. Jackson JL, Lesho E, Peterson C. 2000. Zinc and the common cold: A meta-analysis revisited. J Nutr 130: S1512­S1515. Korant BD, Kauer JE, Butterworth BE. 1974. Zinc ions inhibit replication of rhinoviruses. Nature 248:588­590. Maestroni GJM, Conti A. 1989. Beta-endorphin and dynorphin mimic the circadian immunoen- hancing and anti-stress effects of melatonin. J Immunopharmacol 11:333­340. Mahalanabis D, Chowdhury A, Jana S, Bhattacharya MK, Chakrabarti MK. 2002. Zinc supplementa- tion as adjunct therapy in children with measles accompanied by pneumonia: A double-blind, randomized controlled trial. Am J Clin Nutr 76:604­607. Mahalanabis D, Lahiri M, Dilip P. 2004. Randomized, double-blind, placebo-controlled clinical trial of the efficacy of treatment with zinc or vitamin A in infants and young children with severe acute lower respiratory infection. Am J Clin Nutr 79:430­436. Muller O, Becher H, van Zweeden AB. 2001. Effect of zinc supplementation on malaria and other causes of morbidity in West African children: Randomised double blind placebo controlled trial. Brit Med J 322:1­5. Murray MJ, Murray AB, Murray MB, Murray CJ. 1978. The adverse effect of iron repletion on the course of certain infections. Brit Med J 2:1113­1115. Naveh Y, Lightman A, Zinder O. 1982. Effect of diarrhea on serum zinc concentrations in infants and children. J Pediatr 101:730­732. Oppenheimer SJ. 2001. Iron and its relation to immunity and infectious disease. J Nutr 131:16S­ 633S. Oppenheimer SJ, Gibson FD, Macfarlane SB. 1986. Iron supplementation increases prevalence and effects of malaria: report on clinical studies in Papua New Guinea. Trans R Soc Trop Med Hyg 80:603­612. Peterson PK, Chao CC, Molitor T. 1991. Stress and pathogenesis of infectious disease. Rev Infect Dis 13:710­720. Prasad AS, Fitzgerald JT, Bao B, Beck FWJ, Chandrasekar PH. 2000. Duration of symptoms and plasma cytokine levels in patients with the common cold treated with zinc acetate. Ann Intern Med 133:245­252. Rodriguez E, Diaz C. 1995. Iron, copper and zinc levels in urine: relationship to various individual factors. J Trace Elem Med Biol 9:200­209. Roy S.K., Tomkins A. M., & Akramuzzaman SM (1997) Randomised controlled trial of zinc supple- mentation in malnourished Bangladeshi children with acute diarrhoea. Arch Dis Child 77: 196­200. Ruz M, Solomons NW. 1990. Mineral excretion during acute, dehydrating diarrhea treated with oral rehydration therapy. Pediatr Res 27:170­175.

370 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Sachdev HPS, Mittal NK, Mittal SK, Yadav HS. 1988. A controlled trial on utility of oral zinc supplementation in acute dehydrating diarrhea in infants. J Pediatr Gastroenterol Nutr 7:877­ 881. Sachdev HPS, Mittal NK, Yadav HS. 1990. Oral zinc supplementation in persistent diarrhoea in infants. Ann Trop Paediatr 10:63­69. Sazawal S, Black RE, Bhan MK. 1995. Zinc supplementation in young children with acute diarrhea in India. N Engl J Med 333:839­844. Sazawal S, Black RE, Bhan MK.1997. Efficacy of zinc supplementation in reducing the incidence and prevalence of acute diarrhea--a community-based, double-blind, controlled trial. Am J Clin Nutr 66:413­418. Sazawal S, Black RE, Jalla S. 1998. Zinc supplementation reduces the incidence of acute lower respiratory infections in infants and preschool children: A double-blind, controlled trial. Pedi- atrics 102:1­5. Sazawal S, Black RE, Menon VP. 2001. Zinc supplementation in infants born small for gestational age reduces mortality: A prospective, randomized, controlled trial. Pediatrics 108:1280­1286. Sempertegui F, Estrella B, Correa E. 1996. Effects of short-term zinc supplementation on cellular immunity, respiratory symptoms, and growth of malnourished Ecuadorian children. Eur J Clin Nutr 50:42­46. Shankar AH, Prasad AS. 1998. Zinc and immune function: The biological basis of altered resistance to infection. Am J Clin Nutr 68:447S­463S. Shankar AH, Fishman S, Goodman S, Stoltzfus RJ. 1998. Iron supplements and Plasmodium falci- parum morbidity: A meta-analysis of controlled clinical trials of iron supplementation in ma- larious areas. Background paper for the ILSI-INACG. Shankar AH, Genton B, Baisor M. 2000. The influence of zinc supplementation on morbidity due to Plasmodium falciparum: A randomized trial in preschool children in Papua New Guinea. Am J Trop Med Hyg 62:663­669. Smith AW, Hendrickse RG, Harrison C, Hayes RJ, Greenwood BM. 1989. The effects on malaria of treatment of iron-deficiency anaemia with oral iron in Gambian children. Ann Trop Paediatr 9:17­23. Sommerfelt H, Robberstad B, Stand T, Black RE. 2004. Cost-effectiveness of zinc as adjunct therapy for acute childhood diarrhoea in developing countries. Bull World Health Organ 82:523­531. Stoltzfus RJ, Chwaya HM, Montresor A. 2000. Malaria, hookworms and recent fever are related to anemia and iron status indicators in 0- to 5-y old Zanzibari children and these relationships change with age. J Nutr 130:1724­1733. Strand TA, Adhikari RK, Chandyo RK, Sharma PR, Sommerfelt H. 2004. Predictors of plasma zinc concentration in children with acute diarrhea. Am J Clin Nutr 79:451­456. Voors AW, Stewart GT, Gutekunst RR, Moldow CF, Jenkins CD. 1968. Respiratory infection in Marine recruits. Influence of personal characteristics. Am Rev Respir Dis 98:801­809. Waller MF, Haymes EM. 1996. The effects of heat and exercise on sweat iron loss. Med Sci Sports Exerc 28:197­203. Weinberg ED. 1978. Iron and infection. Microbiol Rev 42:45­66. Wolman SL, Anderson GH, Marliss EB, Jeejeebhoy KN. 1979. Zinc in total parenteral nutrition: Requirements and metabolic effects. Gastroenterology 76:458­467. Zinc Against Plasmodium Study Group. 2002. Effect of zinc on the treatment of Plasmodium falciparum malaria in children: A randomized controlled trial. Am J Clin Nutr 76:805­812. Zinc Investigators' Collaborative Group. 1999. Prevention of diarrhea and pneumonia by zinc supple- mentation in children in developing countries: Pooled analysis of randomized controlled trials. J Pediatr 135:689­697. Zinc Investigators' Collaborative Group. 2000. Therapeutic effects of oral zinc in acute and persis- tent diarrhea in children in developing countries: Pooled analysis of randomized controlled trials. Am J Clin Nutr 72:1516­1522.

APPENDIX B 371 Copper, Zinc, and Immunity Susan S. Percival University of Florida, Gainesville INTRODUCTION When the body is invaded by a foreign agent, the immune response follows a sequential, methodological and functional process: recognition of that foreign agent, response of the immune cells and the eradication of the foreign agent (Box B-4). Each immune cell has a unique way of recognizing foreign agents, and must distinguish between self and non-self. Not much is known about nutri- tional influences on the recognition function. Immune cells must respond once the foreign invader is recognized. Such responses include traveling to the site of infection, proliferating and differentiating. Cytokine synthesis and secretion are also part of the cellular response and drive much of the subsequent reactions. More research has been performed regarding nutritional influences on these cel- lular processes compared to the amount of research on the recognition process. BOX B-4 Sequential, Methodological, and Functional Scheme of Immunity Recognition of Foreign Agents T-Cell Receptor B-cell membrane bound receptor Toll-like receptors Major Histocompatibility Complex, class I and II Co-stimulatory molecules Co-inhibitory molecules Cellular Responses Chemotaxis Proliferation Differentiation Cytokine signaling Memory Apoptosis Eradication Functions Phagocytosis Oxidative burst Antigen processing and presentation Antibody production Cell killing (cytolysis) MCH = Major Histocompatibility Complex

372 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL One of the cellular processes that has relatively more information relating nu- trient status to immunity is proliferation of peripheral blood mononuclear cells (PBMC). Cells also develop memory and undergo apoptosis to down regulate the response. Finally, the third and last step is eradication of the foreign agent. Engulfing and digesting microorganisms, processing and presentation of anti- gen, antibody synthesis and cell perforation as some of the eradication mecha- nisms. Research has also been done in regards to nutrient deficiencies and eradi- cation functions. All nutrients are critical for an appropriate immune response. At rest, pre- sumably the immune system can optimally function at Recommended Dietary Allowances (RDA) levels of nutrient intake. It is not known, however, if or how much nutrient requirements change to evoke an optimum response. Immunity should be regarded as a continuum. We are always infected, but if immuno-surveillance is optimum, then we do not show symptoms. Only when a microorganism has found a way to overcome the system, do we get symptoms such as fevers or rashes. If immunity has been compromised due to inadequate nutrients or other factors, then it is easily overwhelmed and we constantly have symptoms associated with illness. However, on the other side of the continuum, if the immune system remains continually stimulated, chronic disease such as heart disease, autoimmunity, rheumatoid arthritis and some cancers may result. While much of the research and discussion centers on peripheral blood cells, it should be remembered that the gut is an important immune organ, constantly sampling lumen contents, processing and presenting antigen, and having effector cell activity, cytotoxic activity and barrier function. More research is needed on the role of nutrients in the gut's immune function, particularly the barrier function. IMMUNE SYSTEM: INDICATORS OF FUNCTIONALITY Historically, PBMC proliferation has been widely used as an indicator of immune status. But how much of a decline in PBMC proliferation is required to negatively impact the host? Murasko and colleagues studied PBMC proliferation as a predictor of mortality in the elderly. In a group of people who had a low response to mitogen, a greater percent of that group died in subsequent years than those that had a high or mid response (Table B-13; Murasko and Goonewardene, 1990; Murasko et al., 1987). The low responders were very low having lost 75­93 percent of the PBMC proliferative response compared to young controls. In another study, individuals who had post-surgical complications had a significantly lower PBMC proliferation value at baseline pre-surgery than those who did not have post-surgical complications (Takagi et al., 2001). Those that had post-surgical complications had 40 percent lower response to phytohaemag- glutinin (PHA) and 35 percent lower response to ConA compare to the group without complications. The level of the reductions in PBMC proliferation sug- gests that overall host response is negatively impacted when PBMC proliferation

APPENDIX B 373 TABLE B-13 Correlations Between Mortality and PBMC Proliferation Mortality (4.5 year PHA Response ConA Response PWM Response follow-up) Age % dead units % reduction units % reduction units % reduction Young NA 184 162 56 Elderly Low 33% 23 87% 12 93% 14 75% Mid 16% 70 62% 43 73% 43 23% High 11% 135 27% 103 37% 60 -7% NOTE: PHA = phytohaemagglutinin; ConA = concanavalin A; PWM = pokeweed mitogen SOURCE: Adapted from Murasko et al. (1990). is moderately reduced. In a study on energy intake deficit and high intensity exercise, PBMC proliferation was reduced 35 to 75 percent of the groups' base- line values (Kramer et al., 1997). This reduction was associated abscesses and other visual signs of impaired immunity (personal communication, K. Friedl and A. Young, U.S. Army Research Institute for Environmental Medicine, June 13, 2005). COPPER AND IMMUNITY: RESEARCH IN HUMANS It is known that copper has an important role in immunity from multiple models, although the exact mechanism of copper's function is not known. Evi- dence from individuals with the genetic disorder, Menkes, or those who have been on extended total parenteral nutrition (TPN) solutions without copper dem- onstrate functional immune changes. These models, however, may have other nutritional, genetic, or immunological issues that were not measured in the re- search. Rodent models, not only weanling animals, but perinatal and marginal models have been used to study copper deficiency and immunity. Rodent models on marginal copper diets (Hopkins and Failla, 1995) and swine models (Bala et al., 1992) show impairment in immunity without changes in copper dependent enzymes or serum copper levels. This confirms that biomarkers of copper status are urgently needed (see Keen and Uriu-Adams 2005 in this Appendix). A metabolic study of copper depletion was performed in healthy humans. Eleven humans were placed on a diet containing 0.66 mg of copper per day for 24 days and then further reduced to 0.38 mg/day until day 66 and then repleted with 2.5 mg per day until day 90 (Kelley et al., 1995). Peripheral blood mono- nuclear cell (PBMC) proliferation was impaired at the end of 0.38 mg/day but not at the end of 0.66 mg/day. The impairment was about two-third of the baseline values. These values did not return to baseline levels at the end of the repletion period.

374 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL The biological significance of a 33 percent reduction is not known. In the study where post-surgical complications were noted, reductions of 35­45 per- cent of control values were associated with host complications. It is possible that the reduction caused by copper deficiency impacts overall host resistance, but this is not known. A reduction in serum interleukin (IL)-2 receptor concentrations by the end of the 0.38 mg/day period were observed (Kelley et al., 1995). Levels did not return to baseline after repletion. White blood cell numbers were not affected. Neutrophil copper concentrations were reduced by the end of 0.66 mg/day, fur- ther reduced at the end of 0.38 mg/day and did not return to baseline values after repletion (Table B-14) (Turnlund et al., 1997). Neutrophil copper concentration remained at about 66 percent of the baseline values at the end of the repletion phase. Phagocytic activity of the neutrophil was not affected by these copper changes, and no other neutrophil functional indices were measured. High copper intake (7 mg) for 5 months resulted in some statistical changes in certain immune functional assays (Turnlund, et al., 2004) but the biological significance of these changes is not known (Table B-15). Antibody titer response to three flu viruses appeared lower, although only the response to one strain was TABLE B-14 Neutrophil Copper Concentrations Day Concentration, means ± SD 0 125 ± 61a Mid MP2 91 ± 42b End MP2 54 ± 27c End MP3 79 ± 35b,c NOTE: MP = metabolic period. a, b, cValues having different letters are significantly different at p < 0.05. SOURCE: Turnlund et al. (1997). TABLE B-15 Immune Changes and High Copper Intake Indicator Before After Neutrophils (× 109/L) 3.3 ± 0.2 3.2 ± 0.2* Lymphocytes (× 109/L) 2.14 ± 0.06 2.48 ± 0.06* IL-2R (pg/mL) 33.1 ± 3.5 27.3 ± 2.2* IL-6 (pg/mL) 1.4 ± 0.5 2.3 ± 0.6 NOTE: pg = picogram. * Significantly different from levels before copper supplementation, P < 0.05. SOURCE: Turnlund et al. (2004).

APPENDIX B 375 TABLE B-16 Changes in Vaccination Titers After High Copper Intake Vax titers (fold increase) Controls Experimental Beijing 47 ± 9 14 ± 4* Sydney 92 ± 55 14 ± 4 Harben 32 ± 14 12 ± 6 *Significantly different from control subjects, P < 0.05. SOURCE: Turnlund et al. (2004). statistically lower (Table B-16). However, individuals averaged a 14 fold titer increase and immunologists consider a 4-fold increase to be responsive to the vaccination. ZINC AND IMMUNITY: RESEARCH IN HUMANS Like copper, many models of zinc and immunity have been reported. A genetic disorder of zinc metabolism, acrodermatitis enteropathica, showed changes in immunity. Likewise, the elderly, hemodialysis patients, and people in developing countries all show immune changes that respond to zinc supplemen- tation. It is difficult to interpret these studies since multiple nutrients may be impacted and differences in genetics and diseases may confound the results. The models that have shown an impact on immunity in a human population by zinc deficiency are not germane to the military. Having said that, the indices of im- munity that are reduced in zinc deficiency are drawn from those publications. As shown in Box B-5 almost all measured indices of immunity are impacted nega- tively by zinc deficiency. In a controlled metabolic study of zinc depletion, eight men consumed 13.7 mg/day for 5 wk, were reduced to 4.6 mg/day for 10 weeks and then repleted for 5 weeks with 13.7 mg/day (Pinna et al., 2002). No changes were observed for plasma zinc, or two zinc-requiring enzymes, alkaline phosphatase or 5'nucleoti- dase throughout the course of the study. Leukocytes remained constant in num- ber and in their percent distribution. Neutrophil superoxide generation did not change. Proliferation of PBMC was significantly reduced by about 25­30 percent at the end of the depletion and did not return to baseline values after 5 wk of repletion. It is not clear whether this reduction will impact the host's overall immunity. Secretion of INF- and tumor necrosis factor (TNF)- were not al- tered. IL-2 receptor secretion was impaired at suboptimal levels of PHA stimula- tion, but not at maximal levels of stimulation.

376 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL BOX B-5 Reductions in Immune Parameters in Zinc Deficient Models Recognition of Foreign Agents Major histocompatibility complex Receptors with zinc-finger domains Cellular Response NK Chemotaxis Neutrophils Cytokine secretion Macrophages Proliferation T cell Cytokine secretion Proliferation B cell Differentiation Memory Gut Barrier function Prolonged parasite survival (mice) Eradication Functions Phagocytosis Cytotoxic function Oxidative burst Antibody production Although PBMC proliferation did not return to normal by the end of reple- tion, the authors believe that zinc deficiency may be prolonged due to the long lifespan of lymphocytes. They also suggested that it did not return to baseline values due to stresses associated with living in a metabolic ward. If additional stress in already compromised zinc status results in immune status changes, this would be relevant to the military. More research is needed. However, the sub- jects did not exhibit increased rates of infection and illness and all other immune parameters remained at baseline levels suggests that immunity is largely upheld during a marginal zinc intake for relatively short period of time. During stress, zinc appears to be redistributed rather than lost from the body. Isotope tracer studies suggest that when plasma zinc goes down, it is taken up by the liver, thymus and bone marrow, important immune organs (Huber and Cousins, 1988). However, the plasma is not the only organ with reduced zinc levels, but concentrations of zinc in skin, bone and intestine are also reduced. Due to the gut's important role in immunity, more research is needed to under- stand zinc's role in gut immunity. There is no evidence for increased susceptibility to specific diseases with low zinc status although many organisms have been studied and evidence shows an increase susceptibility to a broad range of infectious diseases with the follow- ing etiological agents:

APPENDIX B 377 · Virus (Herpes simplex) · Bacteria (Listeria, Salmonella, Mycobacterium tuberculosis) · Protozaoan parasites (trypanasoma, Toxoplasma gondii, Plasmodium) · Candida albicans · Helminthes (tricinella, schistosoma) Similarly, there is no evidence that suggests zinc deficiency promotes one type of infection over another. There is also no evidence that zinc is required by microorganisms and the acute phase response is to prevent organisms from using zinc. Levels of zinc in the plasma do not get low enough to prevent microorganisms from using it. Also, those levels return to normal rapidly--by about 24 hours. CONCLUSIONS Human models of copper or zinc depletion indicate that PBMC proliferation may be impacted by low levels of consumption. Other indices of immune func- tion do not seem to be affected under these metabolic conditions and the length of time studied. The PBMC proliferation rates in both cases did not return to pre- study levels. The extrapolation of these observations to infectious disease resis- tance is not straightforward, and depends upon the nature of the microbe, its own nutrient needs, and the relative importance of innate, as opposed to immuno- logic, defense mechanisms. Research areas that need further investigation are (1) the impact of nutrients on the recognition function of immunity; (2) gut function, particularly barrier function and survival of foreign invaders; (3) immunity as impacted by stress and by heavy exercise, coupled with the potential for marginal nutrient deficien- cies; (4) overall host resistance to infection needs to be correlated with PBMC proliferation; (5) the need for a great level of nutrients during a major immune response. REFERENCES Bala S, Lunney JK, Failla ML. 1992. Effects of copper deficiency on T-cell mitogenic responsive- ness and phenotypic profile of blood mononuclear cells from swine. Am J Vet Res 53(7):1231­ 1235. Hopkins RG, Failla ML. 1995. Chronic intake of a marginally low copper diet impairs in vitro activities of lymphocytes and neutrophils from male rats despite minimal impact on conven- tional indicators of copper status. J Nutr 125(10):2658­2668. Huber KL, Cousins RJ. 1988. Maternal zinc deprivation and interleukin-1 influence metallothionein gene expression and zinc metabolism of rats. J Nutr 118(12):1570­1576. Kelley DS, Daudu PA, Taylor PC, Mackey BE, Turnlund JR. 1995. Effects of low-copper diets on human immune response. Am J Clin Nutr 62(2):412­416.

378 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Kramer TR, Moore RJ, Shippee RL, Friedl KE, Martinez-Lopez L, Chan MM, Askew EW. 1997. Effects of food restriction in military training on T-lymphocyte responses. Int J Sports Med 18 (Suppl 1):S84­S90. Murasko DM, Goonewardene IM. 1990. T-cell function in aging: Mechanisms of decline. Annu Rev Gerontol Geriatr 10:71­96. Murasko DM, Weiner P, Kaye D. 1987. Decline in mitogen induced proliferation of lymphocytes with increasing age. Clin Exp Immunol 70(2):440­448. Murasko, DM, Gold, M.J., Hessen, M.T. and Kaye, D. 1990. Immune reactivity, morbidity, and mortality of elderly humans. Aging Immun and Infect Disease 2:171­179. Pinna K, Kelley DS, Taylor PC, King JC. 2002. Immune functions are maintained in healthy men with low zinc intake. J Nutr 132(7):2033­2036. Erratum in: J Nutr 2002 Nov;132(11):3431. Takagi K, Yamamori H, Morishima Y, Toyoda Y, Nakajima N, Tashiro T. 2001. Preoperative immunosuppression: Its relationship with high morbidity and mortality in patients receiving thoracic esophagectomy. Nutrition 17(1):13­17. Turnlund JR, Scott KC, Peiffer GL, Jang AM, Keyes WR, Keen CL, Sakanashi TM. 1997. Copper status of young men consuming a low-copper diet. Am J Clin Nutr 65(1):72­78. Turnlund JR, Jacob RA, Keen CL, Strain JJ, Kelley DS, Domek JM, Keyes WR, Ensunsa JL, Lykkesfeldt J, Coulter J. 2004. Long-term high copper intake: Effects on indexes of copper status, antioxidant status, and immune function in young men. Am J Clin Nutr 79(6):1037­ 1044. Impact of Nutritional Deficiencies and Psychological Stress on the Innate Immune Response and Viral Pathogenesis John F. Sheridan, Patricia A. Sheridan, and Melinda A. Beck Ohio State University, Columbus INTRODUCTION Many nutrients have been demonstrated to be essential to maintain an ad- equate immune system able to defend against infectious diseases. When in garri- son training or combat operations, the stress, and often negative energy balance, may contribute to impairment of the immune system. The risk of contracting an infectious disease, foodborne or otherwise, needs to be managed in part by ad- equate nutrition. This paper provides scientific evidence that demonstrates the importance of an appropriate diet, especially selenium intake, in maintaining an optimal immune defense system, especially when faced with stressful situations. Research presented from animal (mice) studies explores the potential rela- tionship between selenium deficiency and infection by enteroviruses, in particu- lar enteroviruses that can cause Keshan disease. Studies that explain the mecha- nisms by which the combination of selenium deficiency and enterovirus infection result in disease are described in detail. Further evidence that explains the onset of disease results from studies conducted to examine the host immune response of selenium deficiency versus selenium adequate mice in response to an experi- mental influenza viral infection.

APPENDIX B 379 COXSACKIE VIRUS B3 AND SELENIUM DEFICIENCY The discovery that the cardiomyopathy know as Keshan disease has a dual etiology that involves both a deficiency of the essential trace mineral selenium as well as an infection with an enterovirus provides the impetus for studies of the relationships between nutrition and viral infection. Enterovirus isolates from pa- tients with Keshan disease in a selenium-deficient area of China were predomi- nantly coxsackievirus group B. Thus, these viruses may contribute to the pathol- ogy of Keshan disease, as coxsackie B viruses are known etiologic agents of myocarditis (Peng et al., 2000). The possible relationship between selenium deficiency and infection with coxsackievirus was explored further using an experimental animal model. Viru- lent strains of coxsackievirus B3 (CVB3) induce myocarditis in infected mice, although avirulent strains, such as CVB3/0, do not induce disease even though the avirulent strain replicates in heart muscle. To determine if a deficiency in selenium could influence the ability of an avirulent virus to cause disease, wean- ling mice were fed a diet that was either deficient or replete in selenium for 4 weeks prior to infection with the avirulent CVB3/0. Mice fed the selenium- sufficient diet did not develop cardiac inflammation, the hallmark of myocardi- tis. In contrast, the Se-deficient mice developed moderate to severe myocarditis (Beck et al., 1994a). To determine if the increase in virulence was due to host factors alone, or a result of genome changes, the virus was isolated from the hearts of selenium-deficient mice and passed back into selenium-adequate mice. If the induction of myocarditis in the selenium-deficient mice was due to host factors alone, then infecting the selenium-adequate mice with this viral isolate would not be expected to cause myocarditis. However, selenium-adequate mice infected with virus isolated from selenium-deficient mice developed myocar- ditis, while selenium-adequate mice infected with virus isolated from other selenium-adequate mice did not develop myocarditis (Beck et al., 1994a). These findings suggested that CVB3/0 virus that replicated in a selenium-deficient host underwent a genomic change. To confirm this finding, CVB3/0 viral isolates obtained from selenium-adequate and selenium-deficient hosts were sequenced. Sequencing of the multiple viral isolates obtained from infected selenium- adequate and selenium-deficient mice confirmed that a viral genome change had occurred (Beck et al., 1995). Six nucleotide changes between the original CVB3/0 strain and the virus isolated from the selenium-deficient mice were found, whereas no changes were found in the genome of virus isolated from selenium- adequate mice. The mutations persisted, and myocarditis developed, when the newly virulent virus infected naïve selenium-adequate mice. Therefore, replica- tion of an avirulent coxsackievirus in the selenium-deficient host led to specific viral mutations that resulted in altered viral virulence. Once these mutations occurred, normal selenium-adequate mice were susceptible to myocarditis fol- lowing infection by the newly pathogenic virus.

380 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL CVB3 MUTATIONS AND OXIDATIVE STRESS Because selenium plays in important role in several antioxidant enzymes, a deficiency in selenium can lead to increased oxidative stress of the host. In order to determine if oxidative stress associated with selenium-deficiency can induce changes in a viral genome, glutathione peroxidase 1 (Gpx-1) knockout mice were used. Selenium is an essential component of the antioxidant enzyme Gpx-1. When selenium is limited in a diet Gpx-1 activity declines (Whanger and Butler, 1998). In order to determine if a decrease in Gpx-1 activity was a crucial step in selenium-associated changes in virulence, Gpx-1 knockout mice were infected with CVB3/0. Similar to selenium-deficient mice, the Gpx-1 knockout mice de- veloped myocarditis, whereas infected wildtype mice did not (Beck et al., 1998). The virus isolated from knockout mice with myocarditis demonstrated mutation to the cardiovirulent genotype at seven nucleotide positions, of which six were identical to the mutations found in virus isolated from selenium-deficient mice (Beck et al., 1998). These results suggest that the change in viral genome in infected selenium-deficient mice is due to increased oxidative stress as a conse- quence of a deficiency in antioxidant protection. Further evidence that increased oxidative stress is the driving force for the viral mutations is provided by the finding that CVB3/0 infected mice deficient in vitamin E, a lipid soluble antioxi- dant that functions differently from Gpx-1, also develop myocarditis due to a change in the viral genome (Beck et al., 1994b). HOST NUTRITIONAL STATUS AND INFLUENZA VIRUS INFECTION Influenza virus is a leading cause of morbidity and mortality in the U.S. According to the Centers for Disease Control, influenza kills more than 36,000 people annually and is responsible for more than 114,000 hospitalizations (CDC, 2004). Influenza is a single stranded-RNA virus with a segmented genome. It belongs to the Orthomyxoviridae family of viruses. To determine whether selenium deficiency had an effect on a family of viruses other than enteroviruses, weanling mice were fed a diet either deficient or adequate in selenium. After 4 weeks, mice were infected with influenza A/ Bangkok/1/79 (H3N2), a strain that causes mild pneumonitis in normal mice. At all time points examined, Se-deficient mice had greater lung pathology than selenium-adequate mice. Additionally, the lung pathology persisted longer in the selenium-deficient mice (Beck et al., 2001). Three segments of the influenza viral genome were sequenced from selenium- adequate and selenium-deficient mice; hemagglutinin (HA), neuraminidase (NA) and the matrix gene (M). All three proteins have been associated with virulence. The HA and NA proteins are responsible for viral entry and exit from the infected cell, respectively. The M gene, which codes for both M1 and M2 proteins, is

APPENDIX B 381 associated with viral replication. Mutations in the M gene were consistently found in virus recovered from the selenium-deficient mice. As with the coxsackievirus studies, when this virulent influenza viral isolate was passed back into selenium- adequate mice, enhanced lung pathology was observed (Nelson et al., 2001). There- fore, similar to what was found for coxsackievirus B3, host selenium deficiency leads to increased viral mutation in the influenza virus genome, resulting in a more virulent phenotype. In order to understand the mechanisms by which host nutritional status pro- motes viral mutation, we also examined the host's innate immune response. The innate immune response plays an important role in controlling the replication of influenza, and in directing the magnitude of the subsequent adaptive immune response. As a critical part of the innate immune response, IFN- and IFN- are produced by infected cells. These anti-viral cytokines protect uninfected cells from becoming infected by acting in an autocrine or paracrine manner to inhibit viral replication and increase expression of the major histocompatibility com- plex (MHC) class I molecules and natural killer (NK) cell cytotoxicity (Bogdan, 2000; Sinigaglia et al., 1999). Similar to interferon (IFN)- and IFN-, IFN- is a potent antiviral cytokine produced early after infection by NK cells. Additionally, IFN- activates macrophages, increases MHC expression, antigen processing and directs the sub- sequent cell-mediated immune response (Huang et al., 1993; Ruby and Ramshaw, 1991). In these studies, weanling male C57Bl/6 mice were fed either selenium- adequate or selenium-deficient diets for 4 weeks and subsequently infected with influenza A Bangkok/1/79 virus. In response to the influenza infection, selenium- adequate mice upregulated expression of IFN-, IFN- and IFN- 24h post infection (p.i). Although selenium-deficient mice increased IFN-, IFN- and IFN- expression after infection, expression of these genes was 2­4 fold lower than in the Se-adequate mice (Sheridan, et al., 2005). These data suggest that in response to an influenza viral infection, selenium deficiency impairs the early, anti-viral cytokine response. How these changes in gene expression may correlate with the emergence of the viral mutations is currently under investigation. STRESS AND INFLUENZA INFECTION The response to stress, whether physical or psychological, involves a variety of adaptive neuroendocrine mechanisms designed to restore homeostasis (Selye, 1936). Although these neuroendocrine responses are designed to restore homeo- stasis, the mammalian response to stress involves the release of immunomo- dulatory hormones and peptides that influence the host's response to infection. Stress-induced activation of the hypothalamic-pituitary-adrenal axis and sympa- thetic nervous system results in the release of glucocorticoids, catecholamines and opioids that modulate various aspects of innate immunity. Natural killer cell responses, a major element of human and animal natural resistance to infection,

382 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL is affected by stress responses (Bonneau et al., 1991; Campbell et al., 2001; Coe et al., 2002; Dobbs et al., 1996; Hermann et al., 1995). During an influenza viral infection, NK cells play an important role in the early, innate defense (Stein-Streilein and Guffee, 1986). They respond within 2­3 days of infection to kill virus-infected cells and to produce cytokines that begin to initiate and enhance the subsequent, specific anti-viral responses (Kos and Engelman, 1996). The study described here was designed to extend previous findings to a translational model where the consequences of stress-induced modu- lation of NK cell function could be examined in the context of an infectious disease. Mice were subjected to repeated, daily cycles of restraint (RST) and then infected intranasally with influenza A/PR8 virus. In this model, NK cells are an important component of the innate immune response involved in control- ling viral replication and limiting the spread of virus (Kos and Engelman, 1996; Stein-Streilein and Guffee, 1986). Daily cycles of restraint significantly modu- lated NK cell trafficking and cytolytic activity and contributed to elevated virus replication. Daily cycles of restraint suppressed two key chemokines necessary for peak NK activity, monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-1, in lungs of virus-infected mice. Suppression of these chemokines correlated with reduced NK cell number and activity in the lungs (Hunzecker et al., 2004). Reduced NK activity resulted in enhanced patho- physiology. This suggests that in stressed mice, NK cells are not appropriately called to the lungs to fight infection. Interleukin-12, a cytokine critical during the innate response, is an antigen presenting cell-derived cytokine that stimulates NK cells to secrete interferon IFN- and also augments the proliferation and cytolytic activity of NK cells (Fehniger et al., 1999). The stress due to RST also contributed to a decrease in IL-12 gene expression during the course of the influenza infection. These data suggest that not only does RST diminish the recruitment of NK cells to the sites of viral replication, but RST also inhibits the expression of several key genes involved in controlling NK cell effector function. Although influenza infections are resolved by the adaptive T cell response, the innate NK response is important for controlling influenza replication prior to activation of the antigen-specific response. CONCLUSIONS These studies demonstrate that selenium-deficiency and other nutritional changes that lead to increased host oxidative stress result in changes in the viral genome of two different RNA viruses. The viruses that emerge are more virulent and induce significant pathology even in a nutritionally intact host. Together, these data demonstrate that host nutritional status can influence not only the host response to the pathogen, but can also influence the genetic make-up of the viral genome. This last point is extremely important as it represents a new paradigm

APPENDIX B 383 for understand host-pathogen relationships. Developing countries are sites of widespread nutritional deficiencies; they are also areas of emergence of new viral diseases as well as old diseases with new properties. Our data also demonstrate that mice fed selenium-deficient diets have low- ered innate host defenses to influenza infection. Mice that are stressed and in- fected with an influenza virus also have lowered innate immune responses to influenza infection. As the innate immune response is critical to controlling viral infections and directing the subsequent cell-mediated immune response, alter- ations in the response may have profound consequences. New research indicates that oxidative stress may link psychological stress with alterations in immune response (Neigh et al., 2005). We propose that both nutritional deficiencies and psychological stress share a final common pathway of increased oxidative stress that impacts the host's innate defenses. The combination of nutritional defi- ciency with psychological stress may result in even greater oxidative stress and thus increased impact on host immune function and viral mutation rates. There- fore, adequate nutritional protection against oxidative stress is necessary to im- prove immune function and decrease the possibility of viral mutation. REFERENCES Beck M, Kolbeck P, Rohr L, Shi Q, Morris V, Levander O. 1994a. Benign human enterovirus becomes virulent in selenium-deficient mice. J Med Virol 43:166­170. Beck M, Kolbeck P, Rohr L, Shi Q, Morris V, Levander O. 1994b. Vitamin E deficiency intensifies the myocardial injury of coxsackievirus B3 infection of mice. J Nutr 124:345­358. Beck M, Shi Q, Morris V, Levander O. 1995. Rapid genomic evolution of a non-virulent coxsackie- virus B3 in selenium-deficient mice results in selection of identical virulent isolates. Nat Med 1:433­436. Beck MA, Esworthy RS, Ho Y, Chu F. 1998. Glutathione peroxidase protects mice from viral- induced myocarditis. FASEB J 12:1143­1149. Beck M, Nelson HK, Shi Q, Van Dael P, Schiffrin EJ, Blum S, Barclay D, Levander OA. 2001. Selenium deficiency increases the pathology of an influenza virus infection. FASEB J 15:1481­ 1483. Bogdan C. 2000. The function of type I interferons in antimicrobial immunity. Curr Opin Immunol 12:419­424. Bonneau RH, Sheridan JF, Feng NG, Glaser R. 1991. Stress-induced effects on cell-mediated innate and adaptive memory components of the murine immune response to herpes simplex virus infection. Brain Behav Immun 5:274­295. Campbell T, Meagher MW, Sieve A, Scott B, Storts R, Welsh TH, Welsh CJR. 2001. The Effects of Restraint Stress on the Neuropathogenesis of Theiler's Virus Infection: I. Acute Disease. Brain Behav Immun 15:235­254. Centers for Disease Control and Prevention. 2004. Prevalence of overweight and obesity among adults with diagnosed diabetes--United States, 1988­1994 and 1999­2002. MMWR Morb Mortal Wkly Rep 53:1066­1068. Coe CL, Kramer M, Kirschbaum C, Netter P, Fuchs E. 2002. Prenatal stress diminishes the cytokine response of leukocytes to endotoxin stimulation in juvenile rhesus monkeys. J Clin Endocrinol Metab 87:675­681.

384 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Dobbs CM, Feng N, Beck M, Sheridan JF. 1996. Neuroendocrine regulation of cytokine production during experimental influenza viral infection: Effects of restraint stress-induced elevation in endogenous Ecorticosterone. J Immunol 157:1870­1877. Fehniger TA, Shah MH, Turner MJ, VanDeusen JB, Whitman SP, Cooper MA, Suzuki K, Wechser M, Goodsaid F, Caligiuri MA. 1999. Differential cytokine and chemokine gene expression by human NK cells following activation with IL-18 or IL-15 in combination with IL-12: Implica- tions for the innate immune response. J Immunol 162:4511­4520. Hermann G, Beck FM, Sheridan JF. 1995. Stress-induced glucocorticoid response modulates mono- nuclear cell trafficking during an experimental influenza viral infection. J Neuroimmunol 56: 179­186. Huang S, Hendriks W, Althage A, Hemmi S, Bluethmann H, Kamijo R, Vilcek J, Zinkernagel RM, Aguet M. 1993. Immune response in mice that lack the interferon-gamma receptor. Science 259:1742­1745. Hunzeker J, Padgett DA, Sheridan PA, Dhabhar FS, Sheridan JF. 2004. Modulation of natural killer cell activity by restraint stress during an influenza A/PR8 infection in mice. Brain Behav Immun 18:526­535. Kos FJ, Engleman EG. 1996. Role of Natural Killer Cells in the Generation of Influenza Virus- Specific Cytotoxic T Cells. Cell Immunol 173:1­6. Neigh GN, Samuelsson AR, Bowers SL, Nelson RJ. 2005. 3-Aminobenzamide prevents restraint- evoked immunocompromise. Brain Behav Immun 19:351­356. Peng T, Li Y, Yang Y, Niu C, Morgan-Capner P, Archard LC, Zhang H. 2000. Characterization of Enterovirus Isolates from Patients with Heart Muscle Disease in a Selenium-Deficient Area of China. J Clin Microbiol 38:3538­3543. Ruby J, Ramshaw I. 1991. The antiviral activity of immune CD8+ T cells is dependent on interferon- gamma. Lymphokine Cytokine Res 10:353­358. Selye H. 1936. A syndrome produced by diverse nocous agents. Nature (Lond) 138:32. Sheridan, P.A., Sheridan, J.F., Beck, M.A. 2005. Selenium deficiency alters the innate immune re- sponse during an influenza infection in mice. FASEB J. 19(4):A443. Sinigaglia F, D'Ambrosio D, Rogge L. 1999. Type I interferons and the Th1/Th2 paradigm. Dev Comp Immunol 23:657­663. Stein-Streilein J, Guffee J. 1986. In vivo treatment of mice and hamsters with antibodies to asialo GM1 increases morbidity and mortality to pulmonary influenza infection. J Immunol 136:1435­ 1441. Whanger P, Butler J. 1998. Effects of various dietary levels of selenium as selenite or selenomethionine on tissue selenium levels and glutathione peroxidase activity in rats. J Nutr 118:846­852. The Influence of Minerals on Muscle Injury and Recovery Joseph G. Cannon Medical College of Georgia, Augusta MECHANISMS OF INJURY AND REPAIR Muscle damage can be caused by overloading, atrophy, ischemia/reperfusion, toxins, blunt impact, freezing, or lacerations. The general response to injury is stereotyped, regardless of the mode of damage. However, the nature of the initial damaging event at the cellular level, and any foreign material (such as infectious

APPENDIX B 385 microorganisms or toxins) within the tissue, can affect the types of cells recruited to the site of injury. For example, ischemia itself may cause little damage to muscle but activates the endothelium such that upon reperfusion, neutrophils contacting the endothelium release reactive oxygen species that can cause extensive damage. In contrast, mechanical overloading of muscle fibers causes direct damage to the structural proteins of the sarcomeres, but the magnitude of damage and time course of repair are unaffected in animals that have been depleted of neutrophils (Lowe et al., 1995). The presence of microbes in the wound draws in neutrophils and wound healing is usually delayed until the microbes are cleared by the phagocytic and cytotoxic activities of the neutrophils and macrophages. The recruitment and activation of macrophages and fibroblasts are common responses to all modes of muscle injury, in fact common to all types of tissue injury. The sequence of events in muscle repair and regeneration illustrates the function of these cells (adapted from Bischoff, 1994). Cellular and Molecular Events Following Muscle Injury An initial injury disrupts cell membranes and extracellular matrix. Liberated intracellular factors and matrix fragments bind to scavenger receptors on resi- dent macrophages. Cytokines such as interleukin-1 beta (IL-1) are secreted by the macrophages. These cytokines activate endothelial cells to express adhesion molecules (selectins) that facilitate leukocyte extravasation. The leukocytes se- crete matrix metalloproteinases (MMPs) that break down endothelial basement membranes and extracellular matrix, allowing the cells to migrate out of the vasculature and into the damaged area. Twenty-four human MMPs have been identified: all possess a modular struc- tures, including a common catalytic domain containing a zinc binding site. They are pro-enzymes activated by proteolytic cleavage (Lee and Murphy, 2004). MMP-2 and MMP-9 cleave type IV collagen, which is a major constituent of basement membranes and extracellular matrix. MMP-9 is a major product of macrophages and neutrophils (Goetzl et al., 1996). Monocytes recruited from the bloodstream differentiate into macrophages and phagocytize damaged tissue. Most damage is cleared within a few days, but macrophages remain in the area secreting chemoattractants and growth factors that recruit and activate fibroblasts and muscle precursor (satellite) cells. These satellite cells also secrete a matrix metalloproteinase to facilitate their migration, in this case MMP-2 is produced. Following myotoxin injury, MMP-9 was ex- pressed early during inflammatory cell infiltration, while MMP-2 coincided with regeneration of fibers (Carmeli et al., 2004). Matrix metalloproteinases also acti- vate latent forms of cytokines: MMP-2 and -9 activate transforming growth fac- tor beta (TGF), IL-1 and tumor necrosis factor (TNF), whereas MMP-1 and -3 release basic fibroblast growth factor (FGF-2) bound to extracellular matrix (McCawley and Matrisian, 2001).

386 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL After arriving at the site of injury, the satellite cells proliferate and then fuse together into myotubes that mature into new muscle fibers. Insulin-like growth factor-I (IGF-I) is an important stimulus for fusion and differentiation. In muscle, as in other tissues, much of the zinc is bound to metallothionein. While activated satellite cells are proliferating, the Zn/metallothionein complexes are localized in the nuclei. Upon fusion and differentiation, the complexes migrate to the cytosol (Apostolova et al., 2000). Meanwhile, fibroblasts produce collagen and other proteoglycans for reconstruction of basement membranes and extracellular matrix and the original tissue structure is restored. Adhesion molecules, matrix metalloproteinases, macrophages, fibroblasts perform similar functions in the repair of skin, bone and other organs. However instead of satellite cells, new skin tissue is formed by epidermal cells and new bone is formed by osteoblasts. The function of these effector molecules and cells is under paracrine regulation by locally-produced cytokines and growth factors. INFLUENCE OF ZINC ON INDIVIDUAL EFFECTORS OF WOUND HEALING In zinc deficiency, heart and skeletal muscle zinc concentrations are un- changed, but drop in plasma, liver, and bone. The normal plasma zinc concentra- tion is about 15 µmole/L, with 84 percent bound to albumin, 15 percent bound to 2macroglobulin and 1 percent bound to amino acids. Zinc deficiency reduces osteoblast activity, collagen and proteoglycan synthesis, and platelet aggregation (Rude and Shils, 2006). Zinc promotes antioxidant state by: (1) preserving metallothionein (as de- scribed below), (2) serving as a component of superoxide dismutase, and (3) interfering with iron-mediated free radical formation. Zinc also blocks caspase-6, a mediator of apoptosis (Tapiero and Tew, 2003). Metallothionein Four isoforms of metallothionein are known: MT-1 and MT-2, which are ubiquitous, and MT-3 and MT-4, which are located in the brain and skin. Each metallothionein molecule binds 7 zinc atoms. Zinc induces the mRNA for apo- metallothionein (thionein), which is susceptible to proteolysis until zinc-binding confers resistance. Physical trauma, glucocorticoids, IL-1, IL-6, and reactive oxygen species induce metallothionein, which is thought to provide protection against oxidative damage and to serve as a reservoir of zinc that can be donated to "zinc finger" transcription factors and zinc-dependent enzymes (Davis and Cousins, 2000). Monocyte metallothionein mRNA correlates with zinc intake in adequate diets (Cao and Cousins, 2000). Topical application of zinc oxide in- creases metallothionein expression in rat skin wounds (Lansdown, 2002).

APPENDIX B 387 Matrix Metalloproteinases Matrix metalloproteinase gene expression does not appear to be affected by mild zinc deficiency or excess, based on dietary studies in mice (Moore et al., 2003). In these experiments, whole thymus was digested, mRNA was harvested and 48,000 transcripts were analyzed. Zinc metalloenzymes and zinc-finger tran- scription factors not affected. Nevertheless, matrix metalloproteinase activity does appear to be dependent upon zinc availability. For example, tetracycline analogs inhibited MMP activity in vitro in proportion to their ability to bind zinc and the inhibition could be partially antagonized by excess zinc (Ryan et al., 2001). In the same report, oral administration of analogs to streptozotocin- diabetic rats reduced MMP-9 activity in the skin. A zinc chelating protein re- leased by activated neutrophils, calprotectin, may serve as an endogenous regu- lator of MMP activity (Isaksen and Fagerhol, 2001). Topical application of zinc oxide increases MMP activity in pig skin wounds (Agren, 1993). Cytokines and Growth Factors There is no compelling evidence that zinc deficiency adversely affects cyto- kine production. In a study of five healthy subjects (23­38 years old), zinc defi- ciency was induced by a diet of soy-based protein and a phytic acid supplement to reduce zinc bioavailability. Total dietary zinc was 2­3.5 mg/day for 20­24 weeks. The subjects were then repleted with zinc acetate (25­50 mg/day) for 8­12 weeks. Peripheral blood mononuclear cells were isolated for analysis of cytokine secretion at baseline, end of the depletion phase, and end of the reple- tion phase. Zinc depletion resulted in significant reductions in interferon-gamma (IFN) and TNF secretion, compared to baseline and repletion conditions, but had no significant influence on IL-1, IL-4, IL-10 or IL-6 secretion (Beck et al., 1997). In a study of shorter duration, 8 healthy men (27­47 yrs) were placed on a zinc restricted diet (4.6 mg/day) for 10 weeks. No significant effect was ob- served on phytohemagglutinin-induced TNF or IFN secretion by peripheral blood mononuclear cells nor on phorbol myristic acetate-induced neutrophil su- peroxide production (Pinna et al., 2002). On the other hand, IGF-I may be significantly affected by zinc availability. Serum IGF-I correlated with zinc intake in adequate diets (Devine et al., 1998) whereas zinc deficiency reduced serum IGF-I concentration (Cossack, 1991) and zinc supplementation increased it (Imamoglu et al., 2005). Topical application of zinc oxide increased IGF-I mRNA by 50 percent in pig skin wounds (Tarnow et al., 1994). In vitro, zinc decreased affinity of IGF binding protein-5 and in- creased the affinity of myoblast IGF receptor type 1 for both IGF-I and IGF-II (McCusker and Novakofski, 2003). This, in effect, increases the bioavailability of IGF-I to the muscle cell, one of the regulatory factors in cell growth and multiplication during muscle repair after injury.

388 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Phagocytic Activity The limited evidence available suggests that zinc supplementation may have a negative effect on the function of phagocytic cells. In one study, 11 healthy adult men given 300 mg/day zinc sulfate orally for 6 weeks. Plasma zinc increased from 83 to 200 µg/dL by the 6th week. Neutrophil chemotactic migration and phagocy- tosis were reduced by ~50 percent, whereas bactericidal capacity (percent viable ingested bacteria at 2 hours) was unchanged. No data regarding incidence of infec- tion were reported (Chandra, 1984). In another study, 39 marasmic infants (7­8 months old) were given formula with (1.9 mg/kg/day) or without (0.35 mg/kg/day) supplemental zinc for 105 days (Schlesinger et al., 1993). Monocyte phagocytosis and fungicidal activity were significantly less in the supplemented group. Further- more, the supplemented group had more than 2 times as many days of impetigo episodes as the control group (1.3 ± 1.1 versus 0.55 ± 0.8, P < 0.02). INFLUENCE OF ZINC ON INTEGRATED WOUND HEALING Very little information exists regarding the influence of zinc on overall wound healing. In one study, groups of mice were placed on a normal or low- protein diet for 4 weeks prior to abdominal surgery (Nezu et al., 1999). After surgery, the mice were further subgrouped by placement on total parenteral nu- trition with either normal zinc content or no zinc for 6 days. No differences were observed in bursting pressure of the anastomosed intestine. Low protein groups had ~33 percent, non-significant reduction in tensile strength of sutured abdomi- nal wall muscle, but no influence of zinc was observed. Low protein caused a ~50 percent reduction in tensile strength of sutured skin. Zinc deficiency reduced skin tensile strength at the suture by 35 percent in normal protein condition and by 69 percent (P < 0.05) in low protein condition. Perhaps the most extensively studied application of zinc to humans relates to the potential effectiveness of oral zinc in healing arterial or venous leg ulcers. The conclusions of two Cochrane Systematic reviews (Wilkinson and Hawke, 1998, 2005) are that oral zinc has little effect, except perhaps a small benefit for individuals with low serum zinc concentrations. CONCLUSIONS The complete mechanism by which skeletal muscle initiates and completes a repair process is not well deciphered. The number of synchronized events is numerous and involved many cells and signaling factors whose functions re- mained unknown. During the process proteins that depend on zinc as a modula- tor of function or synthesis, such as methalothionin, metalloproteinases, are known to play a role but the relationship between zinc status or zinc intake and their function during muscle repair is unknown. Likewise, no response on cyto-

APPENDIX B 389 kine production has been observed as a result of zinc deficiency in humans. Only IGF-1 levels in serum and bioavailability to muscle cells appears to be affected by zinc status, zinc deficiency being related to lower serum concentrations of IGF-1. With so few suggestive data regarding the involvement of zinc or any other minerals in muscle repair processes, there currently appears to be little justifica- tion for increasing dietary intakes of minerals based on the potential for im- proved wound healing or muscle repair that may occur during the high intensity exercise or other traumatic injuries potentially occurring during military garrison training. REFERENCES Agren MS. 1993. Zinc oxide increases degradation of collagen in necrotic wound tissue. Br J Dermatol 129:221­223. Apostolova MD, Ivanova IA, Cherian MG. 2000. Signal transduction pathways and nuclear transloca- tion of zinc and metallothionein during differentiation of myoblasts. Biochem Cell Biol 78:27­37. Beck FW, Prasad AS, Kaplan J, Fitzgerald J, Brewer G. 1997. Changes in cytokine production and T cell subpopulations in experimentally induced zinc-deficient humans. Am J Physiol 272:E1002­ E1007. Bischoff R. 1994. The satellite cell and muscle regeneration. In: Engel AG, Franzini-Armstrong C., eds. Myology. New York: McGraw-Hill, Inc. 1:97­118. Cao J, Cousins RJ. 2000. Metallothionein mRNA in monocytes and peripheral blood mononuclear cells and in cells from dried blood spots increases after zinc supplementation of men. J Nutr 130:2180­2187. Carmeli E, Moas M, Reznick AZ, Coleman R. 2004. Matrix metalloproteinases and skeletal muscle: A brief review. Muscle Nerve 29:191­197. Chandra RK. 1984. Excessive intake of zinc impairs immune responses. JAMA 252:1443­1446. Cossack ZT. 1991. Decline in somatomedin-C (insulin-like growth factor-I) with experimentally induced zinc deficiency in human subjects. Clin Nutr 10:284­291. Davis SR, Cousins RJ. 2000. Metallothionein expression in animals: A physiological perspective on function. J Nutr 130:1085­1088. Devine A, Rosen C, Mohan S, Baylink D, Prince RL. 1998. Effects of zinc and other nutritional factors on insulin-like growth factor I and insulin-like growth factor binding proteins in post- menopausal women. Am J Clin Nutr 68:200­206. Goetzl EJ, Banda MJ, Leppert D. 1996. Matrix metalloproteinases in immunity. J Immunol 156:1­4. Imamoglu S, Bereket A, Turan S, Taga Y, Haklar G. 2005. Effect of zinc supplementation on growth hormone secretion, IGF-I, IGFBP-3, somatomedin generation, alkaline phosphatase, osteocalcin and growth in prepubertal children with idiopathic short stature. J Pediatr Endocrinol Metab 18:69­74. Isaksen B, Fagerhol MK. 2001. Calprotectin inhibits matrix metalloproteinases by sequestration of zinc. Clin Pathol Mo Pathol 54:2289­2292. Lansdown ABG. 2002. Metallothioneins: Potential therapeutic aids for wound healing in the skin. Wound Rep Reg 10:130­132. Lee MH, Murphy G. 2004. Matrix metalloproteinases at a glance. J Cell Sci 117:4015­4016. Lowe DA, Warren GL, Ingalls CP, Boorstein DB, Armstrong RB. 1995. Muscle function and protein metabolism after initiation of eccentric contraction-induced injury. J Appl Physiol 79:1260­ 1270.

390 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL McCawley LJ, Matrisian LM. 2001. Matrix metalloproteinases: They're not just for matrix anymore! Curr Opin Cell Biol 13:534­540. McCusker RH, Novakofski J. 2003. Zinc partitions insulin-like growth factors (IGFs) from soluble IGF binding protein (IGFBP)-5 to the cell surface receptors of BC3H-1 muscle cells. J Cell Physiol 197:388­399. Moore JB, Blanchard RK, Cousins RJ. 2003. Dietary zinc modulates gene expression in murine thymus: Results from a comprehensive differential display screening. Proc Natl Acad Sci 100:3883­3888. Nezu R, Takagi Y, Ito T, Matsuda H, Okada A, 1999. The importance of total parenteral nutrition- associated tissue zinc distribution in wound healing. Surg Today 29:34­41. Pinna K, Kelley DS, Taylor PC, King JC. 2002. Immune functions are maintained in healthy men with low zinc intake. J Nutr 132:2033­2036. Rude RK, Shils ME. 2006. Magnesium. In: Modern Nutrition in Health and Disease. In: Shils ME, Shike M, Ross AC, Caballero B, Cousins RJ. Philadelphia, PA: Lippincott, Williams and Wilkins. Pp. 223­247. Ryan ME, Usman A, Ramamurthy NS, Golub LM, Greenwald RA. 2001. Excessive matrix metal- loproteinase activity in diabetes: Inhibition by tetracycline analogues with zinc reactivity. Curr Med Chem 8:305­316. Schlesinger L, Arevalo M, Arredondo S, Lonnerdal B, Stekel A. 1993. Zinc supplementation impairs monocyte function. Acta Paediatr 82:734­738. Tapiero H, Tew KD. 2003. Trace elements in human physiology and pathology: Zinc and metal- lothioneins. Biomed Pharmacother 57:399­411. Tarnow P, Agren MS, Steenfos H, Jansson JO. 1994. Topical zinc oxide treatment increases endog- enous gene expression of insulin-like growth factor-I in granulation tissue from porcine wounds. Scand J Plast Reconstr Hand Surg 28:255­259. Wilkinson EAJ, Hawke CI. 1998. Does oral zinc aid the healing of chronic leg ulcers? A systematic literature review. Arch Dermatol 134:1556­1560. Wilkinson EAJ, Hawke CI. 2005. Oral zinc for arterial and venous leg ulcers. Cochrane Database, Accession number: 00075320-100000000-00387. Physical Activity and Nutrition: Effects on Bone Turnover, Bone Mass, and Stress Fracture Jeri W. Nieves Helen Hayes Hospital and Columbia University, West Haverstraw, New York INTRODUCTION Bone mass accumulates throughout childhood and adolescence until peak bone mass is reached during the third decade of life (Heaney et al., 2000; Weaver, 2002), at a time when many individuals enter military service. When higher bone mineral density (BMD) is attained at a young age (peak bone mass) there is a subsequent reduction in the risk of childhood fractures, stress fractures, and possibly osteoporosis and osteoporotic fractures later in life (Goulding et al., 2001; Marx et al., 2001; Melton et al., 1998, 2003; Myburgh et al., 1990;

APPENDIX B 391 Torgerson et al., 1996). Studies indicate that a larger bone size is also related to a reduced risk of fracture. Genetic factors account for between 60­80 percent of the variance in peak bone mass and bone size (McGuigan et al., 2002; Mitchell et al., 2003; Nguyen et al., 2003; Orwoll et al., 2001). However, an individual may not achieve their genetically determined bone mass/size, if environmental and lifestyle conditions are not permissive. High levels of physical activity and adequate calcium intake have been shown to improve accrual of peak bone mass, although data in males are limited (HHS, 2004; Klesges et al., 1996; Moiso et al., 2004; Molgaard et al., 2001). BONE HEALTH IN THE MILITARY: A U.S. MILITARY ACADEMY STUDY A recent study was conducted with military academy students, both men and women, to investigate the relationship between exercise and bone health and diet, particularly milk consumption and bone health. Results show that there is an association between eating disorders and bone health as well as between negative energy balance and bone health in women cadets. Those women with abnormal menstrual function had more bone loss compare with women with normal menstrual function, possibly due to the effects of excess exercise in menstrual function. In men cadets, milk consumption was associated with min- eral content of bone and the benefits of exercise for bone health where only seen with higher intakes of milk. This study follows up previous studies with military personnel. Results from this recent study are presented in detail in the sections that follow. Other relevant studies are also described. In this study, college aged subjects were recruited from the United States Military Academy (USMA) Class of 2002, West Point, NY. Exercise and milk intake were assessed by self-administered questionnaires. Eating disorders were determined using the Eating Disorders Inventory (EDI). Female cadets reported menstrual function for the prior year. The outcome measurements used as indica- tors of bone health were peripheral quantitative computed tomography (QTC) and tibial length, Peripheral QCT (Stratec XCT-2000; Germany) was used to image a single slice at the two-third distal tibia. The distal third of the tibia was determined by a manual measurement of tibial length between the base of the patella and the styloid process to the closest centimeter. Peripheral QCT provides bone density (volumetric or true density), as well as bone size and geometry including cortical thickness, periosteal circumference, and endosteal circumference. EXERCISE AND BONE MASS AND SIZE In the recent Military Academy study, described above prior exercise in males was significantly correlated to tibial content (r = 0.23; p < 0.001) and there was a significant 5 percent difference in total tibial content between those in the

392 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL highest exercise (> 11 hours per week) group versus all others (p < 0.001). Male cadets in the lowest exercise group when compared to those in the highest exer- cise groups had 7 percent lower cortical thickness (p < 0.04), and 3 percent smaller periosteal circumference (p < 0.04; Ruffing et al., in review). Although exercise is beneficial to bone health, excessive exercise can lead to abnormal menstrual function, which may have a negative impact on bone health. In the Military academy study, female cadets who had regular menstrual cycles during the first year in the academy gained bone in the spine and total hip as compared to women who had fewer than 6 menstrual cycles per year who lost bone mass at the spine and hip (p-value < 0.05; Nieves et al., 2000). CALORIC INTAKE AND BONE MASS Several studies show that athletes with energy expenditures in excess of caloric intake have low BMD; however, this is often confounded by abnormal menstrual function (Cobb et al., 2003). Even in male distance runners, without hormonal disturbances, bone mass is lower than in controls if energy balance is negative; it is hypothesized to result from an energy intake that is lower than required (Hetland et al., 1993). Clearly caloric restriction for weight loss is re- lated to bone loss, this was demonstrated in a review by Shapses and Cifuentes (2004). In USMA female military cadets, BMD losses over 4 years occurred at both the spine and hip in cadets with sub-clinical eating disorders (EDI highest quartile) whereas there was no change in BMD or gains in those women in the lower quartiles of EDI (p < 0.05; Nieves et al., 2005). CALCIUM, MILK, AND BONE MASS The most frequently studied mineral in relation to bone health is calcium. There have been 10 of 11 controlled trials of calcium supplementation (300­ 1,000 mg/day) that have shown a positive relationship, with BMD increases from 1­6 percent depending on the study duration and skeletal site measured (Bonjour et al., 2001; Chan et al., 1995; Chevalley et al., 2005; Dibba et al., 2000; Johnston et al., 1992; Lee et al., 1993; Matkovic et al., 2004; Merrilees et al., 2000; Nowson et al., 1997; Rosen, 2003). When calcium supplementation was discontinued some studies report gain in bone health was lost (Johnston et al., 1992; Lee et al., 1993) whereas in others the benefit remained (Bongour et al., 2001; Dibba et al., 2000; Matkovic et al., 2004). A review in premenopausal women (Welten et al., 1995) suggested that calcium supplementation to a total calcium intake from 1,500 to 2,500 mg/day produces an annual mean percentage difference in bone mass of 1.3 percent at multiple skeletal sites. In some studies, milk supplementation has been evaluated rather than cal- cium alone. In an earlier study of milk supplementation in young adults there were significantly greater increases in total body bone mineral content and total

APPENDIX B 393 body bone mineral density as compared to young adults given a placebo (Cadogan et al., 1997). In the recent study of male military cadets described above, milk consumption was related to tibial mineral content and there was a 6 percent higher tibial content between those consuming more than 3 glasses of milk per day as compared to those consuming 1 or fewer glasses (p < 0.03; Ruffing et al., in review). Daily milk consumption was positively associated with cortical thick- ness and periosteal circumference, such that cortical thickness was significantly greater in the highest milk intake categories compared to the lowest milk intake group (p < 0.04; p < 0.01). There was a significant interaction between milk intake and prior exercise in relation to cortical thickness (Ruffing et al., in review). Males who exercise more only showed a skeletal benefit of the exercise if milk intake was greater than one glass per day. OTHER MINERALS AND BONE MASS The data relating trace minerals to bone health are very limited. There have been limited studies evaluating phosphorus intake and bone health in young adults. Individuals with a healthy diet who consume the RDA for phosphorus are unlikely to have any negative impact on bone health. It is likely that the relation- ships between calcium, protein, and phosphorus intakes are what are important for bone health (Lemke et al., 1998). Low intakes in the face of high calcium intake from supplements may create a relative phosphorus deficiency because availability of phosphorus decreases as calcium supplement intake increases (Heaney and Nordin, 2002). Magnesium deficiency, if severe, will disturb cal- cium homeostasis leading to impaired PTH (parathyroid hormone) secretion and a lack of skeletal and renal response to PTH leading to hypocalcemia. There are also some data that suggest that magnesium intakes are inversely related to bone resorption. However, in a cross-over study of 26 females aged 20­28 years, who were given 240 mg magnesium per day had no effect on bone turnover (Doyle et al., 1999). By contrast, magnesium administration (360 mg) in 12 males age 27­ 36, resulted in an initial significant suppression of bone formation and resorp- tion, that was transient and lasted for 10 days out of 30 days (Dimai et al., 1998). Several cross-sectional studies have noted a positive association between magnesium intake and BMD in premenopausal women (Angus et al., 1988; Freudenheim et al., 1986; Houtkooper et al., 1995; New et al., 1997, 2000; Wang et al., 1997; Yano et al., 1985), although not all (Michaelsson et al., 1995). These inconsistent results may be related to the difficulty in trying to discriminate the effect of a diet deficient in magnesium versus other deficiencies that also may impact BMD. The concentration of zinc in bone is higher than in most other tissues and can be quickly depleted. Zinc has a structural role in the bone matrix where it complexes with fluoride into the hydroxyapetitie crystals of the bone. One study related low zinc levels to forearm bone mineral in premenopausal women (Angus et al., 1988). Zinc may diminish bone resorption and stimulate

394 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL bone formation but these data is mostly the result of animal studies (Hosea et al., 1999). In rat studies zinc has been shown at the site of fracture healing, but there are no clinical studies on the role of zinc in fracture healing. Copper deficiency has negative effects on bone but there is inconclusive evidence regarding any bone benefits of increasing copper intake (Roughead and Lukaski, 2003). There are limited data and no large trial data regarding the role of boron in bone health. Strontium has only been studied in very large doses (up to 1­2 g/day) and these high strontium intakes may be beneficial for bone and may prevent bone loss in older women (Reginster et al., 2002). Silicon defi- ciency has negative effects on bone and silicon intake was positively related to bone density of the hip in some (men and young women) but not postmeno- pausal women (Jugdaohsingh et al., 2004). BONE TURNOVER, STRESS FRACTURES, AND EXERCISE The impact of exercise on PTH and bone turnover has been studied in sev- eral small studies and the results are not always in agreement and it appears that the type of exercise, duration, timing of samples all can influence the results (Eliakim et al., 1997; Karlsson et al., 2003). Probably one of the most relevant papers for this topic was an evaluation of changes in dioxypyridinoline (DPD) during Marine Recruit Training (n = 158 females and 58 males). Increases in bone resorption were found to correspond to an increase in miles of weight bearing exercise performed during training. The authors reported significant bone resorption at the end of Marine recruit training and attributed this to an accumula- tion of weight bearing exercise as well as an increase in marching miles (Sheehan et al., 2003). It is possible that basic training will lead to increased resorption followed by increased formation and that the window between increments in these two processes may be the period of greatest risk of stress fractures. In the study of marine recruits (Sheehan et al., 2003) weekly DPD levels for females with and without stress fractures were evaluated and, although the differences were not significant, there was a trend toward higher levels of urinary DPD in female recruits with stress fractures as compared to female recruits without stress fracture, which indicates bone resorption might be related to stress fractures. If bone resorption was excessive as reported and if it were related to excess risk for stress fracture, then it would be expected that prophylactic treatment with risedronate would reduce the incidence of stress fracture during military training. When subjected to strains higher than usual bone will remodel to repair microdamage and to strengthen the bone. In a randomized controlled trial for 15 weeks, 324 new infantry recruits in Israel were given risedronate, a bisphos- phonate that reduces bone resorption or a placebo. There was no significant difference between drug and placebo for tibial, femoral, metatarsal, or total stress fracture incidence (Milgrom et al., 2004). It is possible that calcium and vitamin D supplementation may be capable of

APPENDIX B 395 preventing stress fractures in female naval recruits; Dr. Joan Lappe, at Creighton University, is currently evaluating this hypothesis. In this trial the female naval recruits are provided supplementation with 2,000 mg calcium and 800 IU vita- min D (Oscal+D) given as 2 doses with meals for 8 weeks basic training (n = 5,200). There are no increases in adverse effects at this dose and by the end of 2005 the study should be completed (Personal communication, J. Lappe, Creigh- ton University). SUMMARY The studies presented here point to a need for a clearer understanding of the impact of exercise and other factors on bone turnover. Some of the conclusions from this discussion are that an adequate caloric intake must be maintained to meet energy expenditure or bone loss may occur. Higher bone losses might also occurred due to dermal losses during exercise. Bone loss will be exaggerated in females with loss of menstrual function or eating disorders. Micro-fracture re- pair is also dependent on calcium intake. To counteract any excess in bone turnover and meet the demands of the skeleton during intense activity calcium higher than the AI of 1,000 mg may be needed with intense exercise. Intakes of 1,500­2,500 mg/day may be needed to maintain or increase BMD in younger recruits since peak bone mass may not yet be achieved. Basic training appears to first lead to increased resorption (perhaps to compensate for calcium loss due to sweat or negative energy balance), but this is followed by increased formation (stimulated by intense training) and the window between increments in these two processes may be the period of greatest risk of stress fractures. More data are clearly needed to understand the role of nutrition in stress fracture occurrence. Milk or similar products have the potential to provide cal- cium as well as other important nutrients but until now, data on calcium and stress fracture are limited with two negative and one positive studies. Ongoing research on the potential benefits of higher calcium intakes indicates that supple- ments of up to 2,000 mg of calcium are safe. Results from this study will be forthcoming soon and should shed more light on the prevention of stress frac- tures by calcium. REFERENCES Angus RM, Sambrook PN, Pocock NA, Eisman JA. 1988. Dietary intake and bone mineral density. Bone Miner 4(3):265­277. Angus RM, Pocock NA, Eisman JA. 1998. Nutritional intake of pre- and postmenopausal Australian women with special reference to calcium. Eur J Clin Nutr 42(7):617­625. Bonjour JP, Chevalley T, Ammann P, Slosman D, Rizzoli R. 2001. Gain in bone mineral mass in prepubertal girls 3.5 years after discontinuation of calcium supplementation: A follow-up study. Lancet 358(9289):1208­1212.

396 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Cadogan J, Eastell R, Jones N, Barker ME. 1997. Milk intake and bone mineral acquisition in adolescent girls: Randomised, controlled intervention trial. BMJ 15(7118):1255­1260. Chan GM, Hoffman K, McMurry M. 1995. Effects of dairy products on bone and body composition in pubertal girls. J Pediatr 126(4):551­556. Chevalley T, Bonjour JP, Ferrari S, Hans D, Rizzoli R. 2005. Skeletal site selectivity in the effects of calcium supplementation on areal bone mineral density gain: A randomized, double- blind, placebo-controlled trial in prepubertal boys. J Clin Endocrinol Metab 90(6):3342­ 3349. Cobb KL, Bachrach, LK, Greendale G, Marcus R, Neer R, Nieves J, Sowers MF, Brown BW, Gopalakrishnan G, Luetters C, Tanner HK, Ward B, Kelsey JL. 2003. Disordered eating, men- strual irregularity, and bone mineral density in female distance runners. Medicine and Science in Sports and Exercise 35(5):711­719. Dibba B, Prentice A, Ceesay M, Stirling DM, Cole TJ, Poskitt EM. 2000. Effect of calcium supple- mentation on bone mineral accretion in gambian children accustomed to a low-calcium diet. Am J Clin Nutr 71(2):544­549. Dimai HP, Porta S, Wirnsberger G, Lindschinger M, Pamperl I, Dobnig H, Wilders-Truschnig M, Lau KH. 1998. Daily oral magnesium supplementation suppresses bone turnover in young adult males. J Clin Endocrinol Metab 83(8):2742­2748. Doyle L, Flynn A, Cashman K. 1999. The effect of magnesium supplementation on biochemical markers of bone metabolism or blood pressure in healthy young adult females. Eur J Clin Nutr 53(4):255­261. Eliakim A, Raisz LG, Brasel JA, Cooper DM. 1997. Evidence for increased bone formation follow- ing a brief endurance-type training intervention in adolescent males. J Bone Miner Res 12(10): 1708­1713. Freudenheim JL, Johnson NE, Smith EL. 1986. Relationships between usual nutrient intake and bone-mineral content of women 35­65 years of age: Longitudinal and cross-sectional analysis. Am J Clin Nutr 44(6):863­876. Goulding A, Jones IE, Taylor RW, Williams SM, Manning PJ. 2001. Bone mineral density and body composition in boys with distal forearm fractures: A dual-energy x-ray absorptiometry study. J Pediatr 139(4):509­515. Heaney RP, Nordin BE. 2002. Calcium effects on phosphorus absorption: Implications for the pre- vention and co-therapy of osteoporosis. J Am Coll Nutr 21(3):239­244. Heaney RP, Abrams S, Dawson-Hughes B, Looker A, Marcus R, Matkovic V, Weaver C. 2000. Peak bone mass. Osteoporos Int 11(12):985­1009. Hetland ML, Haarbo J, Christiansen C. 1993. Low bone mass and high bone turnover in male long distance runners. J Clin Endocrinol Metab 77(3):770­775. Hosea HJ, Taylor CG, Wood T, Mollard R, Weiler HA. 2004. Zinc-deficient rats have more limited bone recovery during repletion than diet-restricted rats. Exp Biol Med (Maywood) 229(4):303­311. Houtkooper LB, Ritenbaugh C, Aickin M, Lohman TG, Going SB, Weber JL, Greaves KA, Boyden TW, Pamenter RW, Hall MC. 1995. Nutrients, body composition and exercise are related to change in bone mineral density in premenopausal women. J Nutr 125(5):1229­1237. Johnston CC Jr, Miller JZ, Slemenda CW, Reister TK, Hui S, Christian JC, Peacock M. 1992. Calcium supplementation and increases in bone mineral density in children. N Engl J Med 327(2):82­87. Jugdaohsingh R, Tucker KL, Qiao N, Cupples LA, Kiel DP, Powell JJ. 2004. Dietary silicon intake is positively associated with bone mineral density in men and premenopausal women of the Framingham Offspring cohort. J Bone Miner Res 19(2):297­307. Karlsson KM, Karlsson C, Ahlborg HG, Valdimarsson O, Ljunghall S, Obrant KJ. 2003. Bone turnover responses to changed physical activity. Calcif Tissue Int 72(6):675­680. Klesges RC, Ward KD, Shelton ML, Applegate WB, Cantler ED, Palmieri GM, Harmon K, Davis J. 1996. Changes in bone mineral content in male athletes. Mechanisms of action and intervention effects. JAMA 276(3):226­230.

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398 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Nieves J, Zion M, Ruffing J, Garrett P, Lindsay R, Cosman F. 2005. Eating Disorders and Amenor- rhea are Strong Predictors of Change in Bone Mass in Physically Active Young Females. 27th Annual Meeting of the American Society for Bone and Mineral Research (ASBMR) Nashville, TN. Nguyen TV, Livshits G, Center JR, Yakovenko K, Eisman JA. 2003. Genetic determination of bone mineral density: Evidence for a major gene. J Clin Endocrinol Metab 88(8):3614­3620. Nowson CA, Green RM, Hopper JL, Sherwin AJ, Young D, Kaymakci B, Guest CS, Smid M, Larkins RG, Wark JD. 1997. A co-twin study of the effect of calcium supplementation on bone density during adolescence. Osteoporos Int 7(3):219­225. Orwoll ES, Belknap JK, Klein RF. 2001. Gender specificity in the genetic determinants of peak bone mass. J Bone Miner Res 16(11):1962­1971. Reginster JY, Deroisy R, Dougados M, Jupsin I, Colette J, Roux C. 2002. Prevention of early postmenopausal bone loss by strontium ranelate: The randomized, two-year, double-masked, dose-ranging, placebo-controlled PREVOS trial. Osteoporos Int 13(12):925­931. Rosen CJ. 2003. Insulin-like growth factor I and calcium balance: Evolving concepts of an evolu- tionary process. Endocrinology 144(11):4679­4681. Roughead ZK, Lukaski HC. 2003. Inadequate copper intake reduces serum insulin-like growth factor- I and bone strength in growing rats fed graded amounts of copper and zinc. Nutr 133(2):442­ 448. Ruffing JA, Cosman F, Zion M, Tendy S, Garrett P, Lindsay R, Nieves J. In Press. Exercise level and milk intake are positively related to bone mass and size at weight bearing sites. Sheehan KM, Murphy MM, Reynolds K, Creedon JF, White J, Kazel M. 2003. The response of a bone resorption marker to marine recruit training. Mil Med 168(10):797­801. Torgerson DJ, Campbell MK, Thomas RE, Reid DM. 1996. Prediction of perimenopausal fractures by bone mineral density and other risk factors. J Bone Miner Res 11(2):293­297. U.S. Department of Health and Human Services. 2004. Bone Health and Osteoporosis: A Report of the Surgeon General. U.S. Department of Health and Human Services, Office of the Surgeon General. Wang MC, Luz Villa M, Marcus R, Kelsey JL. 1997. Associations of vitamin C, calcium and protein with bone mass in postmenopausal Mexican American women. Osteoporos Int 7(6):533­538. Weaver CM. 2002. Adolescence: The period of dramatic bone growth. Endocrine 17(1):43­48. Yano K, Heilbrun LK, Wasnich RD, Hankin JH, Vogel JM. 1985. The relationship between diet and bone mineral content of multiple skeletal sites in elderly Japanese-American men and women living in Hawaii. Am J Clin Nutr 42(5):877­888. Zelten DC, Kemper HC, Post GB, van Staveren WA. 1995. A meta-analysis of the effect of calcium intake on bone mass in young and middle aged females and males. J Nutr 125(11):2802­2813. Evaluating Nutritional Effects on Cognitive Function in Warfighters: Lessons Learned Harris R. Lieberman U.S. Army Research Institute of Environmental Medicine, Natick, Massachusetts INTRODUCTION There is substantial information in the scientific literature regarding the ef- fects of various nutritional interventions on cognitive performance. Some of this

APPENDIX B 399 research was specifically conducted to address issues of military relevance. Most of the literature in the area addresses the acute effects of nutritional factors on cognitive function; only a few studies have examined the chronic effects of variations in diet or administration of dietary supplements for more than a day. To date, only a few nutritional interventions have been shown to alter cognitive performance in young, healthy, adequately nourished humans. From an evolu- tionary perspective, this is to be expected since it would not be desirable for short term variations in the diet to change behavioral functions, except those associated with seeking or consuming food. Consequently, perhaps the most important lesson to be learned from this literature is that subtle variations in the diet, or treatment with many of the dietary supplements purported to alter cogni- tive function, do not have observable effects on cognitive state. This paper is intended to serve, in part, as the basis for recommendations to be made by the Committee on Mineral Requirements for Cognitive and Physical Performance of Military Personnel. This committee was charged with making recommendations on mineral requirements for military personnel engaged in military operations and in garrison. The committee was asked to consider main- tenance and/or improvement of cognitive and physical performance when for- mulating military mineral requirements. Therefore, they provided several ques- tions to scientists working in the area of military nutrition. This paper addresses the decrements in cognitive function and behavior that result from stressors faced by soldiers during training and combat operations. In particular, it addresses how cognitive function and mood are uniquely affected by military operations. It also addresses selection of appropriate methods to assess warfighter cognitive state and the use of such methods in military nutrition research studies. This paper is organized in a question and answer format to directly address the questions posed by the committee. GENERAL QUESTIONS Are there specific effects of high levels of physical and emotional stress on cognitive function or behavior? There are devastating effects of physical and psychological stress on cogni- tive function and behavior. These adverse effects have been documented in many studies conducted with various military populations and in some limited situa- tions, with civilian volunteers (Haslam, 1984; Lieberman et al., 2005b; Ruby et al., 2002). There is no clear consensus regarding the contribution of each type of stressor to the overall degradation in cognitive performance seen following ei- ther acute or chronic operational (military) stress. Nevertheless, in operations or training conducted to simulate combat by U.S. infantry and special operations units, as well as studies conducted with soldiers from other nations, severe dec- rements in cognitive performance inevitably are observed after two days or less of sustained operational stress (Banderet and Stokes, 1980; Haslam, 1984;

400 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL 25 Task 20 Response Time in the 15 on Reaction 10 Latency Degradation 5 Percent Four-Choice 0 0.10 % Hypoglycemia SEALS Rangers Blood Alcohol (2.6 mmol l-1) 72-hr 72-hr FIGURE B-26 Percent degradation from baseline in mean response time on the Four- Choice Visual Reaction Time Test for the Ranger and SEAL studies (Lieberman et al., 2005; Lieberman et al., 2002b) compared with the effects of clinical hypoglycemia (2.6 mmol · L­1) and a blood alcohol level of 0.10 percent, which is legally drunk in many localities (Strachan et al., 2001; Tiplady et al., 2001). In both studies with the SEALS and Rangers, volunteers were exposed to various stressors prior to cognitive testing including substantial sleep deprivation, environmental exposures, and psychological stress. The data from the SEAL trainees are only from those who received placebo treatment, not caffeine (Modified from Lieberman et al., 2005b). McLellan et al., 2003). Typically, such short term operational stress includes sleep deprivation and, in many instances, exposure to environmental extremes and inadequate hydration and nutrition (Lieberman et al., 2005a; Opstad, 1994; Opstad et al., 1978). In several studies we have conducted in the field (Lieberman et al., 2002b, 2005a), in simulated combat-like scenarios, decrements in war- fighter cognitive performance observed have exceeded those documented in stud- ies of civilian volunteers who are legally drunk or suffering from clinical hypo- glycemia (Figure B-26; Lieberman et al., 2005b; Strachan et al., 2001; Tiplady et al., 2001). Severe cognitive decrements are seen in most cognitive functions assessed in such studies, including simple and choice reaction time, logical rea- soning, learning memory and vigilance. For a more detailed discussion see Lieberman et al. (2005b). In studies where the principal "stressor" is inadequate nutrition there does not appear to be substantial adverse effects on cognitive performance, even after a month of substantial caloric deprivation (Lieberman, 1999; Lieberman et al.,

APPENDIX B 401 1997). This is consistent with the limited civilian literature in this area, in par- ticular with the classic Minnesota Starvation studies (Keys et al., 1950). Is there any information regarding a "benefit" of altering nutritional sta- tus to improve cognitive behavior in individuals in the military? A number of studies have demonstrated that caffeine can enhance certain aspects of warfighter cognitive and physical performance in rested and sleep deprived volunteers (Amendola et al., 1998; Fine et al., 1994; Kamimori et al., 2003; LaJambe et al., 2005; Lieberman et al., 2002b; McLellan et al., 2004, 2005). In rested volunteers, the effects of caffeine in moderate doses are largely confined to tasks requiring sustained vigilance. They can also be observed on mood questionnaires that assess alertness, a mood state associated with changes in vigilance (for recent reviews see Institute of Medicine, 2001; Lieberman, 2003; Smith, 2005). These effects can also be observed in simulations of military activities such as sentry duty, where vigilance is a key factor (Johnson and Merullo, 2000). In sleep deprived volunteers, the effects of caffeine are observed not only on tests of vigilance and sentry duty, but on a wider variety of cognitive functions including learning, short term memory, and reasoning (Figure B-27; Kamimori et al., 2003; Lieberman et al., 2002b). It has been hypothesized that the more widespread cognitive effects of caffeine in sleep deprived individuals are not directly the result of caffeine's modulation of these higher order cogni- tive functions, but are secondary to the compound's ability to maintain alert- ness (Lieberman, 2003). An ad-hoc committee of the Committee on Military Nutrition Research has reviewed the literature in this area and concluded, ". . . caffeine in the range of 100 to 600 mg is effective in increasing speed of reaction time without affecting accuracy and in improving performance on visual and audio vigilance tasks" (Institute of Medicine, 2001). A nutrient that has been extensively studied for its possible beneficial ef- fects during acutely stressful environments is the amino acid tyrosine, a dietary precursor for the synthesis of dopamine and norepinephrine in the brain (for a recent review see Deijen, 2005). Studies conducted at a number of civilian and military laboratories suggest that high doses of tyrosine, greater than those pres- ent in single serving of foods, mitigate some of the adverse effects of acute stress on cognitive performance (Ahlers et al., 1994; Banderet and Lieberman, 1989; Magill et al., 2003). There are also some data suggesting that substantial doses of carbohydrate in beverage form can enhance cognitive performance of soldiers engaged in aerobic activities and who are not receiving fully adequate energy from rations (Figure B-28; Lieberman et al., 2002a). There is no question that carbohydrate supplementation enhances aerobic performance as demonstrated by numerous civilian and several military studies (for a recent review see Montain and Young, 2003). For a recent review of the cognitive literature in this area, particularly with regard to military nutrition issues, see Lieberman, 2003.

402 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL 160 A. VISUAL VIGILANCE 140 B. FOUR-CHOICE REACTION TIME 140 120 120 100 Correct Hits 100 80 Latency Premature Errors 80 60 Time-Out Errors 60 40 Hits 40 False Alarms 20 Response Time 20 0 140 C. MATCHING TO SAMPLE 160 D. REPEATED ACQUISITION Placebo 120 140 100 From 120 80 100 60 80 Change 40 60 Correct Responses 20 Response Time 40 Incorrect Responses Time-Out Errors Time-to-Completion 0 Percent 20 180 E. POMS & SSS 160 F. RIFLE MARKSMANSHIP Tension 160 140 Depression Anger 140 120 120 100 100 80 80 60 DCM Vigor SGT Fatigue 60 40 STIME Confusion MISS SSS 40 20 Placebo 100 mg 200 mg 300 mg Placebo 100 mg 200 mg 300 mg Caffeine Dose FIGURE B-27 Percentage change in performance and mood following varying doses of caffeine compared to placebo treatment 1 hr after caffeine administration. Navy SEAL trainees undergoing Hell Week training were given 100, 200, or 300 mg of caffeine or placebo. The treatments were administered after 72 hours of sleep deprivation and expo- sure to stressors such as running, lifting, paddling inflatable boats, swimming, and calis- thenics, as well as psychological stressors. At both 1 and 8 hours after caffeine adminis- tration, subjects were given several cognitive tests. Significant improvement was seen on many of the tests in a dose-dependent manner. (A) Percentage change from placebo on measures of visual vigilance. (B) Percentage change from placebo on the four-choice visual reaction time test. (C) Percentage change from placebo on the matching-to-sample test. (D) Percentage change on the repeated acquisition test. (E) Percentage change from placebo on the 6 sub-scales of the Profile of Mood States (POMS) and Stanford Sleepi- ness Scale (SSS). (F) Percentage change from placebo on measures of rifle marksman- ship. Overall, caffeine appeared to be most effective at a dose of 200 mg, although on some tasks the optimal dose was 300 mg. NOTE: DCM = Distance from Center of Mass; MISS = Percent of Targets Missed; SGT = Shot Group Tightness; STIME = Sighting Time SOURCE: Lieberman et al. (2002a).

APPENDIX B 403 5 % IMPROVEMENT FROM PLACEBO 4 50% 25% 38% 50% 3 (s) 2 1 Baseline 0 From -1 38% Vigilance -2 Difference -3 -4 Mean Placebo, n = 29 -5 6 % CHO, n = 36 -6 12 % CHO, n = 28 -7 March 30min Rest Run1 4hr Rest Run2 Activity FIGURE B-28 Mean (± SEM) differences from baseline in auditory vigilance reaction times over the 10 hour on study designed to evaluate the effects of carbohydrate on cognitive and physical performance (Lieberman et al., 2002a). This study examined the effects of carbohydrate supplementation on cognitive function during a day of sustained aerobic activity. Army Rangers received either 6 percent (by volume) carbohydrate, 12 percent (by volume) carbohydrate, or placebo beverage in 6 doses, in addition to two ration meals. During the day, volunteers completed a 19.3-km march, two 4.8-km runs, and live-fire marksmanship exercises. Each subject's vigilance was tested continuously by an ambulatory monitor worn on the nonpreferred wrist (Lieberman and Coffey, 2000; Lieberman et al., 2005c). Performance on the monitor was summed over 5 time periods that corresponded to the activity taking place. Because the values plotted are differences from baseline values, the higher the number on the y axis, the better the performance. Carbohydrate treatment had a positive, dose-dependent effect on mean reaction time vigi- lance difference scores. The interaction between treatment and time was also significant and reaction time was shorter with carbohydrate treatment that with placebo at every time period. Percent improvement on the 12 percent carbohydrate beverage relative to placebo treatment during the same activity is also provided above each activity. SOURCE: Lieberman et al. (2002a). SPECIFIC QUESTIONS What cognitive functions are important when soldiers are engaged in mili- tary operations and in garrison? What behaviors are important for performance when soldiers are engaged in military operations and in garrison?

404 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL These two closely related questions are best addressed together. Cognitive functions are hypothetical constructs formulated to describe mental processes. When such processes are converted into an action, a behavior occurs. Therefore, from a practical perspective it is only the behavior that can be measured and is operationally relevant. Even the most concrete thought (cognitive process) can- not be observed unless some behavior occurs, except indirectly with technolo- gies, such as brain scanning, that assess physiological correlates of mental activi- ties. When behavioral psychologists study cognitive processes, they are in fact studying the behaviors which they hypothesize are the result of specific cogni- tive processes. The relationship between cognitive processes and the behavior that results has to be inferred, and is always complex. Even simple tasks require multiple cognitive functions. For example, a seemingly simple cognitive test, such as visual choice reaction time, which produces a very simple behavior, pushing a button, requires a complex cascade of cognitive processes (Lieberman, 2003). Motivation to perform the task, allocation of attention, visual perception, visual processing, decision making, short and long term memory (recalling which stimu- lus is correct and recalling the instructions for the test), sensory-motor integra- tion, and motor control are some of the cognitive processes required to perform the task. Given the behavioral complexity of such a simple task, attempting to objectively and quantitatively specify the critical cognitive parameters associ- ated with the thousands of tasks associated with military operations in the field and garrison is, at present, impossible. There are hundreds of military specialties, each with hundreds of individual behaviors associated with performance of the job. Military performance is not unique in this regard, as few, if any of the behavioral processes required to adequately perform most civilian occupations have been specified. In response to this complexity, psychologists have attempted to identify some key cognitive capabilities that appear to govern many aspects of human behavior and develop tests to assess them. Functions that can be assessed by cognitive tests include: sensation, perception, vigilance, attention, learning, mem- ory, language, fine and gross motor performance, decision making, and complex mental processes, such as mathematical reasoning and face recognition. Cogni- tive psychologists who study military performance have attempted to choose functions from these categories that they believe are the most important for optimal military performance. In many instances, computerized cognitive test batteries can be administered to military populations to collect this information rapidly and with minimal hardware. The cognitive functions that seem to be frequently selected across various military laboratories, presumably because they are thought to be most important for warfighter operational performance, in- clude: psychomotor tasks like simple and choice reaction time, vigilance, atten- tion, short-term memory, and logical reasoning. More complex behaviors are also assessed such as marksmanship and performance in simulators of real world

APPENDIX B 405 tasks. Performance on the more abstract and cognitive tests often predict perfor- mance on more realistic tasks (for examples see McLellan et al., 2003). Is there evidence from military studies that decrements in cognitive functions or behaviors occur in the field and may be related to mineral deficiencies? As discussed above in response to the first general question, cognitive per- formance rapidly degrades in operational scenarios designed to simulate combat. The loss of cognitive function associated with actual combat has been termed the `fog of war' although the term has more general implications (Clausewitz, 1993; Kiesling, 2001; Lieberman et al., 2005b; Opstad et al., 1978). In developed countries, it seems unlikely that mineral deficiencies are associated with decre- ments in cognitive function on the battlefield or in garrison. An exception may be women, who are at greater risk of deficient mineral status than men. Of particular concern is iron status, due to the prevalence of iron deficiency and borderline iron deficiency seen in the young female population and in military population samples. Since iron status appears to be associated with physical and cognitive performance decrements there may be operational consequences in some women (Beard, 1995, 2003; Beard et al., 1995; Bruner et al., 1996; Cline et al., 1998; McClung et al., 2006). What makes military personnel unique regarding the use of tests for cog- nitive function and behavior? Military personnel are exposed to a great many physical and cognitive stres- sors that are unique to their profession. Some examples include: fear associated with severe injury or loss of life for the individual or his close associates in combat; long periods of time away from home; environmental stressors such as heat, cold and high altitude; acute and chronic sleep deprivation; exposure to environmental toxins unique to military operations such as depleted uranium, as well as more common pollutants; possible exposure to chemical and biologi- cal agents, and the simultaneous or consecutive combination of many of these stressors. What are the advantages and disadvantages of using self-report, such as the Profile of Mood States, to measure mood, fatigue, cognitive function, etc.? Mood questionnaires are frequently used to assess the effects of dietary constituents on behavioral state in civilian and military populations (this section has been adapted with minor modifications from Lieberman, 2005). When they are adequately standardized and have acceptable psychometric properties, mood questionnaires are accepted as valid measures of mental states and can be admin- istered in a few minutes, unlike cognitive test batteries. This is an important advantage for military field studies since it is usually difficult for volunteers to spend a great deal of time taking comprehensive cognitive test batteries. One of the most widely employed mood questionnaires in nutrition-behavior research is the Profile of Mood States (POMS). This questionnaire provides six individual subscales: vigor, fatigue, tension, depression, anxiety, and anger, as well as an

406 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL overall indication of mood state (McNair et al., 1971). Depending on the particu- lar mood assessed, they can be highly correlated with tests of cognitive function (Bolmont et al., 2000; Glenville and Broughton, 1978; Nicholson and Stone, 1986). Although mood questionnaires do not have the cachet of tests of cogni- tive performance because they are mistakenly not considered to be objective, they are useful for documenting and explaining the effects of dietary constituents on human behavior. In fact, the distinction between an objective and a subjective test is more a matter of appearance than reality. Cognitive performance tests seem to be objective because they apparently bypass the emotional content of the behavior assessed, but in fact, when standardized, both cognitive performance and mood questionnaires are objective and reliable measures of particular as- pects of human behavior. Each type of test requires the subject to make a re- sponse to a particular sequence of standardized stimuli. Mood questionnaires are valuable in studies of dietary constituents as they explain and validate the results of cognitive performance tests. Furthermore, they can provide evidence that a dietary treatment or diet has effects not readily detected by cognitive tests. For example, caffeine alters a variety of mood states in a manner that would not be expected given its stimulant-like actions. Mood questionnaires have shown that in normal volunteers, given moderate doses of caffeine, depression and hostility are reduced and clarity of mind, imagination, and energy increase (Amendola et al., 1998; Leathwood and Pollet, 1982). This demonstrates the importance of administering mood questionnaires as comple- mentary tests to explain certain cognition outcomes. In general, if consistent effects are observed using different measurement techniques, such as performance tests and mood questionnaires, then the results of a study are more likely to be valid. As noted above, caffeine consistently affects tests of vigilance and closely related mood states such as vigor and fa- tigue (Lieberman, 2003; Smith, 2002). Drugs that are stimulants, like amphet- amine, also have consistent effects on these outcomes (Magill et al., 2003). Use of mood questionnaires can also provide information regarding confounding fac- tors, such as unanticipated changes in fatigue, anxiety, or depression of volun- teers during testing. Unfortunately, there is an unfounded, preconceived belief that standardized tests of mood state are less objective than tests of cognitive performance. In particular, military policy makers should consider results from such mood states questionnaires as valid data that provides pertinent information and together with cognitive tests results, can be used to develop strategies to improve military performance. In fact, cognitive tests can sometimes be worse predictors of actual military performance than mood questionnaires, as they are rarely standardized and appropriately validated in young, healthy populations. Can one extrapolate data on cognitive function or behavior from the civil- ian population to military personnel? What are study design characteristics or methods that need to be considered when making such extrapolations?

APPENDIX B 407 Due to the unique nature of combat and the special requirements for many military occupations as discussed above, extrapolation from civilian to military populations is difficult. Under limited circumstances such generalization may be possible as a first approximation, but definitive data from appropriate military populations will usually be required. Moreover, such a determination can only be made on a case-by-case basis. Even key design characteristics and optimal methods for studies will vary from case-to-case. For a recent discussion of some of the critical methodological issues in the area of nutrition and behavior see Harris (2005) and Lieberman (2005). A conservative approach is necessary in relating nutrition to behavior across different populations since the effects in question are often modest. SUMMARY There is abundant scientific evidence that the circumstances faced by sol- diers during training and combat operations adversely affect their cognitive func- tion and behavior. Some of these circumstances involve a variety of stressors, from physical to psychological, that impair normal brain function. Valid mea- sures of cognitive performance and behavior are of critical importance to deter- mine factors that alter cognitive domains and to implement strategies that may improve performance. Measuring cognitive functions and behavior is a complex task that can only be conducted when the many domains that constitute cognition and behavior are examined. Therefore, many tests have been devised that explore these domains and have been used and standardized for both civilian and military populations. When developing strategies to improve military performance, policy makers should consider results from both mood states questionnaires and cognitive tests since, when standardized, both types provide pertinent information and compli- ment each other. That is, mood states questionnaires can help understand results from tests cognitive performance. Although studies with the civilian population can initially be useful in explor- ing these issues, extrapolation to military personnel is in most cases challenging due to the differing situations and extreme demands often placed on military per- sonnel. Therefore, it is advisable to duplicate the military environment to the ex- tent possible (i.e., physical environment, physical activity, and other stressors) when conducting research intended to address military requirements. DISCLAIMERS The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. Human subjects participated after giving their free and informed voluntary consent. The investigators adhered to the poli-

408 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL cies for protection of human subjects as prescribed in Army Regulation 70-25, and the research was conducted in adherence with the provisions of 45 CFR Part 46. Citations of commercial organizations and trade names in this report do not constitute an official Department of the Army endorsement or approval of the products or services of these organizations. Approved for public release; distri- bution is unlimited. REFERENCES Ahlers ST, Thomas JR, Schrot J, Shurtleff D. 1994. Tyrosine and glucose modulation of cognitive deficits resulting from cold stress. In: B. Marriott, ed. Food Components to Enhance Perfor- mance. Institute of Medicine. Washington, DC: National Academy Press. Pp. 301­320. Amendola CA, Gabrieli JDE, Lieberman HR. 1998. Caffeine's effects on performance and mood are independent of age and gender. Nutr Neurosci 1:269­280. Banderet LE, Lieberman HR. 1989. Treatment with tyrosine, a neurotransmitter precursor, reduces environmental stress in humans. Brain Res Bull 24:147­150. Banderet LE, Stokes JW. 1980. Simulated, sustained combat operations in the field artillery fire detection center (FOC): A model for evaluating biomedical indices. Technical Report No. T 9/80. Natick, MA: U.S. Army Research Institute of Environmental Medicine. Beard J. 1995. One person's view of iron deficiency, development and cognitive function. Am J Clin Nutr 62:709­710. Beard J. 2003. Iron deficiency alters brain development and functioning. J Nutr 133:1468S­1472S. Beard JL, Feagans L, Frobose C. 1995. Cognitive dysfunction in iron deficient adolescents. FASEB J 9:A975. Bolmont B, Thullier F, Abraini JH. 2000. Relationships between mood states and performances in reaction time, psychomotor ability, and mental efficiency during a 31-day gradual decompres- sion in hypobaric chamber from sea level to 8848 m equivalent altitude. Physiol Behav 71:469. Bruner AB, Joffe A, Duggan AK, Casella JF, Brandt J. 1996. Randomised study of cognitive effects of iron supplementation in non-anaemic iron-deficient adolescent girls. Lancet 348:992­996. Clausewitz C von. 1993. On War. New York: Alfred A. Knopf. Cline AD, Patton JF, Tharion WJ, Strowman SR, Champagne C, Arsenault J, Reynolds KL, Warber JP, Baker-Fulco C, Rood J, Tulley RT, Lieberman HR. 1998. Assessment of the relationship between iron status, dietary intake, performance, and mood state of female army officers in a basic training population. Technical Report No. T98-24. Natick, MA: U.S. Army Research Institute of Environmental Medicine. Deijen JB. 2005. Tyrosine. In: Lieberman HR, Kanarek R, Prasad C, eds. Nutritional Neuroscience. Boca Raton, FL: CRC Press LLC. Pp. 363­381. Fine BJ, Kobrick JL, Lieberman HR, Riley RH, Marlowe B, Tharion WJ. 1994. Effects of caffeine or diphenhydramine on visual vigilance. Psychopharmacology 114:233­238. Glenville M, Broughton R. 1978. Reliability of the Stanford Sleepiness Scale compared to short duration performance tests and the Wilkinson Auditory Vigilance Task. Adv Biosci 21:235. Harris RBS. 2005. Consideration of Experimental Design for Studies in Nutritional Neuroscience. In: Lieberman HR, Kanarek R, Prasad C, eds. Nutritional Neuroscience. Boca Raton, FL: CRC Press LLC. Pp. 11­23. Haslam DR. 1984. The military performance of soldiers in sustained operations. Aviat Space Environ Med 55:216­221. IOM (Institute of Medicine). 2001. Caffeine for the Sustainment of Mental Task Performance: For- mulations for Military Operations. Washington, D.C.: National Academy Press.

APPENDIX B 409 Johnson RF, Merullo DJ. 2000. Caffeine, gender, and sentry duty: Effects of a mild stimulant on vigilance and marksmanship. In: Friedel K, Lieberman HR, Ryan DH, Bray GA, eds. Pen- nington Center Nutrition Series Volume 10: Countermeasures for Battlefield Stressors. Baton Rouge, LA: Louisiana State University Press. Pp. 272­289. Kamimori GH, Johnson D, Thorne D. 2003. Efficacy of multiple caffeine doses for maintenance of vigilance during early morning operations. Sleep 26:A196. Keys A, Brozek J, Henschel A, Mickelsen O, Taylor HL. 1950. The Biology of Human Starvation, Vol. 1. Minneapolis, MN: The University of Minnesota Press. Pp. 1255­1342. Kiesling EC. 2001. On war Without the Fog. Fort Leavenworth, KS: Combined Arms Center. LaJambe CM, Kamimori GH, Belenky G, Balkin TJ. 2005. Caffeine effects on recovery sleep fol- lowing 27 h total sleep deprivation. Aviat Space Environ Med 76:108­113. Leathwood PD, Pollet P. 1982. Diet-induced mood changes in normal populations. J Psychiatric Res 17:147. Lieberman HR. 1999. Amino Acid and Protein Requirements: Cognitive performance, stress, and brain function. In: The Role of Protein and Amino Acids in Sustaining and Enhancing Perfor- mance. Washington, D.C.: National Academy Press. Pp. 289­307. Lieberman HR. 2003. Nutrition, brain function, and cognitive performance. Appetite 40:245­254. Lieberman HR. 2005. Human nutritional neuroscience: fundamental issues. In: Lieberman HR, Kanarek R, Prasad C, eds. Nutritional Neuroscience. Boca Raton, FL: CRC Press LLC. Pp. 3­10. Lieberman HR, Coffey BP. 2000. Preliminary findings from a new device for monitoring perfor- mance and environmental factors in the field. In: Friedel K, Lieberman HR, Ryan DH, Bray GA, eds. Pennington Center Nutrition Series Volume 10: Countermeasures for Battlefield Stres- sors. Baton Rouge, LA: Louisiana State University Press. Pp. 126­159. Lieberman HR, Askew EW, Hoyt RW, Shukitt-Hale B, Sharp MA. 1997. Effects of thirty days of undernutrition on plasma neurotransmitter precursors, other amino acids and behavior. J of Nutr Biochem 8:119­126. Lieberman HR, Falco CM, Slade SS. 2002a. Carbohydrate administration during a day of sustained aerobic activity improves vigilance, assessed with a novel ambulatory monitoring device, and mood. Am J Clin Nutr 76:120­127. Lieberman HR, Tharion WJ, Shukitt-Hale B, Speckman KL, Tulley R. 2002b. Effects of caffeine, sleep loss and stress on cognitive performance and mood during U.S. Navy SEAL training. Psychopharmacology 164:250­261. Lieberman HR, Bathalon GP, Falco CM, Kramer FM, Morgan III CA, Niro P. 2005a. Severe decre- ments in cognition function and mood induced by sleep-loss, heat, dehydration and undernutri- tion during simulated combat. Biol Psych 57:422­429. Lieberman HR, Bathalon GP, Falco CM, Morgan III CA, Niro P, Tharion WJ. 2005b. The fog of war: decrements in cognitive performance and mood associated with combat-like stress. Aviat Space Environ Med 76(7):C7­C14. Lieberman HR, Kramer FM, Montain SJ, Niro P, Young AJ. 2005c. Automated ambulatory assess- ment of cognitive performance, environmental conditions and motor activities during military operations in obiomonitoring for physiological and cognitive performance during military op- erations. Proceeding of SPIE 5797:14­23. Magill RA, Waters WF, Bray GA, Volaufova J, Smith SR, Lieberman HR, et al. 2003. Effects of tyrosine, phentermine, caffeine, d-amphetamine and placebo on cognitive and motor perfor- mance deficits during sleep deprivation. Nutr Neurosci 6:237­246. McClung JP, Marchitelli LJ, Friedl KE, Young AJ. 2006. Prevalence of iron deficiency and iron deficiency anemia among three populations of female military personnel in the U.S. Army. J Am Coll Nutr 25:64­69.

410 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL McLellan TM, Bell DG, Lieberman HR, Kamimori GH. 2003. The impact of caffeine on cognitive and physical performance and marksmanship during sustained operations. Canadian Mil J 4:47­54. McLellan TM, Bell DG, Kamimori GH. 2004. Caffeine improves physical performance during 24 h of active wakefulness. Aviat Space Environ Med 75:666­672. McLellan TM, Kamimori GH, Bell DG, Smith IF, Johnson D, Belenky G. 2005. Caffeine maintains vigilance and marksmanship in simulated urban operations with sleep deprivation. Aviat Space Environ Med 76:39­45. McNair DM, Lorr M, Droppleman LF. 1971. Profile of Mood States Manual. San Diego, CA: Educational and Industrial Testing Service. Montain SJ, Young AJ. 2003. Diet and physical performance. Appetite 40:255­267. Nicholson AN, Stone BM. 1986. Antihistamines: Impaired performance and the tendency to sleep. Eur J Clin Pharmacol 30:27. Opstad K. 1994. Circadian rhythm of hormones is extinguished during prolonged physical stress, sleep and energy deficiency in young men. Eur J Endocrinol 134:56­66. Opstad PK, Ekanger R, Nummestad M, Raabe N. 1978. Performance, mood, and clinical symptoms in men exposed to prolonged, severe physical work and sleep deprivation. Aviat Space Environ Med 49:1065­1073. Ruby BC, Shriver TC, Zderic TW, Sharkey BJ, Burks C, Tysk S. 2002. Total energy expenditure during arduous wildfire suppression. Med Sci Sports Exerc 34:1048­1054. Smith A. 2002. Effects of caffeine on human behavior. Food Chem Toxicol 40:1243. Smith A. 2005. Caffeine. In: Lieberman HR, Kanarek R, Prasad C, eds. Nutritional Neuroscience. Boca Raton, FL: CRC Press LLC. Pp. 341­361. Strachan MW, Deary IJ, Ewing FM, Fergusson SS, Young MJ, Frier BM. 2001. Acute hypoglycemia impairs the functioning of the central but not the peripheral nervous system. Physiol Behav 72:83­92. Tiplady B, Drummond GB, Cameron E, Gray E, Hendry J, Sinclair W, Wright P. 2001. Ethanol, errors, and the speed-accuracy trade-off. Pharmacol Biochem Behav 69:635­641. Iron and Cognitive Performance John L. Beard and Laura E. Murray-Kolb Pennsylvania State University, University Park INTRODUCTION Iron deficiency is the most common single nutrient deficiency in the world (WHO, 2002). Infants, children, and women of reproductive age are the most commonly affected though there is reason to believe there are functional conse- quences to iron deficiency in any individual, regardless of sex, age, and racial background (Beard, 1995). As many as 9­11 percent of women of reproductive age have iron deficiency (Looker et al., 1997); in military personnel > 50 percent of women in basic training were iron deficient (Westphal et al., 1995) suggesting a greater overall prevalence of iron deficiency in females in the military. The prevalence of iron deficiency specific to males and females in the military is not well documented nor is the stability of the prevalence estimates established

APPENDIX B 411 (Westphal et al., 1995). The special demands made upon military personnel in terms of physical and emotional stress may well lead to alterations in nutrient metabolism which in turn can be associated with cognitive and behavioral alter- ations (Cline et al., 1998). The purpose of this review is to present what is known regarding cognitive alterations that can be ascribed to iron deficiency with, or without anemia. MILITARY PERSONNEL Several specific topics need to be addressed within this review: (1) Is there any direct evidence that iron deficiency and iron deficiency anemia alters cognitive functioning? (2) What is the evidence that the additional high physical and emo- tional stresses placed on military personnel will alter the relationship between iron nutritional status and cognitive performance? (3) What is known about the prob- able biological mechanisms for altered cognition in iron deficiency? The existing scientific literature relating iron status to cognition and behavior is almost exclusively in the civilian population with the exception of several recent published reports (Booth, 2003) and one in-house report (Cline et al., 1998). This latter report was issued in 1998 from the U.S. Army Research Institute of Environ- mental Medicine regarding cognitive performance, and physical performance in a group of female officers going through basic training. One-third of the 57 subjects were low in serum ferritin while only seven percent were anemic at entry. After basic training, 64 percent of the women had low ferritin levels despite reported iron intakes > 16 mg/day. Intake of iron was < 80 percent of the military recom- mended dietary allowances in only 8 subjects suggesting dietary intakes of iron during training was close to adequate in most of the soldiers. The increase in prevalence of low ferritin however suggests the physical and emotional demands of basic training did increase iron losses; a finding consistent with the effect of physical training on iron balance (Murray-Kolb et al., 2001). Importantly, there was no relationship between iron status and three nega- tive emotions (tension, depression, anger) or the positive emotion scale (vigor) within the profile of mood states (POMS) battery that was administered. There was a positive correlation between "iron status" and confusion but the report is unclear regarding their definition of iron status in this regard so it is impossible to conclude if anemia or tissue iron deficiency were responsible for this statisti- cally significant relationship. As a group, iron sufficient women did not differ from iron deficient women in any of these behavioral measures. The cognitive task, a four-choice reaction time paradigm, was also not different in iron defi- cient compared to iron sufficient subjects. Other reports on the relationship of iron status to cognition and behavior in military personnel are very sparse and not conclusive (Booth, 2003: Booth et al., 2003). In these two studies on Austra- lian military personnel consuming either the fresh food diet or the combat ration pack, the soldiers had significant declines (approximately 15 percent) in serum

412 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL ferritin and folate as well as a decline in antioxidant status while training during 12 or 23 days. Poor baseline antioxidant status improved in all the soldiers, especially those consuming the combat ration pack possibly due to the vitamin C fortified food items. Emotionally, there was increased fatigue and more negative affect but specific relationships to a micronutrient could not be established in either of these studies. The conclusions from the available studies in military personnel do not support a specific role for changes in iron status being related to mood, behavior, or cognitive performance but the extremely limited knowledge base makes this conclusion very tenuous. CIVILIAN PERSONNEL There are a number of studies performed in adults and adolescents (Table B-17) that have had a focus on the relationship between iron status and cogni- tive or behavioral functioning. There are two cross sectional designs that looked at subscales of the General Health Questionnaire (GHQ) showing that both low ferritin and oral contraceptive use were required to observe a relationship be- tween ferritin and depression (Fordy and Benton, 1994; Rangan et al., 1998). Other work using the Minnesota Multiphasic Personality Inventory (MMPI, the most widely used personality test in the U.S.) test and a set of French fatigue, depression, and anxiety scales showed no effect of iron status on these measures with the exception of a French study in which women with ferritin between 20­50 µg/L showed a 2.2 times greater benefit in terms of fatigue scores compared to women with ferritin > 50 µg/L (Hunt and Penland, 1999; Verdon et al., 2003). The former study was a blinded, placebo intervention trial but only for four weeks, which is unlikely to be sufficient time to replenish pools of essential iron (Youdim et al., 1989). One additional cross sectional study looked at depression in post-partum women and showed a dou- bling in amount of depression in anemic women but the study did not examine other iron status indicators so attribution of depression to iron deficiency ane- mia cannot be done (Corwin et al., 2003). Several blinded intervention and placebo controlled studies in adolescent girls (Bruner et al., 1996) and in women during the post partum period (Beard et al., 2005) provide more sub- stantive data regarding the relationship of iron status to cognition and emo- tions. The iron deficient, but not anemic adolescent girls, that were provided iron supplements showed a significant and substantial improvement in verbal learning and memory but attention performance was unaffected. In contrast, iron deficient anemic mothers given iron for 28 weeks had much less depres- sion and anxiety than anemic mothers given the placebo (Beard et al., 2005). This latter study was conducted in a complex environment of poverty, poor health care, and other confounding issues that comprise a situation of very high "stress," whereas the former study in inner city Baltimore was performed in individuals with much better opportunities for overall quality of life.

APPENDIX B 413 A research design that had a specific focus on iron status and cognition in women of reproductive age was recently completed in the U.S. (Murray Kolb et al., 2005, in revision). One hundred forty-nine women participated in the baseline testing while 113 completed the entire study. Blood samples were collected both before and after 16 weeks of treatment with either an iron supplement (FeSO4) or a placebo (gelatin capsule). Cognitive testing was also conducted both at baseline and after 16 weeks of treatment. The cognitive testing consisted of 8 self-administered and automated com- puterized tasks of basic cognition (Detterman et al., 1990) measuring three do- mains: attention (three tasks), memory (three tasks), and learning (two tasks). These tasks were developed to measure the "modal model" of information pro- cessing which offers the opportunity to test specific aspects of cognition. This model is composed of three memory stores (very short term memory, short term memory, and long term memory) which are served by a stimulus encoding mechanism for input, a retrieval mechanism for output, and an output mecha- nism which executes responses. It also contains an executive functioning mecha- nism which oversees movement of information through the system. Analysis of data was conducted within each domain (memory, attention, and learning) as well as across all domains. Given the large number of variables for both the cognitive testing as well as the hematology measurements, factor analysis was used to reduce the number of variables and the probability of type 1 error. Factor analysis of the hematology variables resulted in four factors, storage, trans- port, pre-anemia, and anemia (Table B-18). Factor analysis of the cognitive vari- ables resulted in two factors, performance and time (Table B-19). While iron re- searchers have traditionally categorized subjects as iron sufficient, iron deficient, or iron deficient anemic, the authors of this study recognize that iron status is a continuous measure and, therefore, should be assessed as such. Thus, to perform the baseline analyses, data were sorted according to each hematological factor (storage, transport, pre-anemia, anemia) and then divided into quintiles. The ex- tremes of the distribution (upper versus lower quintile) were then compared. When data were sorted by the storage factor and analyzed across all cogni- tive domains, a significant difference between the upper and lower quintiles was found with respect to performance but not with respect to time. The opposite was found when the data were sorted by the anemia factor. That is, a difference was found in time needed to complete the tasks while there were no differences with respect to performance. Baseline data were then analyzed by cognitive domain (attention, memory, and learning) and quintiles of haematological factors. Sorting the data by the storage factor revealed a difference in performance for the attention, memory, and learning tasks. Although there was no difference on the time factor for the attention and learning domains, the amount of time needed to complete the memory tasks was significantly different between the upper and lower quintiles. In contrast, when the data were sorted by the anemia factor, those women in the

414 in txt. and iron stress, symbol all attention, Behavioral with on digit or learning acuity treatment, Results (Follow-up) effect depressive symptoms, and IDA normalized variables Iron verbal memory visual No Cognitive and in effect had ferritin Status correlated Hb and depressive overall with 2-fold depressive symptoms effect symptoms Iron Results (baseline) RPMI n/a Anemics No OCA No Between time digit acuity learning span Stress Measures EPDS RPMI Perceived Symbol Visual Verbal Attention Memory CESD GHQ Digit Reaction Attention MMPI Relationship the weeks weeks Length 28 8 on and Focusing partum partum males women post women adolescent girls post women females Group Size 95 81 37 297 365 Adults Young (60 in mg blinded blind; /d) 4 Fe/day) sectional sectional sectional esign placebo control mg placebo control (1,300 FeSO D Double Double Cross Cross Cross Studies B-17 al., al., al., 1994 et et et and and 2005 1996 2003 Benton, Penland, 1999 TABLE Functioning Author Beard Bruner Corwin Fordy Hunt

415 and with in 50 Ravens , < relation. = memory, errors a txt Questionnaire; RPMI status depression treatment. depression ferritin there if alth es; He iron learning, attention; speed iron placebo was 29% 13% Only General = contraceptiv and oral ID GHQ = memory, in ferritin different and depression Scale; OCA not learning IDA Attention, Non-anemic>anemic ID OCA n/a Depression Inventory; Scale. Postnatal scale Personality Anger Cognitive tasks CESD Detterman STAI STAS GHQ Fatigue Depression Anxiety Edinburgh = Multiphasic State-Trait = weeks EPDS weeks 16 4 STAS Scale; Minnesota = Inventory; MMPI Depression women women women Anxiety 149 255 144 anemia; Studies State-Trait ) deficiency 4 = iron/ (80 blind, iron mg sectional FeSO Epidemiological = STAI blinded placebo intervention (60 day) placebo mg for Stratified Cross Double IDA Index; Center = al., al., Matrices deficiency; Kolb 2005 et et CESD al., iron = et 1998 2003 Murray Rangan Verdon NOTE: ID Progressive

416 TABLE B-18 Components of Hematological Factors Factor Name Variables Loaded Storage sFt -sTfR -TfRIX* Body Iron Transport Iron, TfSat Pre-anemia MCV, MCH, MCHC, -RDW Anemia Hb, Hct, RBC NOTE: Hb = hemoglobin; Hct = hematocrit; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration; MCV = mean corpuscular volume; RBC = red blood cells; RDW = red cell distribution width; sFt = serum ferritin; sTfR = serum transferrin receptor; TfRIX = transferrin receptor index; TfSat = transferrin saturation. *Calculated as log(sTfR/sFt). TABLE B-19 Components of Cognitive Factors Domain Performance Factor* Time Factor Attention RT, SD, TT # incorrect RT, SD, TT trial time TT # attempted trials RT, SD, TT decision time RT, SD, TT movement time Memory PR, RC % correct PR, RC, ST trial time ST # incorrect RC, ST decision time RC, ST movement time PR, RC reaction time Learning LR # attempted trials LR trial time LR # blocks achieved LR, PM reaction time LR % correct PM # correct All Domains all of the variables listed above all of the variables listed above NOTE: LR = learning task; PM = progressive matrices task; PR = probed recall task; RC = recogni- tion memory task; RT = reaction time task; SD = stimulus discrimination task; ST = Sternberg memory search task; TT = tachistoscopic threshold task. *For those tasks measuring "negative" performance, the absolute values of the scores were used; therefore, a higher score on the performance factor is always indicative of better performance.

APPENDIX B 417 highest quintile completed the tasks in the attention domain significantly faster than those women in the lowest quintile and approached a significantly faster time for the memory as well as learning domains. On the other hand, performance on the attention, memory, and learning domains was not affected when data were sorted by the anemia factor. After the 16 weeks of supplementation, an improvement in overall perfor- mance on the cognitive tasks was found for those women who significantly improved their ferritin status. In women whose iron status improved, whether due to iron supplementation or other unknown reasons, attention and learning improved more than five fold compared to those whose iron status remained low. This improvement was seven times greater for the memory domain for those whose iron status improved. No differences were observed with respect to time necessary to complete the tasks in those women who improved their ferritin levels versus those who did not. Women who significantly improved their hemoglobin concentration over the 16 weeks of the study also had a significant improvement in processing speed in attention and memory tasks; this was not the case for those women who had no change in hemoglobin concentration. Importantly, no relationship was found with respect to "correctness" on the cognitive tasks and hemoglobin con- centration. The careful screening of subjects for the study identified the anemia as being due to iron deficiency and not other causes. OTHER STUDIES Several other pertinent observations exist that may explain possible biologi- cal mechanisms whereby iron deficiency in adults can alter cognitive and behav- ioral functioning. Variations in iron status, as reflected by variations in serum ferritin, are related to EEG asymmetry (Tucker et al., 1981, 1982, 1984). The authors demonstrated a strong relationship of activity recorded in occipital elec- trodes to variations in plasma ferritin, the studies were not conducted to establish specific relationships between brain regional activity and cognition and iron. The biochemical explanation for these alterations in electrical activity may lie in fundamental alterations in brain energy metabolism with brain iron deficiency (DeUngria et al., 2000) as well as in neurotransmission efficacy and degree of myelination alterations (Beard and Connor, 2003). There are a few examples that have used humans to study the relation ship of brain functioning and iron deficiency. For example, individuals with Restless Legs Syndrome (RLS) have alterations in brain iron content and also have clini- cal manifestions such as aphasic contractions of peripheral limb muscles. Known treatments are L-DOPA or iron relieve the symptoms and in the case of iron supplementation, may actually "cure" the disease in some patients (Earley et al., 2000). Another example of the association between iron status in adults and neural functioning is the treatment of renal dialysis patients with recombinant

418 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL human eytthropoietin and aggressive oral iron therapy; the standard clinical prac- tice in place now for more than a decade and a half. Studies that have examined cognitive performance after treatment often see 8­10 point improvements in the Wechsler global intelligence scores (Temple et al., 1992) and, significant im- provement in cerebral oxygen tension and brain oxygen extraction. This occurs when the hematocrit is moved from around 30 percent up to the normal 45 percent packed red cells and occurs with an associated change in EEG signals and improved attention (Metry et al., 1999; Pickett et al., 1999). However, many other biological factors are changing with the dialysis treatment and compari- sons with healthy age matched controls may provide some strong clues as to specific mechanisms whereby iron treatment of iron deficiency anemia results in improved cognitive performance and emotionality. CONCLUSION Although the few studies with military subjects do not show a clear associa- tion between iron status and cognitive function, the experiments with civilian women reviewed in this report strongly suggest that military personnel with poor iron nutritional status, usually women, may suffer from impaired performance, specifically in domains representing learning, memory, and attention tasks and that they may benefit from higher iron intakes. They may also have more depres- sion and anxiety in situations in which there is "high stress." The evidence data relative to the association between iron status and cognitive performance and mood states need to be collected from appropriate study designs, performed with military personnel under circumstances that paralled the physical, psychological and environmental stressors during military operations and training. REFERENCES Beard JL. 1995. One persons' view of iron deficiency, development, and cognitive function. Am J Clin Nutr 62:709­710. Beard JL, Connor JR. 2003. Iron status and neural functioning. Annual Rev Nutr 23:41­58. Beard JL, Hendricks MK, Perez EM, Murray-Kolb L, Berg A, Vernon-Feagans L, Irlam J, Isaacs W, Sive A, Tomlinson M. 2005. Effects of maternal iron deficiency anemia. Part 1: Maternal emotions and cognition in postpartum iron deficiency. J Nutr 135:267­272. Booth CK. 2003. Combat rations and military performance-do soldiers on active service eat enough? Asia Pac J Clin Nutr 12:S2. Booth CK, Coad RA, Forbes-Ewan CH, Thomson GF, Niro PJ. 2003. The physiological and psycho- logical effects of combat ration feeding during a 12-day training exercise in the tropics. Mil Medicine 168:63­70. Bruner AB, Joffe A, Duggan AK, Casella JF, Brandt J. 1996. Randomized study of cognitive effects of iron supplementation in non-anemic iron-deficient adolescent girls. Lancet 348:992­996. Cline A, Patton JF, Tharion WJ. 1998. Assessment of the relationship between iron status, dietary intake, performance, and mood state of female army officers in a basic training population. USARIEM Technical Report T98-24.

APPENDIX B 419 Corwin EJ, Murray-Kolb L, Beard JL. 2003. Low hemoglobin level is a risk factor for postpartum depression. J Nutr 133(12): 4139­4142. Detterman DK. 1990. CAT: Computerized Cognitive Abilities Tests for research and teaching. MicroPsychol Network 4:51­62. DeUngria M, Rao R, Wobken JD, Luciana M, Nelson CA, Georgieff MK. 2000. Perinatal iron deficiency decreases cytochrome c oxidase (CytOx) activity in selected regions of neonatal rat brain. Pediatr Res 48(2):169­176. Earley CJ, Allen RP, Beard JL, Connor JR. 2000. Insight into the pathophysiology of Restless Legs Syndrome. J Neuroscience Res 62(5):623­628. Fordy J, Benton D. 1994. Does low iron status influence psychological functioning? J Human Nutr Dietetics 7:127­133. Hunt JR, Penland JG. 1999. Iron status and depression in premenopausal women: An MMPI study. Behav Med. 25:61­68. Looker AC, Harris TB, Wahner HW. 1991. Comparing serum ferritin values from different popula- tion surveys. Vital Health Stat 2(111):1­19. Metry G, Wiksotrom B, Valind S, Sandhagen B, Linde T, Beshara B, Langstrom B, Danielson BG. 1999. Effect of normalization of hematocrit on brain circulation and metabolism in hemodialy- sis patients. J Am Soc Nephrol 10:854­863. Murray-Kolb LE, Beard JL, Joseph LJ, Davey SL, Evans WJ, Campbell WW. 2001. Resistance training affects iron status in older men and women. IJSN 11:287­298. Murray-Kolb L, Whitefield K, Beard JL. 2005. Iron status is related to cognitive functioning in young adult women. AmJ Clin Nutrition (in revision). Pickett JL, Theberge DC, Brown WS, Schweitzer SU, Nissenson AR. 1999. Normalizing hematocrit in dialysis patients improves brain function. Am J Kidney Dis 33:1122­1130. Rangan AM, Blight GD, Binns CW. 1998. Iron status and non-specific symptoms of female students. Am College Nutr 17: 351­355. Temple RM, Langan SJ, Deary IJ, Winney RJ. 1992. Recombinant EPO improves cognitive function in chronic haemodialysis patients. Mephrol Dial Transplant 7:240­245. Tucker DM, Sandstead HH. 1981. Spectral electroencephalographic correlates of iron status: Tired blood revisited. Physiol Behav 26:439­449. Tucker DM, Sandstead HH, Swenson RA, Sawler BG, Penland JG. 1982. Longitudinal study of brain function and depletion of iron stores in individual subjects. Physiol Behav 29:737­740. Tucker DM, Sandstead HH, Penland JG, Dawson SL, Milne DB. 1984. Iron status and brain func- tion: Serum ferritin levels associated with asymmetries of cortical electrophysiology and cogni- tive performance. Am J Clin Nutr 39:105­113. Verdon F, Furnand B, Fallab-Stubi CL, Bonaard C, Graff M, Michaud A. 2003. Iron supplementa- tion for unexplained fatigue in non-anemic women: Double blind randomized placebo con- trolled trial. Br Med J 326:1124­1128. Westphal KA, Friedl KE, Sharp MA. 1995. Health, performance, and nutritional status of U.S. Army women during basic combat training. USARIEM Technical Report T96-2. World Health Organization (WHO). 2002. The World Health Report, 2002. Reducing Risks, Promot- ing Healthy Life. [Online]. Available: http://www.who.int/whr/2002/en/whr02_en.pdf [accessed March, 2, 2006]. Youdim MBH, Ben-Schachar D, Yehuda S. 1989. Putative biological mechanisms on the effects of iron deficiency on brain metabolism. Am J Clin Nutr 50:607­617.

420 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Zinc and Other Mineral Nutrients Required for Cognitive Function and Behavior in Military Personnel James G. Penland USDA-ARS Grand Forks Human Nutrition Research Center, Grand Forks, North Dakota INTRODUCTION A recent review of energy requirements of military personnel (Tharion et al., 2005) indicates that energy requirements are higher on average for soldiers than civilians, but vary dramatically depending on factors such as the type of task (e.g., support, training, or combat), the physical environment (e.g., tempera- ture and altitude), and the presence of additional stressors (e.g., extreme physical activity, restricted sleep, or psychological stress). Energy requirements are also higher on average for male than female soldiers, although that difference largely disappears when adjusted for body size and physical activity. During field operations, soldiers may decrease food consumption up to 50 percent resulting in suboptimal intake of energy and micronutrients (Baker-Fulco, 1995; Shippee, 1993), including minerals such as zinc, magnesium, selenium, copper, and phosphorus. In addition to negative affects on immune function and physical performance, failure to meet energy requirements is known to impair psychological function and behavior in both military personnel and civilians (see below for a brief review of those findings). However, little is known about requirements for micronutrients, including minerals such as zinc, iron, and mag- nesium, and the impact of inadequate intakes on military performance. Data from civilians suggest that these nutrients are essential to optimal psychological function and behavior relevant to performance of military duties. Moreover, spe- cific groups of military personnel (e.g., male soldiers involved in first-strike assault operations) may have mineral requirements that exceed those of the ma- jority of military personnel. Repeated assault operations may result in mineral depletion because inadequate intake and increased turnover and losses of miner- als may promote marginal deficiencies under stressful conditions. Also, entry into operations with marginal mineral status may put these soldiers at risk for reduced mineral nutritional status and result in impaired physiological and psy- chological function and performance, particularly when assaults occur repeti- tively without replenishment of depleted mineral reserves. This paper describes the research studies conducted on animals, the civilian population, and military personnel that shed light on questions regarding mineral requirements for military personnel. In particular, the following specific ques- tions have been addressed:

APPENDIX B 421 · What is the evidence that zinc intake and status is related to cognitive function and behavior of soldiers? Does varying zinc intake and status affect the ability of soldiers to perform mental tasks? · What is the evidence that zinc intake and status is related to cognitive function and behavior of civilians? What are the effects of zinc deficiency in cognitive function and behavior? · What are the likely mechanisms of action for zinc effects on cognitive function and behavior? What brain regions are excited or depressed in associa- tion with changes in zinc intake and status? · What other minerals interact with zinc and what is the effect of the inter- action on their homeostasis and roles in neural function? · Does supplementation with zinc affect cognitive function or behavior? Will supplementation with zinc restore or improve mental performance of sol- diers with impairments in cognitive function due to sleep deprivation, energy deficits, physical activity, or other stressors? If so, at what level and for how long is supplementation needed? · What is the evidence that magnesium, selenium, copper, and phosphorus intake or status are related to behavior and cognitive function? Unfortunately, studies with military personnel that would answer to these questions are scarce. Therefore, this paper responds to these questions with avail- able relevant data from studies with civilian adults with supporting evidence from animal studies or studies in children. This paper focuses on zinc, magne- sium, selenium, copper, and phosphorus. The important role of iron nutrition in cognitive function and behavior is discussed elsewhere in this Appendix (Beard, 2005). Although there has been no direct examination of the possible role of specific mineral nutrients in cognitive performance and mood states of soldiers during training and field operations, the effects of caloric restriction, alone and in combination with other stressors, has been studied and a summary of the conclusions is also presented here. The studies presented make evident some of the current limitations of the data collected. For instance, blood biochemical markers of nutritional status do not reveal overt mineral deficiencies among military personnel but this may be explained by a lack of sensitive biochemical markers of mineral status, brief durations of restricted intakes, and mineral mobilization from stores into the blood with increased metabolic demands and loss of body weight. Another major limitation is the need to extrapolate results of mineral nutri- tion studies with civilians to military personnel; that soldiers are subject to mul- tiple and often severe stressors in many different domains (e.g., physical fatigue, extreme environmental conditions, sleep deprivation, food restriction, depres- sion, anxiety, fear). Data from civilian studies that evaluate mineral effects on cognitive function and behavior under conditions of multiple demands (e.g., dual-tasks) and stressors would be most relevant to the military.

422 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL THE EFFECT OF STRESSORS ON COGNITIVE FUNCTION AND BEHAVIOR OF MILITARY PERSONNEL As noted in previous reports from the Committee for Military Nutrition Re- search (e.g., Mays, 1995), underconsumption of military rations for short periods (10­45 days) in the absence of significant environmental, physical, and mental stress reliably reduces weight (up to 6 percent) but produces no meaningful degra- dation of cognitive performance as measured by multiple methods. Moderate lev- els of underconsumption may actually enhance performance. Underconsumption by 50 percent or more may result in subjective reports of impaired concentration and memory, along with non-cognitive behavioral (i.e., mood) changes. When severe caloric restriction is combined with additional stressors, including increased physical exercise, sleep deprivation, and mental stress, cognitive performance may fall by as much as 35 percent within a few days (Shippee et al., 1994). A controlled study (Booth et al., 2003) of the biochemical, physiological and psychological effects of caloric restriction during a 12-day field training exercise in Australian soldiers found weight loss, suppressed immune function (IgA and IL-2R), a decline in iron status (ferritin), dehydration, impaired sleep, increased fatigue, reduced vigor, and increased feelings of confusion in soldiers, regardless of the diet (i.e., diets provided either negative or zero energy balance). However, there was no meaningful impact on physical fitness or cognitive perfor- mance (reaction time, vigilance, perception, and memory). In contrast, Lieberman et al. (2005) recently showed that the combined stressors of sleep deprivation, physical activity, psychological stress, noise, hot and humid temperatures, dehydration, and general undernutrition during a 53-hour Army simulated com- bat exercise led to severe declines in cognitive performance on tasks measuring attention, memory and reasoning, and to deterioration in mood states, including increased confusion, anxiety, depression, and fatigue. Longer periods of exposure to multiple stressors, nutritional, environmental, physical and mental, such as the 60+ days involved in Ranger training, clearly result in impaired cognitive performance, including attention (decoding), per- ception, memory, and reasoning (Mays, 1993). Shippee et al. (1994) found de- creased iron status and increased zinc and copper status in Ranger II; however, dietary intakes were not measured so it is unclear whether the changes were due to decreased intake, inflammation, or some other factor. Pre-existing marginal nutritional status, rest, psychological state, etc., also likely exacerbate the impact of underconsumption, alone or in combination with other stressors typical of field training and combat operations. At least one study (Crowdy et al., 1982) found no significant decrements in cognitive performance (vigilance, math, and coding) when soldiers consumed 47 percent energy expenditure during 12 days of rigorous training exercises in the Tropics. It is possible that relatively brief periods of caloric restriction, similar to sleep restriction and deprivation, do not degrade performance because soldiers

APPENDIX B 423 may sacrifice speed to maintain performance accuracy (i.e., speed-accuracy trade- off) (Belenky et al., 1994; Mays, 1993). EFFECTS OF MINERAL NUTRITION ON COGNITIVE FUNCTION AND BEHAVIOR OF CIVILIANS Controlled studies with civilian adult men and women are not extensive but provide important clues about how specific mineral nutrients may affect cogni- tive performance and mood states of military personnel. Zinc Zinc is required for the structure and activity of more than 300 enzymes (Vallee and Falchuk, 1993). Frederickson et al. (2005) provide a comprehensive discussion of the broad role of zinc in central nervous system function indicating several potential mechanisms linking zinc and cognitive function and behavior. Zinc is concentrated in the cortex and limbic system (especially the hippocam- pus and amygdala). Zinc is contained in glutamatergic neurons, active in the vesicle, synaptic cleft, and post-synaptic neuron. Zinc modulates brain excitabil- ity primarily through its effects on both excitatory and inhibitory receptors (e.g., N-methyl-D-aspartate [NMDA] or, -aminobutyric acid [GABA] receptors). Zinc may also affect cognition and behavior through non-CNS (central nervous sys- tem) actions (Golub et al., 1995). These include involvement in neurotransmitter precursor production in the liver, hormone and growth factor transport and re- ceptor binding, hormone and toxicant metabolism in the liver and testes, and pancreatic insulin production and glucose metabolism. Dreosti (1993) has sug- gested that zinc-deficiency effects on behavior and brain function may result from impaired activity of several zinc-dependent enzymes in the brain. Because zinc functions in all physiological systems, adequate zinc status is essential for optimal physiological and psychological performance. The majority of studies relating zinc intake or status to cognitive function and behavior have been conducted in infants and children. In these groups, im- proved zinc nutrition has consistently benefited psychomotor skills, but has in- consistently affected attention, memory, reasoning, and psychosocial adjustment. In civilian adults, low zinc intakes and status have been related to deficits in memory, perception, attention, and motor skills while zinc supplementation has improved memory (Table B-20). Clinical evaluation of mental status revealed impaired short-term memory, increased emotional lability, and perceptual defi- cits in adults with progressive systemic sclerosis whose zinc status was altered by histidine administration (Henkin et al., 1975). Goldstein and Pfeiffer (1978) administered zinc or a placebo to schizophrenics and assessed brain function by electroencephalogram (EEG) 4 hours after supplementation. There was a greater reduction in EEG amplitude (toward normal) in zinc-supplemented schizo-

424 1996 1984 1991 1995 Adults 1991 2000 2000 2002 1992 2005 2000 performance. Sandstead, Sandstead, Hornbostel, Finley, al., al., al., al., Cook, al., al., 1991 Civilian and et et and et et 1995 and and and et 1988 et in cognitive nda Reference Tucker Penland, Kretsch Penland Darnell Penland Delorme Penland, Benton Hawkes Penland Penland Penland, Penland Behavior nutrition iron and of Mood Latency studies. Disturbance Attention Reasoning Function Memory Energy Sleep Memory Depression Confusion Perception discussion Activity Activity, Depression Depression Positive Total a Activity Memory Restless for a Alpha Total Se, Time, supplementation Theta in Cognitive Memory, Psychomotor, Perception, Memory Memory Perception Psychomotor EEG EEG % Anxiety, Energy RBC Hostility Confusion, GSH-Px, Hostility, Confidence, Sleep Feeling Depression, Short-term Distraction volume Outcome on this placebo in to Beard by Nutrients compared mg/day mg/day b b b µg/day µg/day µg/day paper mg/day 10 mg/day mg/day 315 Mineral Design 356 239 mg/day mg/day 15 14 53 vs. vs. mg/day mg/day µg/day 10­40 3 3 supplement vs. vs. vs. vs. vs. vs. vs. vs. workshop or Study 5 1­4 5 3 30 30 Hypomagnesmia 113 100 13 28 0 1 1 Other the to intake condition. and Refer dietary Zinc placebo Women of details. higher and to included Subjects Men Men Men Women Women Women Men Women Women Men Men Women Women Women Effects additional study, for compared B-20 text lower See of TABLE Mineral Zinc Magnesium Selenium Copper NOTE: Effect Supplementation a b

APPENDIX B 425 phrenics compared to controls, but both groups showed a decrease in EEG activ- ity. Henrotte et al. (1977) found a relationship between low red blood cell (RBC) zinc and photic responsivity, and determined that people with a Type A person- ality are characterized by a higher RBC and urinary zinc than those with Type B personality (Henrotte et al., 1985; Henrotte et al., 1986). Humphries et al. (1989) reported biochemical indices of zinc deficiency in 54 percent of a group of anorexic patients and 40 percent of a group of bulimic patients. Unfortunately, neither of these last two studies reported dietary zinc intakes so it is impossible to draw useful conclusions about associations between zinc intake level and cognitive function from those studies. In healthy men participating in a con- trolled feeding study, Sandstead et al. (1983) found a correlation between plasma zinc and brain electrical activity in three of five subjects; the principal finding was increased left and decreased right hemisphere amplitudes in the occipital lobes in the lower frequencies of the EEG. The meaning of these changes in brain electrophysiology for cognitive function remains unknown. In a zinc depletion experiment with healthy young males, Tucker and Sandstead (1984) found that low serum zinc in these otherwise well-nourished men was correlated with faster but less accurate performance on memory for digits and several perceptual tasks. Darnell and Sandstead (1991) found that 30 mg/day zinc added to a vitamin-mineral supplement improved visual memory, but not verbal memory, in sideropenic women, whereas a vitamin-mineral supplement alone did not. Penland (1991) administered a battery of tasks assessing cognitive processes and psychomotor skills to 14 healthy men, aged 21­38 years, participating in a 6-month, live-in metabolic study of zinc nutrition. The men were fed 1, 2, 3, or 4 mg/day zinc during each of four consecutive 35-day deprivation periods adminis- tered in a random, double blind manner. Contrasted to a control period when the men were fed 10 mg/day zinc, low zinc intakes were associated with poorer perfor- mance on at least one task from each of five functional categories; those categories were, psychomotor function (tracking and connect-the-dots tasks), attention (ori- enting and misdirection tasks), perception (search-count task), memory (letter, shape and cube recognition tasks), and spatial function (maze task). However, a dose-response effect of dietary zinc on performance was not observed. Penland et al. (2002) also found improved performance on memory and reasoning tasks in adult women supplemented with 30 mg/d zinc for 8 weeks. In a double-blind metabolic study of 8 healthy young men (Kretsch et al., 2000), reaction times during word recall were significantly slower when men were fed 4.6 compared to 13.7 mg/day zinc for 70 days. In a double-blind metabolic study of 23 healthy postmenopausal women (Penland et al., 2000), immediate recall of word lists was improved when women were fed a total of 53 compared to 3 mg/day zinc for 90 days, when the diet was also low (< 1 mg/day) in copper. In the only intervention study of elderly with dementia, Potocnik et al. (1997) found "modest" improve- ments on the Mini Mental State Examination in 4 Alzheimer's patients following supplementation with 30 mg/day zinc for 12 months.

426 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Zinc may play a role in the regulation of mood states, particularly depres- sion. Henkin et al. (1975) reported irritability, anger, paranoia and depression in adults made severely zinc deficient by histidine administration (histidine binds zinc normally bound to albumine and depletes tissue zinc levels). Aggett (1989) reported that loss of affect and emotional lability were pronounced in children with acrodermatitis enteropathica and in early zinc deficiency of TPN (total parenteral nutrition) patients. See also Levenson 2005 in this appendix for a detailed review of zinc and depression. Nowak and Szewczyk (2002) provide a recent discussion of possible mechanisms linking zinc and depression. There have been no studies in soldiers or civilians that address whether the effects of stressors on cognition and behavior can be mitigated by varying zinc intakes. There is only one study with rats that points to increased stress re- sponses (corticosterone and anxiety) with zinc deficiency (Chu et al., 2003). There are animal studies that suggest that zinc plays a role in regulating appetite and food consumption; however, studies looking at the possible role of zinc in regulating human food behavior find highly complex food behavior pat- terns, and the role of zinc is still unclear (Shay and Mangian, 2000). Rats begin to reduce their food intake within 3­5 days of severe (< 1 ppm/ day) zinc deprivation and continued deprivation may result in intakes 50 percent that of zinc-adequate controls (Rains and Shay, 1995). Zinc repletion increases intake almost immediately. Most of the reduced intake is in carbohydrates and the pattern of feeding (timing and number of meals) is disrupted (Rains et al., 1998). Both neuropeptide Y release (Levenson, 2003) and leptin (Mantzoros et al., 1998) are reduced during zinc deficiency and are associated with decreased intake and anorexia nervosa. As noted earlier, Humphries et al. (1989) reported biochemical indices of zinc deficiency in 54 percent of a group of anorexic patients and 40 percent of a group of bulimic patients. In summary, data from the few available studies with civilian adults suggest- ing that zinc nutrition may have roles in cognitive function (particularly memory) and mood (particularly depression) is supported also by studies with animals, in- fants and children, and the existing putative mechanisms. At this time however, data are insufficient to identify specific zinc intakes needed to support and maxi- mize cognitive function and behavior in either civilians or military personnel. Magnesium Magnesium is a cofactor in more than 300 enzymes and influences DNA and protein synthesis, intracellular signal transduction, and cell growth and dif- ferentiation (Shils, 1997). Magnesium, thus, is a potentially limiting nutrient for cognitive function and behavior. In the central nervous system, magnesium plays an important role in glutamatergic neurotransmission, inhibiting excitatory NMDA (Cooper et al., 1996), and affects monoaminergic and serotonergic sys- tems (Singewald et al., 2004). Magnesium is also involved in regulation of the

APPENDIX B 427 hypothalmus-pituitary-adrenocortical (HPA) system and corticotropin releasing factor (Murck, 2002). The role of magnesium as an NMDA antagonist and GABA agonist is a likely mechanism responsible for the effects of magnesium on sleep (Held et al., 2002). The relationships between magnesium and mood are linked to increased HPA activity, which is frequently observed in depression and anxiety (Holsboer, 2000). There are no previous studies that have directly correlated the intake or status of magnesium to cognitive function or behavior in soldiers, and few data exist from studies of civilians. Severe magnesium deficiency has been associated with numerous neuro- logical and psychological problems, including convulsions, dizziness, neuromus- cular hyperexcitability (Chvostek and Trousseau signs), hyperemotionality (irri- tability and marked agitation), anxiety, confusion, depression, apathy, loss of appetite, and insomnia (Dubray and Rayssiguier, 1997; Durlach, 1980). Brain function assessed with the EEG has shown increased cortical excitability, char- acterized as diffuse, slow wave activity of the type commonly found in meta- bolic disorders, and "diffuse irritative tracings" in the absence of focal effects, marked by spiked alpha and increased theta activity (Durlach, 1985). Popoviciu et al. (1987) reported a disruption of normal sleep architecture in magnesium- deficient subjects, including greatly reduced deep, slow wave sleep, and de- creased rapid eye movement sleep. However, there are few data on neuropsychological effects of marginal mag- nesium restriction (Table B-20). In an early study that successfully induced magnesium deficiency by dietary restriction, Shils (1969) did not observe any changes in the EEG of subjects fed less than 10 mg/day magnesium for as long as 105 days. However, Shils' study was limited to 7 subjects and EEG analysis was visual rather than quantitative. Contrasting quantitative EEG of athletes (44 male and female kayakists) with low versus normal erythrocyte magnesium, Delorme et al. (1992) found significantly less relative alpha (7.25­12.5 Hz) activity, particularly in the right occipital region, in the low magnesium group. However, magnesium intakes were not experimentally controlled in that study. Penland (1995) fed either 115 or 315 mg/day magnesium for 42 days each to 13 healthy postmenopausal women living on a metabolic research unit; EEG activity (i.e., hyperexcitability) increased, suggesting that relatively short periods of marginal magnesium deprivation can affect brain function. Compared to high dietary magnesium, the low magnesium intake increased total EEG activity in the frontal regions and right temporal and parietal regions, and resulted in frequency- specific increases in left occipital delta (1­3 Hz) activity, theta (4­7 Hz) activity in all but the left temporal region, alpha (8­12 Hz) activity in the right frontal and right temporal regions, and beta (13­18 Hz) activity in the frontal regions. The proportion of theta to total activity in the parietal regions also increased with the low magnesium intake. Increased electrical activity across frequencies and increased theta activity have been associated with neurological disorders,

428 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL behavioral hyperactivity, increasing sleep loss, and impaired cognition, espe- cially memory. Magnesium deficiency leads to reduced offensive and increased defensive behavior in rats (Kantak, 1988) and impaired learning and memory in mice (Bardgett et al., 2005), but there have been no studies showing these effects in humans. Magnesium deficiency in rats also leads to increased pain sensitivity (Begon et al., 2001). Again, this effect has not been investigated in humans. Magnesium may play an important role in regulating sleep. Animal studies have shown that magnesium deficiency increases wakefulness and decreases slow wave sleep (Depoortere et al., 1993) and total sleep time (Poenaru et al., 1984). Intravenous magnesium administration in healthy young men increased EEG activity power in sigma frequencies (11­29 Hz) during non-REM sleep (Murck and Steiger, 1998). Magnesium supplementation (10­30 mmol/day [240 mg/day]) of elderly (60­80 years old) increased EEG power in the delta (1­3 Hz) and sigma frequencies (Held et al., 2002). A recent study found that sleep restriction over a 4 week period resulted in decreased intracellular magnesium in college males (Takase et al., 2004). Magnesium may also be involved in regulating mood states. Many correla- tional studies have shown a positive association between blood magnesium con- centrations and mood disorders, although a few have found either no association or a negative relationship (Imada et al., 2002). However, supplemental and intra- venous magnesium have been effective in treating depression (Murck, 2002). In summary, the importance of magnesium for cognitive function and be- havior has received little attention and is largely unknown. Available data sug- gest that magnesium is involved in regulating brain electrical activity and that increasing intake may benefit sleep modulation and mood states. Selenium Selenium acts through its association with proteins as an antioxidant and a regulator of thyroid hormone metabolism. Selenium-dependent enzymes in the brain are involved in antioxidant defense and redox regulation which may be relevant for neurodegenerative diseases caused by oxidative stress (Schweizer et al., 2004), but a role for selenium in cognitive function and behavior of younger adults has not been investigated. In individuals with low selenium intakes and status who have impaired deiodinase synthesis and activity, reduced conversion of thyroxine (T4) to triiodothyronine (T3) may result in sub-clinical thyroid hor- mone deficiency that impairs mood states (Beckett et al., 1993; Sait-Gonen et al., 2004). In individuals with adequate selenium intake and status, selenium may affect brain function and impair mood states by increasing dopamine turnover (Castano et al., 1997) or increasing the ratio of n-6/n-3 fatty acids (Yao and Reddy, 2005). Low glutathione peroxidase (GSH-Px) activity in women has been associated with elevated fasting glucose and glucose intolerance (Hawkes

APPENDIX B 429 et al., 2004); elevated glucose and glucose intolerance have been associated with depression. There are no previous studies that have directly correlated the intake or status of selenium to cognitive function or behavior in soldiers. However, sev- eral studies have shown an effect of selenium intakes and status on mood states in civilian men and women (Table B-20). Benton and Cook (1991) supplemented 33 women and 17 men with 100 µg/day selenium or a placebo for 5 weeks, in a double-blind crossover design with a 6-month washout period between treat- ments. Higher selenium intakes were related to less anxiety, less depression and more energy as reported on the Profile of Mood States--BiPolar Form (POMS- BI). Penland and Finley (1995) and Finley and Penland (1998) fed 30 healthy men, aged 21­44 years, typical Western diets containing 30 or 230 µg/day sele- nium for 15 week. Men fed high selenium reported less confusion and depres- sion on the POMS-BI over the course of the study. Anxiety, hostility, uncer- tainty and tiredness also appeared to be less with higher selenium intakes, but subject variability was large and the effects were not statistically significant. Within the group fed low selenium, the activity of the selenium enxyme GSH-Px in platelets was significantly correlated with all six mood states measured by the POMS-BI; higher activity was associated with less anxiety, hostility, depression, uncertainty, tiredness, and confusion. Hawkes and Hornbostel (1996) fed 11 healthy men living on a metabolic research unit either 13 or 356 µg/day of selenium for 99 days. Although selenium intakes were not significantly related to mood states by the POMS-BI, a significant positive relationship was found between erythrocyte selenium concentrations and elated (versus depressed) and agreeable (versus hostile) mood states in the group fed low selenium; this find- ing suggests a greater range in mood and selenium status in the low selenium group. Several recent studies show conflicting results. New Zealand adults, 33 fe- males and 18 males, with typically low selenium intakes were supplemented with 0, 10, 20, 30 or 40 µg/day selenium for approximately 6 months (Penland et al., 2005). Monthly POMS-BI tests showed the female group increased agreeableness, confidence and energy, and less total mood disturbance as the study progressed (slope analysis). Males showed no dietary effects on mood states, which may be due to the low statistical power of the small sample size for males. In another study, 60 Chinese men (aged 18­49 years) with low selenium status, baseline plasma selenium concentration was negatively associated with anxiety, depres- sion, tiredness, confusion, and with total mood disturbance (Penland et al., 2006). Plasma selenium concentration was also positively associated with performance on two perceptual tasks, search and matching. However, subsequent food fortifica- tion providing 200 µg/day selenium for 15 weeks markedly improved selenium status but did not improve mood or cognitive performance. In another study (Rayman et al., 2005), supplementation with 100­300 µg/day selenium for 6 months resulted in no significant improvement in mood states; however, subjects

430 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL in that study were between 60­74 years of age, much older than participants in the studies showing a benefit of increased selenium intake for mood. In summary, the importance of selenium for cognitive function and behavior has received little attention and is largely unknown. The limited available data suggest that selenium status is associated with mood states and that increasing selenium intake may benefit mood (Rayman, 2002), but such interventions have not always been effective (Raymon et al., 2005; Shor-Posner et al., 2003). Copper Copper impacts biological function as a catalyst of enzyme activity. It regu- lates iron absorption, neurotransmitter metabolism, antioxidant defense and oxy- gen utilization. Thus, copper status may affect diverse biological functions, in- cluding cognitive function and behavior. The activties of two copper-dependent enzymes may at least partially explain the diverse putative effects of copper intake on memory, mood, and sleep. Dopamine--monooxygenase is required for the synthesis of norepinephrine from dopamine, and Cu/Zn superoxide dismutase protects catecholamines from oxidation by reactive oxygen species (Johnson, 2005). Long-term alterations of the synaptic strength, gene transcrip- tion modulation and other processes are modulated by norepinephrine which supports a role for this neurotransmitter in alterations of neural function and behavior (Berridge and Waterhouse, 2003). Through its role in several enzy- matic functions, adequate intake of copper appears to be important for fully functional cognition and behavior. There are no previous studies that have directly correlated the intake or status of copper to cognitive function or behavior in soldiers, and only two studies from one laboratory have been conducted with civilians. Restricted dietary copper has been associated with impaired verbal memory and disrupted sleep and mood states of women (Table B-20). In a double-blind, metabolic study of 23 healthy postmenopausal women (Penland et al., 2000), short-term memory and immediate recall of a list of words presented verbally worsened when women were fed low compared to high copper (1 versus 3 mg/ day), when the diet was also high (53 mg/day) in zinc. Low copper intakes were also associated with increased difficulty in discriminating between relevant and irrelevant responses. Plasma copper and ceruloplasmin were positively associ- ated with improved verbal memory and long-term memory, and increased clus- tering of verbal material (which indicates improved strategy), but fewer intru- sions (reduced distractions) during recall (Penland et al., 2000). In a depletion-repletion experiment (Penland, 1988; 1989), increased sleep times, longer latency to sleep, and feeling less rested upon awakening as well as increased confusion, depression, and total mood disturbances were reported when dietary copper was low (0.8 versus 2 mg/day). Review of the medical charts of participants in these long-term, live-in metabolic studies for the incidence of

APPENDIX B 431 requests for medication to relieve pain unrelated to injury or illness found in- creased requests for pain medication during periods of low copper intake (un- published data). In summary, the importance of copper for cognitive function and behavior has received little attention and is largely unknown. Two studies in one labora- tory suggest that copper nutrition may affect sleep and memory performance. Phosphorus Inadequate phosphorus intake results in abnormally low serum phosphate levels (hypophosphatemia), resulting in loss of appetite, anemia, muscle weak- ness, bone pain, osteomalacia, increased susceptibility to infection, numbness and tingling of the extremities, and difficulty walking. There are no controlled studies that have directly correlated the intake or status of phosphorus to cogni- tive function or behavior in soldiers or civilians. SUMMARY There are no previous studies relating zinc intake or status to cognitive function or behavior in soldiers. Data from the few available studies with civil- ian adults suggest that zinc nutrition may have roles in cognitive function (par- ticularly memory) and mood (particularly depression). This conclusion is sup- ported by data from studies with animals, infants and children, and reasonable putative mechanisms can be identified. At this time however, data are insuffi- cient to identify specific zinc intakes needed to support and maximize cognitive function and behavior in either civilians or military personnel. The importance of magnesium, selenium, copper, and phosphorus for cogni- tive function and behavior has received little attention and is largely unknown. Available data suggest that magnesium is involved in regulating brain electrical activity and possibly sleep and mood. Increasing selenium intake may benefit mood. Two studies in one laboratory suggest that copper nutrition may affect sleep and memory performance. There are no data to suggest any putative role for phosphorus in cognitive function or behavior. Evidence shows that military personnel fail to consume adequate amounts of zinc and magnesium. These minerals, as well as selenium, copper, and phos- phorus may play important roles in promoting optimal cognitive function and behavior. Limited data on mineral intakes and status of soldiers in various types of training do not provide evidence of overt nutritional deficiencies, but this may be due to a lack of sensitive biochemical markers of nutritional status. It is difficult to discriminate the independent effect of severely restricted energy in- take on potential micronutrient impairments. Nevertheless, cognitive and psy- chological impairments found in civilians with marginal mineral deficits are

432 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL consistent with problems in these areas reported in soldiers during active training and operations. RESEARCH RECOMMENDATIONS There is a need to conduct studies of zinc nutrition and cognitive function and behavior with military personnel in there garrison and in the field, and while engaged in support, training and combat, in the context of moderating variables. These variables include gender, body composition and fitness, task, physical demand, extreme environmental conditions, sleep deprivation, food restriction, and psychological stressors (e.g., depression, anxiety, fear). A direct examina- tion of the role of zinc nutrition in moderating biochemical and physiological responses to stressors of varying types is needed. Further there is a need to determine the possible role of zinc nutrition in regulating the food intake of soldiers. There is also a need to determine the benefits of increasing magnesium, selenium, copper, and phosphorus intakes for cognitive function and behavior in the context of moderating variables affecting military personnel. In particular, there is a need to determine whether increasing magnesium intake will improve sleep, protect against the effects of sleep deprivation, and regulate mood, and whether increasing selenium intake will benefit mood. Exploratory studies on the possible benefits of copper and phosphorus for cognitive function and behav- ior may be undertaken with a lower priority. This information is needed to critically evaluate the adequacy of current rations provided to and consumed by soldiers, and if indicated will provide a foundation for developing new rations that supply specific mineral nutrients re- quired to support optimal cognitive function and behavior of military personnel operating in the presence of multiple demands and stressors with diverse needs. Such information may also be useful to better understanding the determinants of food intake by soldiers (Hirsch and Kramer, 1993). REFERENCES Aggett PJ. 1989. Severe zinc deficiency. In: Mills CF, ed. Zinc in Human Biology. New York: Springer-Verlag. Pp. 259­279. Baker-Fulco CJ. 1995. Overview of dietary intakes during military exercises. In: Marriott BM, ed. Not Eating Enough: Overcoming Underconsumption of Military Operational Rations. Wash- ington, DC: National Academy Press. Pp. 121­149. Bardgett ME, Schultheis PJ, McGill DL, Richmond RE, Wagge JR. 2005. Magnesium deficiency impairs fear conditioning in mice. Brain Res 1038(1):100­106. Beckett GJ, Nicol F, Rae PW, Beech S, Guo Y, Arthur JR. 1993. Effects of combined iodine and selenium deficiency on thyroid hormone metabolism in rats. Am J Clin Nutr 57(2 Suppl):240S­ 243S.

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434 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Hawkes WC, Alkan Z, Lang K, King JC. 2004. Plasma selenium decrease during pregnancy is associated with glucose intolerance. Biol Trace Elem Res 100(1):19­29. Held K, Antonijevic IA, Kunzel H, Uhr M, Wetter TC, Golly IC, Steiger A, Murck H. 2002. Oral Mg(2+) supplementation reverses age-related neuroendocrine and sleep EEG changes in hu- mans. Pharmacopsychiatry 35(4):135­143. Henkin RI, Patten BM, Re PK, Bronzert DA. 1975. A syndrome of acute zinc loss: cerebellar dysfunction, mental changes, anorexia, and taste and smell dysfunction. Arch Neurol 32(11): 745­751. Henrotte JG. 1986. Type A behavior and magnesium metabolism. Magnesium 5(3­4):201­210. Henrotte JG, Veadreaux G, Manod H. 1977. Relations entre re taux du zinc and guin et les modifica- tions de le EEG sous le influence de le nyerpnee chez le homme narmole. [Relationship be- tween blood zinc level and EEG changes under the influence of hyperpnea in normal subjects]. L-Encephale 3(2):159­164. Henrotte JG, Plouin PF, Levy-Leboyer C, Moser G, Sidoroff-Girault N, Franck G, Santarromana M, Pineau M.1985. Blood and urinary magnesium, zinc, calcium, free fatty acids and catechola- mines in type A and type B subjects. J Am Coll Nutr 4(2):165­172. Hirsch ES, Kramer FM. 1993. Situational influences on food intake. In: Marriott BM, ed. Nutritional Needs in Hot Environments: Applications for Military Personnel in Field Operations. Wash- ington, DC: National Academy Press. Pp. 215­243. Holsboer F. 2000. The corticosteroid receptor hypothesis of depression. Neuropsychopharmacology 23(5):477­501. Humphries L, Vivian B, Stuart M, McClain CJ. 1989. Zinc deficiency and eating disorders. J Clin Psychiatry 50(12):456­459. Imada Y, Yoshioka S, Ueda T, Katayama S, Kuno Y, Kawahara R. 2002. Relationships between serum magnesium levels and clinical background factors in patients with mood disorders. Psychiatry Clin Neurosci 56(5):509­514. Johnson WT. 2005. Copper and brain function. In: Lieberman HR, Kanarek RB, Prasad C, eds. Nutritional Neuroscience. Boca Raton, FL: Taylor & Francis. Pp. 289­305. Kantak KM. 1988. Magnesium deficiency alters aggressive behavior and catecholamine function. Behav Neurosci 102(2):304­311. Kretsch MJ, Fong, AKH, Penland JG, Sutherland B, King JC. 2000. Cognitive effects of adaptation to a low zinc diet in healthy men. In: Roussel AM, Favier AE, Anderson RA, eds. Trace elements in man and animals--10: Proceedings of the Tenth International Symposium on Trace Elements in Man and Animals. New York: Kluwer Academic/Plenum Publishers. Pp. 999­1001. Levenson CW. 2003. Zinc regulation of food intake: New insights on the role of neuropeptide Y. Nutr Rev 61(7):247­249. Lieberman HR, Bathalon GP, Falco CM, Kramer FM, Morgan CA, Niro P. 2005. Severe decrements in cognitive function and mood induced by sleep loss, heat, dehydration, and undernutrition during simulated combat. Biol Psychiatry 57(4):422­429. Mantzoros CS, Prasad AS, Beck FW, Grabowski S, Kaplan J, Adair C, Brewer GJ. 1998. Zinc may regulate serum leptin concentrations in humans. J Am Coll Nutr 17(3):270­275. Mays MZ. 1993. Cognitive function in a sustained multi-stressor environment. In: Marriott BM, ed. Review of the Results of Nutritional Intervention, U.S. Army Ranger Training Class 11/92 (Ranger II). Washington, DC: National Academy Press. Pp. 199­214. Mays MZ. 1995. Impact of underconsumption on cognitive performance. In: Marriott BM, ed. Not Eating Enough: Overcoming Underconsumption of Military Operational Rations. Washington, DC: National Academy Press. Pp. 285­302. Murck H. 2002. Magnesium and affective disorders. Nutr Neursci 5(6):375­389. Murck H, Steiger A. 1998. Mg2+ reduces ACTH secretion and enhances spindle power without changing delta power during sleep in men--possible therapeutic implications. Psychopharma- cology [Berlin] 137(3):247­252.

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436 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Shils ME. 1997. Magnesium. In: O'Dell BL, Sunde RA, eds. Handbook of Nutritionally Essential Mineral Elements. New York: Marcel Dekker. Pp. 117­152. Shippee RL. 1993. Nutritional status and immune function of Ranger trainees given increased caloric intake. In: Marriott BM, ed. Review of the Results of Nutritional Intervention, U.S. Army Ranger Training Class 11/92 (Ranger II). Washington, DC: National Academy Press. Pp. 86­104. Shippee R, Friedl K, Kramer T, Mays M, Popp K, Askew EW, Fairbrother B, Hoyt R, Vogel J, Marchitelli L, Frykman P, Martinez-Lopez L, Bernton E, Kramer M, Tulley R, Rood J, Delaney J, Jezior D, Arsenault J, Nindl B, Galloway R, Hoover D. 1994. Nutritional and Immunological Assessment of Ranger Students with Increased Caloric Intake. Technical Report T95-5. Natick, MA: U.S. Army Research Institute of Environmental Medicine. Shor-Posner G, Lecusay R, Miguez MJ, MorenoBlack G, Zhang G, Rodriguez N. 2003. Psychologi- cal burden in the era of HAART: Impact of selenium therapy. Intl J Psychiatry Med 33:55­69. Singewald N, Sinner C, Hetzenauer A, Sartori SB, Murck H. 2004. Magnesium-deficient diet alters depression- and anxiety-related behavior in mice­influence of desipramine and Hypericum perforatum extract. Neuropharmacology 47(8):1189­97. Takase B, Akima T, Satomura K, Ohsuzu F, Mastui T, Ishihara M, Kurita A. 2004. Effects of chronic sleep deprivation on autonomic activity by examining heart rate variability, plasma catechola- mine, and intracellular magnesium levels. Biomed Pharmacother 58(1 Suppl):S35­S39. Tharion WJ, Lieberman HR, Montain SJ, Young AJ, Baker-Fulco CJ, Delany JP, Hoyt RW. 2005. Energy requirements of military personnel. Appetite 44(1):47­65. Tucker DM, Sandstead HH. 1984. Neuropsychological function in experimental zinc deficiency in humans. In: Frederickson CJ, Howell GA, Kasarskis EJ, eds. The Neurobiology of Zinc. Part B: Deficiency, Toxicity, and Pathology. New York: Alan R. Liss. Pp. 139­152. Vallee BL, Falchuk KH. 1993. The biochemical basis of zinc physiology. Physiol Rev 73(1):79­118. Yao JK, Reddy RD. 2005. Metabolic investigation in psychiatric disorders. Mol Neurobiol 31(1­3): 193­204. Zinc, Magnesium, and Copper Requirements and Exercise Henry C. Lukaski USDA-ARS Grand Forks Human Nutrition Research Center, Grand Forks, North Dakota INTRODUCTION Soldiers are expected to perform physically and mentally at high levels despite concurrent exposure to environmental and operational stressors. During deploy- ment, military personnel may reduce food intake up to 50 percent resulting in suboptimal intakes of energy and micronutrients (Baker-Fulco, 1995). Short dura- tion (< 1 week) decreases in energy and macronutrient intakes are suggested to have minor effects on components of physical performance in young men (Taylor et al., 1957) unless body weight, and hence muscle, loss is significant (5­10 per- cent) and then strength is reduced (Friedl, 1995). Montain and Young (2003) re- cently concluded that decrements in maximal oxygen consumption occur with short-term energy restriction and that these decrements have implications for per- formance impairments during non-weight bearing tasks. The impact of reduced

APPENDIX B 437 intake of micronutrients, specifically minerals, and of increased mineral losses on the physical performance of soldiers remains to be determined. Surveys of mineral intakes by soldiers are scarce but data from a few studies suggest that their intakes of some minerals are inadequate. The limited surveys report that zinc and magnesium intakes of male soldiers during training and in garrison are less than those recommended by the Institute of Medicine (IOM, 2001) (for review see Lukaski and Penland, 2006). Although these findings sug- gest the potential for sub-clinical zinc and magnesium deficits, corresponding biochemical assessments of nutritional status did not reveal evidence of deple- tion possibly because of mineral mobilization in association with increased pro- tein catabolism (Lukaski, 2005 in this Appendix) or the lack of sensitive biomarkers of marginal deficiencies. Thus, the impact of these short-term de- creased intakes of minerals per se on physical performance is unknown. There are no data available on copper intakes by military personnel. This review examines the current knowledge about zinc and magnesium and copper needs of soldiers in training and operations. It summarizes civilian studies conducted to assess the effects of exercise on circulating zinc and magnesium con- centrations and describes redistribution of minerals in the body and their excretion in the urine. It also reviews the effects of restricted intakes of zinc and magnesium on components of physical performance and describes the time-course of depletion and repletion with supplemental zinc and magnesium. Information on copper intakes and losses of military personnel is not available. However, new information de- scribing the effects of dietary copper on energy use during exercise is provided. The adequacy of the current Meal, Ready-to-Eat (MRE) and the First Strike Ration (FSR) to meet zinc and magnesium requirements of soldiers in training is evaluated. EXERCISE AND MINERAL METABOLISM Zinc, magnesium, and copper play key roles in supporting physiological functions during physical activity. These minerals express their biological activi- ties as metalloproteins and co-factors for enzymes. Via their role as enzyme cofactors, they regulate energy metabolism, integrate physiological systems, fa- cilitate cellular energy production, coordinate the balance between aerobic and anaerobic energy metabolism, and provide defense against free radicals pro- duced during periods of increased energy production (Lukaski, 2004). Exercise Effects on Mineral Metabolism Exercise is a provocative stressor that causes redistribution of certain miner- als (Oh et al., 1978). This finding stimulated the investigation of the effects of exercise and diet on circulating zinc and magnesium.

438 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Zinc Maximal exercise induces hemoconcentration or reduction of plasma vol- ume that increases plasma zinc concentrations. Young men fed zinc in amounts of 4 (low), 9 (adequate), and 34 (luxuriant) mg/day underwent progressive, maxi- mal exercise tests on cycle ergometers after an overnight fast (Lukaski et al., 1984). Regardless of zinc intake, plasma zinc concentrations increased signifi- cantly immediately after exercise. However, pre- and post-exercise plasma zinc concentrations were significantly less when dietary zinc was low (4 mg/day) compared to the adequate and luxuriant intakes. To control for plasma volume reduction during intense exercise, plasma zinc concentrations were corrected with a previously validated method (Van Beaumont et al., 1973). Low, com- pared to adequate or supplemental, dietary zinc was associated with a significant decrease in total plasma zinc content. A significant relationship was found be- tween total plasma zinc content and change in body zinc retention. Plasma zinc content paralleled changes in zinc balance during periods of low, adequate, and supplemental zinc intake. This finding suggested that zinc mobilization, assessed indirectly by using the total plasma zinc content, was impaired when dietary zinc was restricted. Other studies have extended these observations. Plasma zinc concentrations of men who completed a 6-mile run increased immediately after exercise (Anderson et al., 1984) which is consistent with other reports (Cordova and Alvarez-Man, 1995). Other investigators reported, however, no change in plasma zinc concentration in men who completed a 10-mile run (van Rij et al., 1986). Differences in the timing of phlebotomies and uncontrolled fluid intakes explain the divergent observations (Keen, 1993). Two hours after completion of the runs, however, plasma zinc concentrations decreased significantly to pre-exercise values in all runners (Anderson et al., 1984; Cordova and Alvarez-Mon, 1995; van Rij et al., 1986). Similar findings were reported by Singh et al. (1994) who studied changes in plasma zinc concentrations after a standardized, prolonged endurance run of trained male runners supplemented with 50 mg/day of zinc or placebo for 6 days each in a double-blind cross-over study. These findings indi- cate that high-intensity exercise promotes mobilization of zinc from body stores. Also, recovery after exercise (2 hours) is associated with a sequestration of minerals, particularly zinc, probably as a result of metallothionein induction in liver and kidney (Oh et al., 1978). There is some evidence that physically active adults have disrupted tissue pools of zinc. Trained female runners had intakes of zinc (10 mg/day), consistent with the current recommendation (IOM, 2001), but significantly reduced plasma zinc concentrations and significantly increased urinary zinc losses compared to age-matched untrained women with a similar intake of zinc (Deuster et al., 1989). In response to a 65Zn infusion, the area under the plasma 65Zn curve was signifi- cantly reduced in the trained women (35 versus 44 µmol in 4 hours). This finding

APPENDIX B 439 suggests that zinc in muscle, the largest body pool of zinc, might also be de- pleted in the trained runners. The interaction of dietary zinc and exercise on urinary losses of zinc has been examined. Fourteen physically active men were fed diets high (~19 mg/ day) and low (~4 mg/day) in zinc for 9 weeks in a double-blind, randomized cross-over trial (Lukaski, 2005). Plasma zinc concentrations in both groups in- creased significantly compared to pre-exercise values after 45 minutes of high- intensity exercise. However, the magnitude of the increase was significantly greater when the high-zinc diet was consumed. Urinary zinc losses were signifi- cantly greater on the day of exercise (0.2 versus 0.1 mg/day) when the higher zinc was fed. Parallel changes in urinary nitrogen were observed. These findings indicate that the increased zinc mobilization and excretion night be related to muscle protein catabolism. Intense exercise affects zinc metabolism (Figure B-29); based on the studies described here a hypothesis for the mechanisms of redistribution of zinc with metabolism can be proposed. The initial response is a decrease in plasma volume (hemoconcentration) that appears to increase the concentration of zinc in plasma and serum. Concomitantly, surface losses (sweat and cell sloughing) of water and minerals could also contribute to changes in circulating zinc. During exer- cise, zinc is likely mobilized from soft tissues into the circulation. This response, which is also influenced by the degree of protein breakdown and gluconeogen- Physical activity Plasma volume Zn mobilization Plasma/serum Zn concentration Urinary Zn output Zn sequestration and redistribution FIGURE B-29 Schematic representation of the effect of physical activity on zinc metabolism.

440 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL esis, may contribute to the increased circulating zinc concentration. At least two hours after the cessation of exercise, it appears that zinc redistribution and se- questration into soft tissues begins with the up-regulation of metallothionein synthesis; the half-life of hepatic metallothionein is about six hours (Oh et al., 1978). Urinary output of zinc increases on days of exercise compared to non- exercise periods. Under conditions of adequate zinc intake, homeostatic adapta- tions of increased zinc absorption and decreased excretion in sweat and urine could accommodate for losses associated with physical activity. The extent of the homeostatic adaptation and, therefore, the additional needs for zinc intake during intense exercise is unknown. Military Training and Zinc Metabolism One study, in which soldiers participated in field operations, reported al- tered zinc status (Miyamura et al., 1987). During a 4 week training period, en- ergy intake was unchanged and body weight decreased (~2 kg). Dietary zinc intake (16­19 mg/day) was constant and exceeded military recommendations (U.S. Departments of Army, Navy and Air Force, 2001). However, plasma zinc decreased significantly and urinary zinc losses increased significantly. Although these findings suggest that zinc intake was inadequate to maintain zinc status (see below Adequacy of Zinc in Military Rations), more research is needed to decipher alterations of zinc metabolism during military training before firmly concluding that levels of dietary zinc should be higher than currently established (MDRI = 15 or 12 mg for men and women, respectively). Magnesium Acute bouts of exercise affect magnesium metabolism. Serum magnesium concentration significantly increased immediately after short-duration, high- intensity exercise (Deuster et al., 1987) or prolonged endurance exercise (Deuster and Singh, 1994; Lijnen et al., 1988). Buchman et al. (1998) reported significant decreases (~15 percent) in serum magnesium in men and women following a marathon compared to measurements made on non-exercise days. Urinary mag- nesium outputs decreased during and immediately following strenuous exercise (Buchman et al., 1998) but increased during the next 24 hours (Deuster et al., 1987; Lijnen et al., 1988). Thus, exercise intensity and duration alter magnesium homeostasis. Surface losses of magnesium during exercise can be appreciable. Men per- forming controlled work for 8 hours on cycle ergometers in the heat (37.8°C) lost 15­18 mg of magnesium daily in sweat and cellular exfoliation collected from a lower arm site (Consolazio et al., 1963). Surface losses of magnesium accounted for 4­5 percent of daily magnesium intake and 10­15 percent of total magnesium excretion.

APPENDIX B 441 Exercise Energy Lipolysis production/use Mg+2 Mg+2 Adipose Muscle tissue Plasma tissue FIGURE B-30 Magnesium (Mg) fluxes during aerobic exercise. Solid arrows indicate magnesium movement from plasma into specific tissues during aerobic exercise. Abbre- viated arrows show redistribution of magnesium from soft tissues into the circulation. SOURCE: Adapted from Resina et al. (1995). Changes in circulating and urinary magnesium suggest redistribution among body pools (Figure B-30). Resina et al. (1995) proposed that aerobic exercise promotes a relocation of magnesium from plasma into adipose tissue to enhance lipolysis and into working muscle to maintain activity of magnesium-containing enzymes. Thus, magnesium concentrations in serum may decline because of redistribution and sweat losses. After the cessation of exercise, magnesium re distributes from soft tissues into the plasma with concomitant increases in uri- nary magnesium output. MINERAL DEPLETION AND PHYSICAL PERFORMANCE Evidence is accumulating that intakes of zinc and magnesium in amounts less than recommended are associated with impaired physical performance. Lim- ited findings suggest that supplementation of these minerals can enhance re- sponses to physical training. Differences in experimental designs pose difficul- ties in the interpretation of some findings. Zinc Observational studies report decreased physical performance in people with low zinc status (Table B-21). Muscular strength and power were significantly less in adolescents (Brun et al., 1995) and men (Khaled et al., 1997), respec-

442 1988 1999 al., 1997 et al., 1995 al., et 2005 al., et et Loan Reference Krotkiewski Brun Khaled Van Lukaski, exercise 2 and CO E submaximal V and & effects 2 capacity O during Zn ,HR 2 2 strength strength power strength work peak O CO volume. Low ventilatory = Measures e V feeding feeding uptake; Performance Design Cross-over Observational Observational Controlled Controlled oxygen = 2 and O rate; Status heart or indicator = HR status Intake or Zn Zn mg/day Zn Zn mg/day output; 12 19 retention (Zn) mg/day intake serum serum vs. serum vs. plasma Zn Zn 135 1 4 dioxide Zinc carbon B-21 = 2 CO athletes TABLE Sample Women Adolescents Male Men Men NOTE:

APPENDIX B 443 tively, with low serum zinc compared to age- and sex-matched controls with serum zinc concentrations in the range of normal values. Controlled studies with different amounts of dietary zinc demonstrate physi- ological impairments with inadequate zinc intake. Men fed 1 compared to 12 mg of zinc daily had significantly decreased serum zinc concentrations and marked reductions in upper and lower body strength and work capacity (Van Loan et al., 1999). Similarly, cardiorespiratory function (heart and ventilation rates) as well as oxygen consumption and carbon dioxide production were significantly altered in physically active men consuming diets containing 3.7 compared to 18.7 mg zinc daily (Lukaski, 2005). Measures of zinc status (plasma zinc concentration, erythrocyte carbonic anhydrase activity and zinc retention) decreased when the low-zinc diet was fed. Zinc supplementation during physical training resulted in significant gains in muscular strength. Older women supplemented with zinc (135 mg/day) compared to placebo in a double-blind, cross-over trial experienced greater gains in strength after 4 weeks of strength training (Krotkiewski et al., 1982). These findings are questionable because neither total dietary zinc intake nor zinc status was assessed. To add to the lack of clear evidence of benefits of supplementation, other studies suggest that supplementation with zinc does not improve performance (Sing et al., 1992, 1994, 1999; Telford et al., 1992; Weight et al., 1988). Magnesium Magnesium status has been related to physical performance in a few groups of athletes. Observational studies of male, collegiate athletes (Lukaski et al., 1983) and adolescent swimmers (Conn et al., 1988) found significant correla- tions between peak oxygen uptake and plasma or serum magnesium concentra- tions. Dietary magnesium also was a significant predictor of swim performance in male and female collegiate swimmers (Lukaski, 1995). Magnesium supplementation of physically active adults improved metabolic responses to training and during controlled exercise (Table B-22). Young men supplemented with magnesium (250 mg/day) in a blinded-randomized trial and enrolled in resistance training increased power significantly more than placebo- treated controls (Brilla and Haley, 1992). Physically active men (Ripari et al., 1989) and collegians (Brilla and Gunther, 1995) had significantly reduced heart rate, decreased oxygen consumption or increased endurance time, and decreased oxygen uptake during submaximal exercise, with magnesium supplementation (250 mg/day). Although these findings suggest a benefit of magnesium supple- mentation, they fail to indicate if functional improvements are limited to indi- viduals with reduced magnesium intake or status. Studies of individuals with low magnesium status demonstrate the benefit of increased magnesium intake on performance measures. Elite rowers with serum magnesium concentrations at the low end of the range of normal values and

444 2003 1995 1992 1989 1993 Nielsen, al., Haley, Gunther, et al., & & & et Reference Ripari Brilla Golf Brilla Lukaski E V gain and E effects HR, submax V Mg and ,HR, volume. 2 2 2 HR, strength O O endurance O during exercise Low ventilatory = Exercise E V to rate; a a a a feeding heart = Responses HR Design Supplement Supplement Supplement Supplement Controlled and output; Intake dioxide (Mg) carbon mg/d 360 = 2 450 507 vs. trial. 540 322 CO intake, vs. vs. vs. vs. 360 Mg 250 250 Placebo 290 153 vs. uptake; Magnesium supplementation oxygen B-22 = 2 Women O & TABLE Sample Men Men Women Men Women NOTE: Double-blind a

APPENDIX B 445 supplemented with 250 mg of magnesium daily used significantly less oxygen during submaximal rowing ergometer tests compared to placebo (Golf et al., 1994). Also, postmenopausal women fed diets containing 330­360 compared to 150 mg of magnesium responded with increased muscle and erythrocyte magne- sium concentrations, and magnesium retention; they had reduced heart rate, ven- tilation rate, and oxygen use during exercise (Lukaski and Nielsen, 2003). Thus, magnesium supplementation of people with sub-optimal magnesium status, re- gardless of physical activity level (e.g., trained or untrained), improves physi- ological function during exercise and magnesium nutritional status. Conversely, magnesium supplementation (365 mg/day for 10 weeks) of marathon runners with normal magnesium status had no beneficial effects on performance or bio- chemical indicators of magnesium nutritional status (Terblanche et al., 1992). Copper Only recently has there been interest in determining the effect of copper intake on energy metabolism and performance. The mechanisms of action appar- ently is reduced activity of a copper-containing enzyme, cytochrome c oxidase (CCO). Dietary copper restriction results in metabolic and functional impair- ments. In rodents, copper deprivation consistently resulted in decreased activity of CCO in brain, liver, heart, skeletal muscle (Prohaska, 1990) with the greatest reductions in muscle (Reeves et al., 2005). Davidson et al. (1993) found signifi- cantly decreased CCO activity in the soleus muscle and a blunted response to submaximal, aerobic training of adult, male Long-Evans rats fed a diet low compared to adequate in copper (< 1 versus 6 mg/kg diet). Exercise training was associated with a significantly reduced increase in muscle CCO in the copper- restricted compared to copper-adequate animals. The copper-deprived rats were unable to complete the exercise training regimen. Physically active young men fed copper at the recommended level of intake (0.9 mg/day; IOM, 2001) had adverse responses to submaximal exercise (Lukaski, submitted). Compared to a higher intake (1.6 mg/day), muscle cytochrome c oxidase activity decreased sig- nificantly when dietary copper was 0.9 mg/day. Plasma lactate concentration increased significantly with the low compared to the higher dietary copper. These findings suggest that copper needs of active men may exceed the recommended dietary copper intake for the general public. FACTORS CONTRIBUTING TO ZINC, MAGNESIUM AND COPPER DEPLETION Many factors contribute to the depletion of body minerals and can lead to sub-clinical deficiency states. Intakes of minerals at the Recommended Dietary Allowance (RDA) minimize the probability of nutritional inadequacy. Thus, the RDA is a target intake for an individual (IOM, 2001).

446 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Increased losses of minerals can affect mineral nutritional status. Exposure to conditions that increase urinary, fecal or surface losses, including cell slough- ing and sweat, can reduce body stores of minerals. Conditions that decrease gastrointestinal transit time, such as diarrhea, increase fecal losses of minerals. Similarly, factors that increase urinary output of minerals, including catabolism of muscle and use of diuretics, promote excretion of minerals. Restricted intake of minerals, however, generally decreases mineral losses in urine. Environmental conditions that increase surface losses may adversely impact mineral balance or retention. Under thermoneutral conditions, whole-body sur- face losses of zinc, copper, and iron were 0.5, 0.34, and 0.33 mg/day or 4, 26, and 2 percent, respectively, of dietary intake (Jacob et al., 1981). Exposure to a hot climate increased losses of minerals in arm sweat. Consolazio et al. (1964) reported zinc and copper losses of 2.2 and 1.5 mg/day or 19 and 48 percent of daily intake, respectively, during approximately 8 hours of exposure to 38°C daily during a 16-day heat exposure study and limited exercise (30 min/day). Similarly, iron and magnesium arm surface losses were 1 and 17 mg/day or 5 and 6 percent, respectively (Consolazio et al., 1963). Although data on whole- body surface losses of minerals during periods of heavy physical activity are lacking, one study (DeRuisseau et al., 2002) reported that arm sweat losses of zinc were 9 and 8 percent of the RDA for men and women, respectively, during a 2 hour period of controlled exercise. For a more detailed discussion on mineral sweat losses with exercise, see Haymes (2005) in this appendix. The principal factor in precipitating copper depletion is intake less than needed to accomodate losses in urine, feces and sweat. Urinary copper losses tend to be small and negligible. Adaptation in absorption is the principal homeo- static regulation of copper status (Klevay et al., 1984). Although whole-body losses of copper in sweat appear to be small (~0.35 mg/day), they represent more than 25 percent of daily copper intake in healthy men (Jacob et al., 1981). This value is less than that previously reported by Consolazio et al. (1963) for copper losses in arm sweat. Regardless of collection site, surface loss of copper is appreciable. ADEQUACY OF ZINC IN MILITARY RATIONS An evaluation of the adequacy of zinc intake for military personnel is lim- ited by the lack of comprehensive data on mineral intake and losses. Thus, an assessment requires compilation of information from different sources. The prin- cipal source of data is a report from Miyamura et al. (1987) who studied male soldiers participating in a 34 day intensive training exercise. The soldiers experi- enced a significant decrease in plasma zinc concentration and a significant in- crease in urinary zinc loss despite a daily zinc intake of 17 mg. Zinc losses could include about 1 mg/day in urine (Miyamura et al., 1987), 2.3 mg/day in sweat (Consolazio et al., 1964) and 12 mg in feces, assuming a

APPENDIX B 447 30 percent absorption of dietary zinc (Lukaski, 2005). Thus, estimated zinc losses could total as much as 15.3 mg/day. Assuming an average zinc intake of 17 mg/day (Miyamura et al., 1987) and a loss of 15.3 mg/day, the balance is calculated to be + 1.7 mg/day (17­15.3). According to military regulation, operational rations, including the MRE and FSR, should be planned to provide either 15 or 8 mg of zinc daily, respec- tively (U.S. Departments of Army, Navy and Air Force, 2001). Assuming that the estimated daily losses of zinc could exceed 15 mg/day, the recommended contents of the MRE (15 mg) and FSR (8 mg) would not meet the needs to overcome daily losses. The paucity of information describing intakes and losses of magnesium and copper limit the discussion of the adequacy of levels of these minerals in military rations. As summarized previously (Lukaski and Penland, 2006), intakes of zinc and magnesium by military personnel participating in a variety of operational activities are less than recommended levels. Moreover, estimates of daily copper intakes are not available. Evidence that recomended intakes of magnesium and copper are need for optimal physiological and psychological performance rein- forces the need to ascertain intakes of these minerals among active duty mili- tary personnel. EFFECTS OF MULTI-MINERAL AND VITAMIN SUPPLEMENTS PHYSICAL PERFORMANCE Limited studies have examined the effects of multiple vitamin and mineral supplements in exercising adults and measures of physical performance. Thirty male long distance runners participated in a blinded, cross-over supplementa- tion trial with a washout (3 months) between treatment periods (3 months) to evaluate the effects of supplements containing the RDA or recommended in- take of vitamins and minerals on nutritional status and physical performance (Weight et al., 1988a,b). Blood biochemical measures of nutritional status were within the ranges of normal values before and after supplementation with no adverse effects identified. Active supplementation failed to elicit any improve- ment in peak oxygen uptake, peak running speed, and peak post-exercise lac- tate accumulation. Similarly, 86 Australian male and female elite athletes (bas- ketball, gymnastics, swimming, and rowing) participated in a 7­8 month placebo-controlled trial of a multiple vitamin and mineral supplement that con- tained recommended intakes of the nutrients (Telford et al., 1992a,b). Supple- mentation improved blood concentrations of vitamin B1, B6, B12, and folate in athletes with lower levels at entry with no effects on mineral nutritional status indicators. Supplementation did not improve performance measures. Twenty two physically active men received no benefit in peak aerobic capacity, endur- ance capacity or muscle strength from a high potency (e.g., exceeding recom- mended intake levels) multivitamin-mineral supplement compared to placebo

448 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL (Singh et al., 1992). In each study, dietary intake of vitamins and minerals was consistent with population recommendations. Thus, consumption of a multiple vitamin and mineral supplement that meets recommended intakes neither ad- versely affects nutritional status nor enhances various physical performance as- sessed by a variety of measures. SUMMARY AND CONCLUSIONS Evidence from controlled feeding studies and supplementation trials demon- strate that measures of physical performance including muscle strength, cardio- respiratory function and energy metabolism are adversely affected when intakes of zinc and magnesium are less than recommended. If military personnel fail to consume adequate amounts of zinc and magnesium, they also may experience impaired performance, although there are inconsistent results regarding the mag- nesium effects. Acute bouts of exercise and heavy physical training increase losses of min- erals in urine and sweat. These losses are appreciable and may exceed 10­20 percent of daily intakes. It is estimated that the contents of the MRE and FSR, as currently composed, are inadequate to meet the potential daily losses of zinc by soldiers during physical training. There is a paucity of data on mineral intakes and losses by soldiers under the stressful conditions of training and adverse environmental conditions. This in- formation is required to develop appropriate recommendations for mineral in- takes, specifically zinc, magnesium, and copper. DISCLAIMERS Mention of a trademark or proprietary product does not constitute a guaran- tee of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area is an equal opportunity/affirmative action employer and all agency services are available without discrimination. REFERENCES Anderson RA, Polansky MM, Bryden NA. 1984. Strenuous running: Acute effects of chromium, copper, zinc, and selected clinical variables in urine and serum of male runners. Biol Trace Elem Res 6:327­336. Baker-Fulco CJ. 1995. Overview of dietary intakes during military exercises. In: Not Eating Enough: Overcoming Underconsumption of Military Operational Rations. Washington, DC: National Academy Press. Brilla LR, Gunther KB. 1995. Effect of magnesium supplementation on exercise time to exhaustion. Med Exerc Nutr Health 4:230­233.

APPENDIX B 449 Brilla LR, Haley TF. 1992. Effect of magnesium supplementation on strength training in humans. J Am Coll Nutr 11:326­329. Brun JF, Dieu-Cambrezy C, Charpiat A, Fons C. Fedou C, Micallef JP, Fussellier M, Bardet L, Orsetti A. 1995. Serum zinc in highly trained adolescent gymnasts. Biol Trace Elem Res 47:273­278. Buchman AL, Keen CL, Commisso J, Killip D, Ou C-N, Rognerud CL, Dennis K, Dunn JK. 1998. The effect of a marathon run on plasma and urine mineral and metal concentrations. J Am Coll Nutr 17(2):124­127. Conn CA, Schemmel RA, Smith BW, Ryder E, Heusner WW, Ku P-K. 1988. Plasma and erythrocyte magnesium concentrations and correlations with maximal oxygen consumption in nine-to- twelve-year-old competitive swimmers. Magnesium 7:27­36. Consolazio CF. 1983. Nutrition and performance. In: Johnson RE, ed. Progess in Food and Nutrition Science, vol 7. Oxford, UK: Pergamon Press. Consolazio CF, Matoush LO, Nelson RA, Harding RS, Canham JE. 1963. Excretion of sodium, potassium, magnesium, and iron in human sweat and the relation of each to balance and re- quirements. J Nutr 79:407­415. Consolazio CF, Nelson RA, Matoush LO, Hughes RC, Urone P. 1964. Trace Mineral Losses in Sweat. Report 284. U.S. Army Medical Research & Nutrition Laboratory. Fitzsimmons Gen- eral Hospital, August 18. Denver, CO. Cordova A, Alvarez-Mon M. 1995. Behavior of zinc in physical exercise: A special reference to immunity and fatigue. Neurosci Biobehav Rev 19(3):439­445. Davidson J, Medeiros DM, Hamlin RL, Jenkins JE. 1993. Submaximal, aerobic exercise training exacerbates the cardiomyopathy of postweanling Cu-depleted rats. Biol Trace Elem Res 38:251­272. DeRuisseau KC, Cheuvront SN, Haymes EM, Sharp RG. 2002. Sweat iron and zinc losses during prolonged exercise. Int J Sport Nutr Exerc Metab 12:428­437. Deuster PA, Singh A. 1993. Responses of plasma magnesium and other cations to fluid replacement. J Am Coll Nutr 12:286­293. Deuster PA, Dolev E, Kyle SB, Anderson RA, Schoomaker EB. 1987. Magnesium homeostasis during high-intensity aerobic exercise in men. J Appl Physiol 62:545­550. Deuster PA, Day BA, Singh A, Douglass L, Moser-Veillon PB. 1989. Zinc status of highly trained women runners and untrained women. Am J Clin Nutr 49:1295­1301. Friedl KE. 1995. When does energy deficit affect soldier physical performance? In: Not Eating Enough: Overcoming Underconsumption of Military Operational Rations. Washington, DC: National Academy Press. Golf SW, Bohmer D, Nowacki PE. 1994. Is magnesium a limiting factor in competitive sport? A summary of relevant literature. In: Golf S, Dralle D, Vecchiet L, eds. Magnesium 1993. London: John Libbey. IOM (Institute of Medicine). 2001. Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Washington, DC: National Academy Press. Jacob RA, Sandstead HH, Munoz JM, Klevay LM, Milne DB. 1981. Whole body surface loss of trace metals in normal males. Am J Clin Nutr 34:1379­1383. Keen CL. 1993. The effect of exercise and heat on mineral metabolism and requirements. In: Nutri- tional Needs in Hot Environments. Washington, DC: National Academy Press. Khaled S, Brun JF, Micallef JP, Bardet L, Cassanas G, Monnier JF, Orsetti A. 1997. Serum zinc and blood rheology in sportsmen (football players). Clin Hemorheol Microcirc 17:47­58. Klevay LM, Inman L, Johnson LK, Lawler M, Mahalko JR, Milne DB, Lukaski HC, Bolonchuk W, Sandstead HH. 1984. Increased cholesterol in plasma in a young man during experimental copper depletion. Metabolism 33:1112­1118.

450 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Krotkiewski M, Gudmundsson M, Backstrom P, Mandroukas K. 1982. Zinc and muscle strength and endurance. Acta Physiol Scand 116:309­311. Lijnen P, Hespel P, Fagard R, Lysens R, Vanden Eynde E, Amery A. 1988. Erythrocyte, plasma, and urinary magnesium in men before and after a marathon. Eur J Appl Physiol 58:252­256. Lukaski HC. 1995. Interactions among indices of mineral element nutriture and physical perfor- mance of swimmers. In: Kies CV, Driskell JA, eds. Sports Nutrition: Minerals and Electrolytes. Boca Raton, FL: CRC Press. Lukaski HC. 2004. Vitamins and minerals: Effects on physical performance. Nutrition 20:632­644. Lukaski HC. 2005. Low dietary zinc decreases erythrocyte carbonic anhydrase activities and impairs cardiorespiratory function in men during exercise. Am J Clin Nutr 81:1045­1051. Lukaski HC. 2006. Protein turnover and mineral metabolism. In: Mineral Requirements for Military Personnel. Washington, DC: The National Academies Press. Lukaski HC, Johnson PE. Submitted. Copper depletion and altered energy metabolism during exer- cise in men fed copper at the recommended intake. Eur J Appl Physiol. Lukaski HC, Nielsen FH. 2002. Dietary magnesium depletion affects metabolic responses during submaximal exercise in postmenopausal women. J Nutr 132:930­935. Lukaski HC, Penland JG. 2006. Zinc, magnesium, copper, iron, selenium, and calcium in assault rations: Roles in promotion of physical and mental performance. In: Nutrient Composition of Rations for Short-Term, High-Intensity Combat Operations. Washington, DC: The National Academies Press Lukaski HC, Bolonchuk WW, Klevay LM, Milne DB, Sandstead HH. 1983. Maximal oxygen con- sumption as related to magnesium, copper and zinc nutriture. Am J Clin Nutr 37:407­415. Lukaski HC, Bolonchuk WW, Klevay LM, Milne DB, Sandstead HH. 1984. Changes in plasma zinc content after exercise in men fed a low-zinc diet. Am J Physiol 247:E88­E93. Miyamura JB, McNutt SW, Lichton IJ, Wenkam NS. 1987. Altered zinc status of soldiers under field conditions. J Am Diet Assoc 87(5):595­597. Montain SJ, Young AJ. 2003. Diet and physical performance. Appetite 40:255­267. Oh SH, Deagen JT, Whanger PD, Weswig PH. 1978. Biological function of metallothionein. V. Its induction in rats by various stressors. Am J Physiol 234:E282­E285. Prohaska JR. 1990. Biochemical changes in copper deficiency. J Nutr Biochem 1:452­461. Reeves PG, DeMars LCS, Johnson WT, Lukaski HC. 2005. Dietary copper deficiency reduces iron absorption and duodenal enterocyte hephaestin protein in male and female rats. J Nutr 135:92­98. Resina A, Gatteschi L, Rubenni MG, Galvan P, Parise G, Tjouroudis N, Viroli L. 1995. Changes in serum and erythrocyte magnesium after training and physical exercise. In: Vecchiet L, ed. Magnesium and Physical Activity. London: Parthenon Publishing. Ripari P, Pieralisi G, Giamberardino MA, Resina A, Vecchiet L. 1989. Effects of magnesium pidolinate on cardiorespiratory submaximal effort parameters. Magnesium Res 2:71­75. Singh A, Moses FM, Deuster PA. 1992. Chronic multivitamin­mineral supplementation does not enhance physical performance. Med Sci Sports Exerc 24(6):726­732. Singh A, Failla ML, Deuster PA. 1994. Exercise­induced changes in immune function: Effects of zinc supplementation. J Appl Physiol 76(6):2298­2303. Taylor HL, Buskirk ER, Brozek J, Anderson JT, Grande F. 1957. Performance capacity and effects of caloric restriction with hard physical work on young men. J Appl Physiol 10:421­429. Telford RD, Catchpole EA, Deakin V, McLeay AC, Plank AW. 1992a. The effect of 7 to 8 months of vitamin/mineral supplementation on the vitamin and mineral status of athletes. Int J Sports Nutr 2(2):123­134. Telford RD, Catchpole EA, Deakin V, McLeay AC, Plank AW. 1992b. The effect of 7 to 8 months of vitamin/mineral supplementation on the athletic performance. Int J Sports Nutr 2(2):135­154. Terblanche S, Noakes TD, Dennis SC, Marais DW, Eckert M. 1992. Int J Sports Nutr 2(2):154­164.

APPENDIX B 451 U.S. Departments of Army, Navy, and Air Force. 2001. Nutrition Standards and Education. AR 40- 25/BUMEDINST 10110.6/AFI 44-141. Washington, DC: U.S. Department of Defense Headquarters. Van Beaumont W, Strand JC, Petrofsky JS, Hipskind SG, Greenleaf JE. 1973. Changes in total plasma content of electrolytes and proteins with maximal exercise. J Appl Physiol 34:102­106. Van Loan MD, Sutherland B, Lowe NM, Turnlund JR, King JC. 1999. The effects of zinc depletion on peak force and total work of knee and shoulder extensor and flexor muscles. Int J Sport Nutr 9(2):125­135. Van Rij AM, Hall MT, Dohm GL, Bray JT, Pories WJ. 1986. Changes in zinc metabolism following exercise in human subjects. Biol Trace Elem Res 10:99­105. Weight LM, Myburgh KH, Noakes TD. 1988a. Vitamin and mineral supplementation: Effect on the running performance of trained athletes. Am J Clin Nutr 47:192­195. Weight LM, Noakes TD, Labadarios D, Graves J, Jacobs P, Berman PA. 1988b. Vitamin and mineral status of trained athletes including the effects OF SUPPLEMENTation. Am J Clin Nutr 47:186­191. The Effects of Iron Deficiency on Physical Performance Jere D. Haas Cornell University, Ithaca, New York INTRODUCTION Iron deficiency is the most prevalent micronutrient deficiency in both the industrialized and non-industrialized world. In the United States iron deficiency affects approximately 3­4 percent of men and 12­14 percent of women between 18 and 45 years, the age of the majority of military personnel in the U.S. (Looker et al., 1997). While the most common consequence of iron deficiency is anemia, or blood hemoglobin concentration below a specified level, the prevalence of anemia underestimates the amount of iron deficiency in the population. WHO (2001) estimates that the prevalence of iron deficiency is more than twice the prevalence of anemia in any given population. Also, while iron deficiency ac- counts for the majority of anemia in the U.S. population, there are numerous additional causes of anemia, including other micronutrient deficiencies. Numerous studies of the effect of iron deficiency on physical performance have been conducted over the past 35 years with conclusive evidence for a causal relationship (Haas and Brownlie, 2001). Most of the evidence from these studies indicates that low hemoglobin concentration and consequent reduced oxygen transport to working muscles is the primary mechanism for reduced performance due to iron deficiency. However, evidence from animal studies and more recently in human studies of non-anemic human subjects suggest that iron deficiency may affect physical performance through other mechanisms. This review addresses the evidence for the effects of iron depletion in non-anemic individuals.

452 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL ASSESSING MODERATE IRON DEFICIENCY There are a number of indicators of iron nutritional status that when used together can reveal a fairly comprehensive picture of the various body iron pools that are significant for understanding the functional consequences of iron defi- ciency. Figure B-31 shows the progress of iron status from normal to deficient states and the course taken by the major indicators of iron status in common use. The body iron stores reflect the functional iron status of an individual. With increased iron loss or decreased iron intake to compensate for losses, the body iron stores decline to a point indicated as "deficient." The best single indicator of the depletion of iron stores is serum ferritin. Hemoglobin concentration does not start to fall until after iron stores are depleted, and anemia is defined as a hemo- globin level that is achieved during the decline in hemopoiesis. There is a stage of body iron depletion when the iron stores are completely depleted but hemo- globin has not yet reached a level that indicates anemia; this is the iron deficient, non-anemic state (IDNA). Another indicator of iron status that is not represented in Figure B-31 is the blood plasma concentration of the soluble transferrin recep- tor (sTfR). It follows a course similar to that for free erythrocyte protoporphyrin (FEP), and increasing levels indicate a increased demand for iron at the muscle Hemoglobin FEP Ferritin TS Fe stores IDNA anemia IDNA=Iron Deficient Non-Anemic FIGURE B-31 Relationship between various indicators of iron status and the body's level of iron stores. SOURCE: Modified from Guthrie and Picciano (1995).

APPENDIX B 453 tissue level which is not being met by circulating iron in the iron depleted indi- vidual. This indicator appears to identify non-anemic individuals who will ben- efit from iron supplementation as it affects physical performance (Brownlie et al., 2004). Cook and colleagues (Cook et al., 2003) have developed an algorithm to estimate total body iron from the log of the ratio of soluble transferrin receptor and serum ferritin. EVIDENCE FOR THE EFFECT OF IRON DEFICIENCY ON PHYSICAL PERFORMANCE IN ANEMIC SUBJECTS Most of the research on iron deficiency effects on physical performance has focused on anemic subjects. This literature has been reviewed extensively by Haas and Brownlie (2001) who conclude that there is considerable evidence to support a direct causal relationship. One of the more recent studies that used an experimental design that included randomization of subjects to consume either an iron supplement or a placebo was conducted by Li and colleagues (Li, 1993; Li et al., 1995), who studied the effects of iron deficiency on work capacity in female Chinese factory workers. They assessed changes in physical performance with the VO2max test, an assessment of aerobic power, after 12 weeks of con- suming either an iron supplement or a placebo. The results are summarized in Figure B-32. Li (1993) reported a 5 percent improvement in VO2max in the iron supplemented group which corresponded to a 13g/L increase in hemoglobin con- centration. There was a range of hemoglobin values in this sample and the great- 2 * * different from 1.95 placebo, p < .05 1.9 /min) Before After (L 1.85 max 2 1.8 VO 1.75 1.7 114 127 Hb (g/L) 115 113 Iron Supplemented Placebo Control FIGURE B-32 VO2max in Chinese female cotton mill workers before and after 12 weeks of iron supplementation. SOURCE: Li (1993, unpublished thesis).

454 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL est effects of iron supplementation on VO2max were seen in the anemic women. The authors also reported an increase in productivity in the workplace after 12 weeks of iron supplementation (Li et al., 1995). There have been a large number of studies of the effects of iron deficiency on physical performance using experimental animals. One of the most interest- ing was conducted by Davies et al. (1982) with post-weaning rats that developed iron deficiency after consuming a low-iron diet and then repleted rapidly by iron therapy. The results are summarized in Figure B-33. This study confirmed previ- ous studies in animals and humans of an improvement in VO2max, which paral- leled an increase in hemoglobin concentration following iron therapy. This study is significant because it also followed changes in another measure of physical performance, endurance capacity, and a measure of tissue oxidative capacity, muscle pyruvate oxidase, throughout the 7-day period following iron repletion. The course of change in endurance lagged behind that of VO2max and paralleled the increase in pyruvate oxidase. These findings indicate that physical perfor- mance may be only partially mediated by the effect of iron deficiency on oxygen transport in the blood, and that tissue iron depletion may also limit performance. FIGURE B-33 The relationship of iron repletion to hemoglobin response, maximal aerobic capacity (VO2max), muscle pyruvate oxidase activity and endurance capacity in rats made iron deficient post-weaning. SOURCE: Davies et al. (1982). Used with permission.

APPENDIX B 455 They also indicate that different types of physical performance tests need to be considered when studying severe compared to moderate iron deficiency. PHYSICAL PERFORMANCE IN IRON DEPLETED NON-ANEMIC SUBJECTS This section reviews evidence for the effects of iron deficiency on physical performance, focused primarily on non-anemic individuals. Before the evidence is described, a brief review is presented of the rationale for why these affects should be observable and important. Rationale The animal experiments represented by Davies et al. (1982) provide partial rationale for exploring relationships between iron depletion and performance in non-anemic human subjects. Further justification exists when one considers that iron plays an import role in muscle metabolism beyond the transport of oxygen by hemoglobin to the tissue sites for energy conversion to muscular work. Figure B-34 presents a list of iron-containing compounds that are affected by body iron depletion. Approximately 5 percent of the body iron is found in iron-containing enzymes and 10 percent is found in myoglobin. Many of these compounds are involved in transformation of chemical to mechanical energy. A second rationale FIGURE B-34 Iron containing compounds that are affected by iron deficiency.

456 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL Tissue oxidative Diet capacity Endurance capacity Depletion Functional Maximal of iron iron Anemia Oxygen power output stores deficiency Transport (VO2max) Blood loss Energetic Tissue oxidative efficiency capacity FIGURE B-35 Conceptual model of iron deficiency effects on physical performance. is the relatively large number of individuals in a population that may be affected by iron deficiency. This is a number that exceeds the prevalence of anemia in the population (WHO, 2001). While the effects of iron depletion without anemia may be less severe than the effects of iron deficiency, there is growing evidence that the impact on physical performance is not trivial. The proposed mechanisms for the action of iron deficiency on physical performance are summarized in Figure B-35. Body iron stores become depleted due to an imbalance of iron loss and dietary gain. When levels of body iron become too low there is the beginning of a functional iron deficiency which affects compounds associated with muscle metabolism but probably does not affect hemoglobin synthesis and oxygen transport in the blood. Under conditions of more severe depletion, hemoglobin synthesis is compromised along with skel- etal muscle compounds resulting iron deficiency anemia (IDA). Under IDA a broad range of physical performance measures are affected. The anemia results in reduced oxygen transport that limits aerobic power, endurance, and muscular energetic efficiency. The reduced levels and activity of iron-dependant, muscu- lar tissue compounds seen in non-anemic, iron deficiency (IDNA) contribute to reduced endurance and energetic efficiency, but do not limit aerobic power, since blood oxygen transport is not compromised. Evidence for Effects of Iron Depletion in Non-Anemic Women While the functional effects of iron deficiency have been well documented in those individuals who are anemic, the effects of IDNA have only recently

APPENDIX B 457 been examined in some detail (Haas and Brownlie, 2001). Three recent iron supplementation trials with iron deficient non-anemic women provided evidence that supports the general conceptual model presented in Figure B-35 (Brutsaert et al., 2003; Hinton et al., 2000; Zhu and Haas, 1998). All of the studies used a similar research design. Female subjects between 18 and 45 years were identi- fied through population screening to be non-anemic (hemoglobin > 120 g/L), but iron depleted (serum ferritin < 20 µg/L). Subjects were randomly assigned to consume either supplemental iron (100 to 135 mg FeSO4/day) or a placebo daily for 6 or 8 weeks, following a double blind protocol. A battery of measures of iron status and various measures of physical performance were assessed at base- line and at the end of the supplementation period. The results of these studies are summarized as follows. Metabolic Response to Exercise Zhu and Haas (1998) studied thirty-seven non-anemic, iron-deficient (fer- ritin < 16 µg/L) university women who consumed 135 mg/day of FeSO4 (50 mg Fe/day) or a placebo for 8 weeks. Physical performance was assessed by VO2max and time to complete a simulated 15-km time trial with a cycle ergometer, an indicator of endurance. Serum ferritin values increased in the supplemented group but hemoglobin did not change, and there was no group difference in VO2max at the end of the trial. While the iron supplemented group did not complete the time trial in less time than the placebo group, they completed the task at a lower percentage of their VO2max (82 versus 88 percent) and with 5.1 percent less energy expended than the placebo group. While one can conclude that iron defi- cient women are less efficient at doing heavy work, it is not known whether these effects of iron deficiency on performance can be observed under less rigor- ous levels of exertion. The next study addresses this question. Energetic Efficiency in Mexican Women The studies described here (Brutsaert et al., 2003; Haas et al., 2002; Seymour, 2002) investigated the effects of iron supplementation for women on outcomes of physical performance while cycling at different intensities. Forty-three non- anemic, iron deficient (ferritin < 20 mg/L), female Mexican office workers and students consumed 18 mg/day of elemental iron as FeSO4 or a placebo for 6 weeks (Haas et al., 2002). Serum ferritin increased while hemoglobin did not change in the iron supplemented group when compare to the placebo group, and estimated VO2max did not differ between the groups after supplementation. Energy cost of performing 30 and 60 watts of work on a cycle ergometer was assessed at baseline and after 6 weeks. At 60 watts the iron supplemented women showed a 5.2 percent lower energy cost to perform the work after 6 weeks of supplementation (Seymour, 2002). As shown in Figure B-36 this resulted in a

458 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL 31 Placebo p < .05 (%) 30.5 iron 30 29.5 Efficiency 29 28.5 Work 28 Baseline Final kcal of external work performed at 60W WE = (kcal expended at 60W) - (kcal expended at 0W) FIGURE B-36 Work efficiency (WE) at 60 watts in Mexican women before and after 6 weeks of iron supplementation. SOURCE: Seymour (2002, unpublished thesis). significantly higher net work efficiency which was related to increased iron in- take and decreases in tissue iron status, based on the soluble transferrin receptor concentration (sTfR). In a sub-sample of 20 women from this study, an addi- tional test of a maximal voluntary static contraction (MVC) on a dynamic knee extension exercise was administered to assess local muscle fatigue (Brutsaert et al., 2003). The iron supplemented women performed the task with significantly less muscle fatigue than the placebo group after 6 weeks of supplementation. Adaptation to Physical Training Several papers have reported on a study of the effects of iron status and supplementation on improvements on performance outcomes due to adaptation to physical training (Brownlie et al., 2002, 2004; Hinton et al., 2000). In this study, 42 non-anemic, iron-deficient university women were randomly assigned to consume either 100mg/day of FeSO4 or a placebo for 6 weeks. In addition, an additional exercise intervention for all subjects consisted on 20 days of aerobic training during the final 4 weeks of the supplementation trial. It was reported that both groups benefited from the training by increasing their VO2max and reducing their times on a simulated 15-km time trial with a cycle ergometer. As shown in Figure B-37, the iron supplemented group improved its time in the time trial by 3.4 minutes compared to a 1.6 minute improvement in the placebo group (Hinton et al., 2000). The effect of iron treatment was mediated by changes in serum ferritin but not by changes in hemoglobin. VO2max also improved

APPENDIX B 459 33 Placebo Iron 32 31 Time trial 30 * Minutes 29 28 27 Baseline Final FIGURE B-37 Improvements in times for women to complete a 15 km bicycle time trial after 4 weeks training while consuming supplemental iron or placebo. Iron group com- pleted final trial 1.6 min faster than placebo group, after adjusting for differences in initial times and work rate, p < 0.05. SOURCE: Hinton et al. (2000). more in the iron supplemented group and the greatest improvement in time-trial time and work efficiency was seen in the iron supplemented women who were most depleted in tissue iron (sTfR) at baseline (Brownlie et al., 2004). From this study one can conclude that iron deficiency reduces the potential benefits of aerobic training in both endurance and VO2max. It remains to be tested whether the effects of iron deficiency on adaptation to aerobic training can be observed in individuals who are already physically fit. CONCLUSIONS We can draw several conclusions from the research literature on physical performance in iron deficiency anemia and from the recent experiments described in this paper on iron depleted non-anemic women: · Iron deficiency anemia (IDA) has clear functional consequences across a wide range of tests of physical work capacity and productivity · The mechanisms for IDA effects on performance include compromise to both oxygen transport and tissue level oxidative capacity · Iron deficiency without anemia (IDNA) is more prevalent than IDA in the general population and carries measurable but less severe consequences to human performance

460 MINERAL REQUIREMENTS FOR MILITARY PERSONNEL · The impact of IDNA is observed for physical endurance rather than aerobic power, and on reducing the ability to adapt to aerobic training. For relevance to physical performance of military personnel, one can conclude: · The results on the effects of iron deficiency anemia on physical perfor- mance should apply to all individuals · The results on moderate iron deficiency without anemia in females should be extrapolated to males who experience a similar degree of iron deficiency and level of fitness · Military personnel should be screened for anemia and body iron status · Iron deficiency should be corrected in the long term by dietary adjust- ments and by mineral and vitamin supplementation in the short term, as condi- tions warrant. Future research should consider assessing the effects of moderate iron defi- ciency on energetic efficiency and adaptation to training in more physically fit subjects and under conditions such as basic training. Additional research should consider assessing dietary iron requirements for military personnel which are based on potential iron loss from heavy exertion as well as additional demands to support physical training and maintenance of high levels of endurance. REFERENCES Brownlie T, Utermohlen V, Hinton PS, Giordano C, Haas JD. 2002. Marginal iron depletion without anemia reduces adaptation to physical training in previously untrained women. Am J Clin Nutr 75:734­742. Brownlie T, Utermohlen V, Hinton PS, Haas JD. 2004. Tissue-iron deficiency without anemia im- pairs endurance adaptation among previously untrained women. Am J Clin Nutr 79:437­443. Brutsaert T, Hernandez-Cordero S, Rivera J, Viola T, Hughes G, Haas JD. 2003. Progressive muscle fatigue during dynamic work in iron deficient Mexican women. Am J Clin Nutr 77:441­448. Cook JD, Flowers H, Skikne BS. 2003. The quantitative assessment of body iron. Blood 101:3359­ 3364. Davies KJA, Maguire JJ, Brooks GA, Dallman PR, Packer L. 1982. Muscle mitochondrial bioener- getics, oxygen supply, and work capacity during dietary iron deficiency and repletion. Am J Physiol 242:E418­E427. Guthrie H, Picciano MF. 1995. Human Nutrition. St. Louis, MO: Mosby-Year Book. Haas JD, Brownlie T. 2001. Iron deficiency and reduced work capacity: A critical review of the research to determine a causal relationship. J Nutr (suppl) 131:676S­688S. Haas JD, Seymour J, Hernandez-Cordero S, deHaene J, Villalpando S, Rivera J. 2002. Iron depletion increases the energy cost of work in non-anemic Mexican women. Am J Phys Anthropol (Suppl) 34:80. Hinton PS, Giordano C, Brownlie T, Haas JD. 2000. Iron supplementation improves endurance after training in iron-deficient, non-anemic women. J Appl Physiol 88:1103­1111.

APPENDIX B 461 Li R. 1993. Functional Consequences of Iron Supplementation in Iron-Deficient, Chinese Female Workers. Unpublished doctoral dissertation. Wageningen, The Netherlands: Wageningen Agri- cultural University. Li R, Chen X, Yan H, Durenberg P, Garby L, Hautvast JGAJ. 1995. Functional consequences of iron supplementation in iron-deficient, female cotton workers in Beijing, China. Am J Clin Nutr 59:908­913. Looker AC, Dallman PR, Carroll MD, Gunter EW, Johnson CL. 1997. Prevalence of iron deficiency in the United States. J Am Med Assoc 277:973­976. Seymour JM. 2002. Iron deficiency decreases efficiency of intermittent exercise in marginally iron deficient non-anemic Mexican women. Unpublished masters thesis. Ithaca NY: Cornell Uni- versity. WHO (World Health Organization). 2001. Iron deficiency Anemia: Assessment, Prevention, and Control. A Guide for Program Managers. Geneva, Switzerland: World Health Organization. Zhu YI, Haas JD. 1998. Altered metabolic response in iron-depleted non-anemic women during a 15-km time trial. J Appl Physiol 84:1768­1775.

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The U.S. Army Health Risk Appraisal group surveyed 400,000 active duty U.S. Army personnel in the late 1990s to determine whether or not those personnel met the dietary objectives of Healthy People 2000 (HP2000), a national agenda for health promotion and disease prevention. As reported by Yore et al. (2000), Army personnel generally did not meet the HP2000 goals for nutrition even though significant progress had been made during 1991-1998. Although the specific aspects of diet that would be relevant to this Committee on Mineral Requirements for Cognitive and Physical Performance of Military Personnel are lacking, the findings from this survey suggest that there are dietary problems in the military population. The potential for adverse effects of marginal mineral deficiencies among soldiers engaged in training or military operations and the prospect of improving military performance through mineral intakes have spurred the military's interest in this area of nutrition.

Mineral Requirements for Military Personnel provides background information on the current knowledge regarding soldiers' eating behaviors as well as on the physical and mental stress caused by military garrison training or operations. This report also offers facts on the mineral content of rations and its intake by military personnel and addresses the potential effects of nutrient deficiencies due to inadequate intake or higher requirements during military operations. Mineral Requirements for Military Personnel provides information and recommendations on the development and uses of MDRIs and a description of strategies to increase intake of specific minerals, whether via usual foods, fortification, or supplementation. This report features a description of the metabolism and needs for selected minerals by military personnel under garrison training, recommendations on mineral intake levels, and an assessment of mineral level adequacy in operational rations. This report also includes a prioritization of the research needed to answer information gaps and details of study designs required to gain such information.

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