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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,