Project Overview and Committee Summary
As the U.S. military faces the twenty-first century, it must contend with changes in the nature of warfare and deployment that have significant implications for individual performance. The more frequent redeployment of soldiers (necessitated by downsizing and by changing military strategies) mandates greater concern for their physical health and well-being and, therefore, the development of cutting-edge techniques for field assessment of health and nutritional status. Such assessment tools must demonstrate reproducibility and reliability in field tests, must be noninvasive, and must cause minimal interference with battlefield operations. Reliance upon techniques that are tied to laboratories must give way to ambulatory assessment. At the same time, the increasingly technological orientation of modern warfare raises the spectre of battles being waged by soldiers seated at computer terminals, with the capability for mass destruction at their fingertips, and necessitates great concern for the assessment and optimization of cognitive performance in those soldiers. Finally,
budgetary constraints, coupled with the need to stay at the forefront of research, dictate that careful consideration be given to identifying the best available and emerging technologies and making priority decisions regarding which ones should be undertaken directly by the military, which deserve investment of funds to foster military applications, and which are best left to the private sector.
THE COMMITTEE'S TASK
As part of its responsibility to the Military Nutrition Division (MND) (currently the Military Nutrition and Biochemical Division) at the U.S. Army Research Institute of Environmental Medicine (USARIEM), the Committee on Military Nutrition Research (CMNR) on many occasions has provided evaluation of both research plans and ongoing research efforts funded by Department of Defense (DoD) appropriations. Examples include 1992 and 1996 reviews of research activities at the Louisiana State University's Pennington Biomedical Research Center, 1991 and 1993 reviews of a nutrition intervention project's results conducted during a U.S. Army Ranger training program, and a 1995 review of issues related to iron status of women enrolled in U.S. Army Basic Combat Training.
In 1994, the CMNR was asked by the MND to identify and evaluate new technologies to determine whether the technologies will provide useful tools to help solve important issues in military nutrition research in the areas identified by MND, USARIEM. The committee was requested: (1) to provide a survey of newly available and emerging techniques for the assessment and optimization of nutritional and physiological status and performance, and (2) to evaluate the potential of these techniques to contribute to future research efforts involving military personnel. In addition, the committee was asked to make recommendations regarding the practicality and the application of such techniques in field settings. The MND asked the CMNR to include in its response the answers to the six questions listed in Table 1-1.
To assist the CMNR in responding to these questions, a workshop was convened on May 22–23, 1995, in Washington, D.C., that included presentations from individuals with expertise in:
techniques of body composition assessment,
tracer techniques for the study of metabolism,
ambulatory techniques for determination of energy expenditure,
molecular and cellular approaches to nutrition,
assessment of immune function, and
functional and behavioral measures of nutritional status.
TABLE 1-1 Questions Posed by the Army
State of the Art
Will the technologies be a significant improvement over current technologies?
Maturity and Availability
How likely are the technologies to mature sufficiently for practical use?
What is the cost/benefit ratio of the new technologies, and how expensive (in both monetary and personnel terms) will they be to employ compared with the importance of the information they will provide?
Are the technologies of such critical value that their development should be supported by DoD funds—such as can be provided by the Small Business Innovative Research program?
Complicating Factors and Methodological Questions
How practical are the technologies? Will they require dedicated personnel and complex, exotic equipment? Will the data provided be difficult to analyze?
Possible Use in the Field
Can the technologies be used in the field (could they be used in the field or used to analyze samples collected in the field)?
As a background to these presentations, a representative of the MND provided an overview of the military nutrition program and its research activities. For the preparation of their presentations, the invited speakers were asked to address the questions posed by the Army. The speakers discussed their presentations with committee members at the workshop and submitted written reports based on their verbal presentations. The committee met after the workshop to discuss the techniques presented and the information provided. Later, the CMNR reviewed the workshop presentations, summarized the information pertinent to each technique, and drew heavily on its collective expertise and the scientific literature to evaluate the potential contribution of each technique to military nutrition research and make recommendations regarding development of capabilities in these areas. In preparing this report, the committee limited its evaluation to technologies discussed at the workshop. The committee's conclusions and recommendations, as well as the responses to the six questions posed by the Army, appear in Chapter 2.
THE CURRENT ARMY PROGRAM AND ITS FUTURE NEEDS
The Army's nutrition research program is driven by the need of the Armed Forces to maintain and enhance performance in all operational environments.
Key to the performance requirements is understanding how nutritional status affects physical and cognitive performance and long-term health. The research program to provide the knowledge about these relationships is discussed by James A. Vogel in Chapter 3 in this volume. Vogel indicates that this program is composed of four main areas:
development of nutritional strategies,
evaluation of operational rations,
establishment of nutritional requirements under diverse field situations, and
assessment of nutritional status of military populations.
In these areas of research, the technical requirements include methods for measurement of changes in body composition, plus the determination of lean mass and fat mass; measurement of protein metabolism; monitoring the metabolism of energy-yielding nutrients; measurement of energy expenditure under a wide variety of environmental conditions; measurement of molecular and cellular changes in nutrient utilization; measurement of immune function (status); measurement of physiological performance; and measurement of cognitive performance.
The unique requirements of the Armed Forces' nutrition research program are the need to make measurements in extremes of environmental conditions (high and low temperatures, high altitude, high and low humidity, noise, vibration, concussion, or combinations thereof) and stress (sleep deprivation, battle fatigue, prolonged physical exertion, dehydration, and underconsumption of nutrients) and the desire to extend performance while holding logistical requirements to a minimum. This research agenda makes consideration of new research technologies very attractive, especially in terms of cost and timeliness. At the same time, consideration must be given to the relative chances of successful outcome and cost or cost/benefit considerations. Relative to this latter consideration, Vogel suggests that attention be given to the relative potential for performance enhancement or the health benefit that is inherent in ration modification, ration supplementation, or other nutrition interventions. Although the relative military benefit of performance outcomes may be beyond the capacity of the CMNR to evaluate, some assessment can be made on the basis of improvement in the variability and certainty of research data.
TECHNIQUES OF BODY COMPOSITION ASSESSMENT
In Chapter 4 of this report, LTC Karl E. Friedl presents an overview of the use of body composition (BC) assessment by the military, the available technologies, and suggestions for future development. More detailed discussions
The composition of the body reflects a number of factors, including the status of energy stores, training level, and other aspects of nutritional and hydration status. Characteristics that may be inferred indirectly from BC assessments include muscular strength, physical performance, and potential risk for musculoskeletal injury (Reynolds et al., 1994).
Body Composition Standards in the Military
The military utilizes the results of BC research and analysis to develop appropriate standards for selection (accession and retention) and for fitness. While the primary goal of military BC standards is to ensure military readiness (physical fitness, performance, and risk of chronic disease) (Friedl, 1992), a secondary goal is to maintain military appearance (Hodgdon et al., 1990), which is considered a part of readiness. The BC standards utilized by the military are specific to age, gender, and branch of military service. In addition to utilization in standards development, BC is used by the military to assess training programs and the risks and benefits of a wide variety of optimization strategies, including physical, nutritional, and pharmacological interventions. The first tier of BC analysis by the U.S. Army consists of a semiannual weight-for-height screen, based on body mass index (BMI, weight/height2) (AR 600-9, 1986). Personnel who exceed the weight-for-height standards are permitted to undergo a second tier of analysis consisting of body fat assessment by anthropometric (circumference) measurement. As Friedl points out, the method of body fat assessment utilized by the Army must be part of a regulation and must be limited to one method that is accurate and does not inadvertently undermine the goals of classification by declaring those with the greatest muscle mass to be overweight or by requiring inappropriate energy restriction to ''make weight."
BC standards utilized by the military are based primarily on the ability to predict body fat from BMI and secondarily on equations that estimate the percentage of body fat from anthropometric measurements (AR 600-9, 1986). Validation of the equations, which differ according to age, gender, and branch, traditionally has been accomplished by comparison with body fat estimations using hydrodensitometry, the "criterion" measure. Hydrodensitometric determination of body fat is based on a two-compartment model of BC (fat and fat-free mass) (Siri, 1961). This method may be unreliable for both technical and theoretical reasons. From a technical standpoint, residual air volume (air left in the body after voluntary expulsion) and body volume measurements are subject to significant error. From a theoretical standpoint, the method relies on assumptions regarding the two compartments' constancy of composition. These latter assumptions are based primarily on data collected either from men (Keys et al., 1950) or guinea pigs (Pace and Rathbun, 1945) and do not account for
changes in hydration level or differences because of race (ethnicity) or gender. The military's equations tend to overestimate body fat in lean individuals and underestimate body fat in overweight individuals (Friedl and Vogel, 1992).
Recent attempts to validate the military's body fat equations have included the use of a four-compartment model combining hydrodensitometry with dualenergy x-ray absorptiometry (DEXA or DXA) to determine bone mineral density and soft tissue mass, according to Friedl (see Chapter 4 in this volume).
Body Composition Assessment Techniques Proposed to Replace Hydrodensitometry as the Criterion Method
Dual-Energy X-Ray Absorptiometry
The use of DXA for BC assessment is reviewed in Chapter 6 by Wendy M. Kohrt. According to Kohrt, the primary clinical application of DXA (which has replaced dual-photon absorptiometry) is the measurement of bone mineral content and bone mineral density (BMD) to assess risk for osteoporosis. As a tool for BMD assessment, DXA is considered highly reliable and precise; however, its validity, particularly for measuring changes in BMD, remains questionable because of interinstrument measurement discrepancies (see Kohrt, Chapter 6 in this volume), as well as intrainstrument errors resulting from changes in other tissue compartments (Schneider and Reiners, 1997).
The use of DXA to measure BC is based on the principle that the composition of an object can be determined by the attenuation of two distinct low-and high-energy x-ray beams. This technique can distinguish three compartments or materials: bone mineral, nonbone lean tissue, and fat (Nord and Payne, 1990). The x-ray attenuation of each pixel is compared to the known attenuation of reference materials (see Kohrt, Chapter 6 in this volume). As a means of measuring BC, DXA appears to be more precise, reproducible, and convenient than hydrodensitometry for both the patient and the investigator (Hansen et al., 1993). In addition, DXA yields information about regional BC (Jensen et al., 1995) and has the advantage of producing results that are independent of ethnic differences. Also, DXA allows measurement of bone, which is one of the compartments with the greatest interindividual variability. With DXA, less reliance needs to be placed upon assumptions regarding the consistency of the chemical composition of fat-free mass (FFM) than is the case with hydrodensitometry; thus, DXA could potentially replace hydrodensitometry as the criterion method for validation of anthropometric measurements of fat, according to Kohrt (see Chapter 6 in this volume).
Other investigators point out that, although DXA shows much promise in its ability to assess fat accurately (Haarbo et al., 1991), the susceptibility of DXA estimations of FFM to changes in hydration status (Formica et al., 1993; Horber et al., 1992) and protein content, and its inability to analyze the composition of soft tissues that lie close to bone (Tothill and Nord, 1995)
demonstrate that more development is necessary. In addition, DXA is expensive (analyzer costs range from $120,000 to $150,000) and cannot be used in the field.
Bioelectrical Impedance Analysis and Other Techniques
Other techniques that have been utilized, particularly in the field, include near-infrared (NIR) spectroscopy, ultrasound, and bioelectric impedance analysis (BIA). Using hydrodensitometric determinations and anthropometric measurements as the standard, NIR spectroscopy has proven no better than simple height and weight measures (Israel et al., 1989), while ultrasound has proven no better than anthropometric measurements (Hodgdon and Fitzgerald, 1987; see Friedl, Chapter 4 in this volume).
BIA, reviewed in Chapter 7 by Wm. Cameron Chumlea and Shumei S. Guo, measures current flow through the body and was first used to assess hydration status (Nyboer, 1959). The measurements are based upon the assumption that the body is a water-and electrolyte-filled cylinder. When BIA is used to assess body composition, there is a requirement to generate mathematical equations that must be validated against some other criterion method of BC assessment.
BIA has the dual advantages of being noninvasive and relying upon equipment that is relatively portable. The use of BIA to assess body composition has, until recently, relied on measurements at a single frequency (of 50 Hz). Such estimates of body composition are often unreliable (Chumlea et al., 1996). Further, single-frequency BIA is not recommended for use in longitudinal studies since it is not a valid indicator of changes in BC within the same individual over time, particularly if the change is slow or is accounted for primarily by a change in fat content (Forbes et al., 1992).
An alternative method of BIA performs measurements at multiple frequencies. This method is an improvement over single-frequency methods in that data are fitted to a theoretical curve, and the frequencies that best fit the curve are used to estimate BC (Chumlea et al., 1996). A third alternative is the use of bioimpedance spectroscopy (BIS), which measures multiple components of impedance over a spectrum of frequencies and shows considerable promise for longitudinal assessment of body composition (Lukaski, 1996).
The placement of electrodes also influences the utility of BIA measurements. While electrodes are routinely placed on the distal extremities, proximal (toward the trunk) placement of electrodes has been shown to improve the precision of body composition assessment, although fluid accumulation in the extremities must be monitored (Lukaski, 1996). Segmental impedance measures, particularly when performed with BIS, have shown promise in the assessment of changes in body fat (Chumlea et al., 1996).
Because BIA is based on the assessment of total body water, its utility is affected by interindividual differences in hydrational status as well as any
factors that influence intraindividual hydrational status, such as fluid intake, physical activity, nutritional status, illness, and environmental factors (Kushner et al., 1996). Among the recommendations of the 1994 National Institutes of Health Technology Assessment Conference on Bioelectrical Impedance Analysis in Body Composition Measurement (NIH, 1996) are that such variables be standardized and controlled and that additional research, validation, and standardization of procedures be performed.
Computerized Axial Tomography and Magnetic Resonance Imaging
The use of computerized axial tomography (CAT) and magnetic resonance imaging (MRI) (also known as nuclear magnetic resonance) for BC assessment is reviewed by Steven Heymsfield, Robert Ross, ZiMian Wang, and David Frager in Chapter 5 in this volume. CAT is based on the ability of tissues of varying composition to attenuate an x-ray beam to differing extents, with image reconstruction based on mathematical techniques such as Fourier analysis. MRI is based on the action of hydrogen nuclei in the presence of a strong magnetic field, which causes the nuclei to align either with or against the field in a predictable manner. Each orientation has a slightly different energy state; oscillation of the magnetic field at a designated frequency causes the nuclei to flip or precess between orientations with a resulting release or absorption of energy (Gadian, 1982). When the magnet is "turned off," the nuclei return to their original energy state (relax), and the released energy generates an image. The nuclear density and relaxation times are tissue-type dependent. Both CAT and MRI produce high-resolution, cross-sectional images of the whole body or body regions. These images are then analyzed by computer to estimate tissue-and organ-level body composition, and algorithms are applied to reconstruct and estimate the total volume of the tissue or system in question (see Heymsfield et al., Chapter 5 in this volume).
While both CAT and MRI permit clear visualization of the boundaries between adipose tissue, muscle, and bone and quantification of all major tissue components, CAT has the relative disadvantages of causing some radiation exposure and being extremely costly, according to Heymsfield and coworkers (see Chapter 5 in this volume). In contrast, MRI has no known health risks and has the added advantage of permitting the calculation of tissue volumes but is expensive. Both CAT and MRI have been validated using phantoms (composed of tissue or analogous materials), laboratory animals, and human cadavers and both appear to be accurate and reproducible, with MRI having the advantage of even greater precision. Because of its accuracy, its ability to provide cross-sectional imaging and make regional and whole-body measures, and its availability and apparent safety, MRI could provide criterion data for generation of equations to predict BC in diverse populations as well as permitting longitudinal and intervention studies that require multiple or serial measures.
According to Heymsfield and coworkers (see Chapter 5 in this volume), the primary disadvantage of MRI is the preclusion from study until recently of individuals with claustrophobia and the very obese. The availability of open MRI facilities will eliminate this problem. Other disadvantages include cost; sensitivity to motion artifacts, especially in the abdominal region; time required to analyze images; and a technical question regarding the (mathematical) conversion of adipose tissue volume to fat mass.
Included among the future trends for these technologies are CAT spiral imaging, which should permit better reconstruction of BC components and decrease required radiation dose (Fishman, 1994); linkage of MRI to magnetic resonance spectroscopy to permit the study of metabolic processes in vivo; and decrease in the time required to take and analyze MRI images because of technical advances (Jolesz, 1992).
Correlation between Body Composition, Health Status, and Physical Performance
The correlation of total body fat content or fat distribution with actual health, nutritional status, physical activity (fitness), and appearance measures is a major concern for the military (see Friedl, Chapter 4 in this volume).
The correlation between intra-abdominal fat stores, abdominal girth, and cardiovascular (CV) disease risk is well known (Larsson et al., 1984; Metropolitan Life Insurance Company, 1937); thus, there may be reason to assess fat content in that region rather than to assess total fat. In men, intra-abdominal fat deposition is highly correlated with abdominal girth (Despres et al., 1991), which is one of the anthropometric measures included in the Army's BC equation. No such relationship exists for women, however, except for the very heaviest women (Kvist et al., 1986; Vogel and Friedl, 1992; Weits et al., 1988). In women with the highest lean body mass (i.e., those with greatest physical strength), percentage of body fat tends to be overestimated by equations that include a measure of abdominal girth (Hodgdon and Beckett, 1984). Because such women also tend to exhibit male-type fat distribution with the same CV risk factors, the abdominal girth that correlates with greatest physical strength also may predispose these women to increased CV risk (Evans et al., 1983). Thus, women whose physical performance is greatest (as measured by lifting and carrying) are those most likely to exceed body fat standards and to carry the greatest health risk.
It is fairly well established that physical strength is independent of body fat (Sharp et al., 1994). The current military (Army) weight control programs do not select for physical performance nor do they predict strength. While the association between total or regional muscle mass and specific measures of physical performance remains unclear, FFM is the BC component most likely to correlate with physical performance as measured by lifting and carrying
capacity (Fitzgerald et al., 1986; Johnson et al., 1994). However, an assessment of FFM is not currently a part of the military BC standards (AR 600-9, 1986).
Because the assessment of changes in nutritional and/or hydration status during military operational activities is one of the primary interests of the MND, considerable effort has been expended to explore the use of BC assessment techniques both in the field and in the laboratory using pre-and posttreatment (exercise) sampling designs to monitor such changes. Unfortunately, as Friedl points out, the standard anthropometric measures of body composition fail to reflect accurately the true changes in weight and composition over time (Ballor and Katch, 1989). In terms of performance, the real factor of interest, and the more practical measure in field settings, is rate of weight loss. While skinfold thickness appears to detect changes in fat mass with greater reliability than does BIA or hydrodensitometry, it is still relatively insensitive for detecting small changes in fat mass (Jebb et al., 1993). Because changes in body weight or exposure to environmental extremes tends to lead to measurable changes in body water content, the use of DXA or hydrodensitometry to assess changes in body composition under such conditions may result in serious measurement artifacts. BIA may provide relatively precise assessments of changes in body water, but interpretation of changes in body composition are problematic (Friedl et al., 1994a, b; Fulco et al., 1992; Westphal et al., 1995). Thus, there is a need for more than one type of measure to assess changes in BC over time.
Body composition assessment is performed by the military for several purposes. The primary purpose is to establish adherence to weight-for-height standards. All branches of the military maintain such standards for active-duty personnel. The reasons for these standards, while somewhat branch specific, include concerns about readiness, appearance, and health. Personnel who fall outside of the weight range for their height must undergo an assessment of body fat by circumferential measurement. The gender-and branch-specific equations that use these circumferential measures to calculate body fat have been validated with the criterion measurement of hydrodensitometry. Because hydrodensitometry is based on assumptions that are not valid in the field (constancy of body compartment composition) and is problematic to perform, other criterion methods are sought by which to validate and, if necessary, refine the military equations. A four-compartment model that relies on DXA to assess bone, soft lean tissue, and fat and requires measurement of body water as well appears to offer such a criterion method for validation of existing equations for routine body composition assessment. Validated BC equations requiring only circumferential measures continue to be used by the military because of practicality and cost.
Body composition measurement also is used by the military to assess the effects of field training exercises and fitness regimens on gain or loss of lean
tissue and fat mass, in a pre-and posttreatment type of design. Although useful in the laboratory, the techniques of DXA, MRI, and CAT were acknowledged to be generally impractical for field use because of their cost and relative immobility. They are also almost as limited in their ability to detect and accurately measure small changes (< 5 kg or 11 lb) in body composition as is hydrodensitometry. The utility of BIA for assessing body composition is still limited by the need for technical improvements aimed at increasing its validity.
Muscle mass is the body compartment most closely predictive of physical performance. Many of the methods described could estimate muscle mass accurately enough to predict performance; however, at present the focus of military BC assessment remains on fat. Technology that would enable measurement of small changes in muscle mass has not yet been developed.
Finally, the military utilizes the measurement of abdominal girth to predict long-term risk for cardiovascular disease in active-duty men but not women.
TRACER TECHNIQUES FOR THE STUDY OF METABOLISM
In this section, the use of stable isotopes for evaluation of metabolic processes is discussed by Dennis M. Bier, Robert R. Wolfe, and V. R. Young and coworkers (see Chapters 8, 9, and 10 in this volume). Gerald I. Shulman's description of nuclear magnetic resonance as a tool to study metabolism follows (see Chapter 11 in this volume). While James P. DeLany uses the doubly labeled water technique to measure energy expenditure (see Chapter 12 in this volume), a more detailed summary of his presentation is included in the following section due to its extensive use in the field.
Use of Isotopic Tracers to Study Metabolism
Stable isotopes are naturally occurring forms of atoms that, by nature of the increased number of neutrons in their nuclei, can be "traced" with mass spectrometry. Because these atoms are found naturally and can be concentrated in major biological pools and fuels, this technology can be used to monitor quantitatively the rate and outcome of metabolic processes in the body precisely and accurately (see Wolfe, Chapter 9 in this volume). Specifically, by monitoring the steady-state tracer dilution curve of a given isotopically labeled molecule (precursor evaluation) within a metabolic pool or the accumulation of a labeled product such as urea, ammonia, or lactate, the processes contributing to the utilization or production of that molecule and the relative rate of those processes can be monitored. This technique currently is used routinely to monitor rate of protein turnover using 15N-glycine or 13C-leucine (both synthesis and breakdown, see Wolfe, Chapter 9 in this volume), absolute energy expenditure using doubly labeled water (water labeled with both 2H and 18O; see committee summary below and DeLany, Chapter 12 in this volume), and the
rate and relative amount of metabolic substrate oxidation and gluconeogenesis using 13C-and 2H-labeled forms of glucose, glycerol, and fatty acids (see Bier and Wolfe, Chapters 8 and 9 in this volume).
The applicability of the use of stable isotopes in field situations depends upon the route of administration of label and the pool to be sampled. Oral administration of label followed by sampling of urine, as is done when monitoring protein turnover or energy expenditure, can be accomplished in the field, with samples saved for analysis in the laboratory. However, intravenous infusion and arterial or venous sampling of label, as is required for measurement of metabolic fuel oxidation, is better suited to the research laboratory.
Dennis M. Bier points out in Chapter 8 that the use of stable isotopes is somewhat more problematic than the use of radioactive isotopes. Because stable isotopes are naturally occurring, corrections must be made in calculations for the "preenrichment" composition of the pool being studied. In addition, the enriched sample may exchange its isotopic atoms for nonlabeled ones and complicate computation of the enrichment above baseline or specific activity (the tracer/tracee ratio or algebraic sum of tracer outflow to cells and unlabeled inflow to the pool from its various sources); recycling of label also is problematic, limiting the time course of most investigations. Finally, labeled molecules often must be derivatized before analysis, and these derivatizations are not trivial. The development of reliable and accurate methods requires dedicated staff and laboratory space.
The use of stable isotopes to study metabolism can be sensitive, however, allowing the monitoring of even single atoms, according to Bier (see Chapter 8 in this volume). Thus, it requires only small samples of the pool of interest for assessment. Further treatment of samples (derivatization and ionization) can increase sensitivity and precision. In addition, the use of multiple isotopes and separation of molecules labeled at more than one site (mass isotopomer distribution analysis) allow further elucidation of the product-precursor relationship, which maximizes the information gathered in any experiment.
The major advantages of the use of stable isotopes are associated with safety. There are no risks for persons involved in the transportation, storage, or hauling of the stable isotopes. It is also environmentally safe, and the high cost of waste management associated with radioisotopes is eliminated. The method is also safe because the isotopes present no health hazard to the subjects, and the sampling is primarily through easily accessed pools of blood, breath, or urine. In addition, as Robert R. Wolfe points out in Chapter 9, the method is less invasive than muscle biopsy or measurements of arteriovenous differences across an organ bed, which require catheterization of both the inflow and outflow to a particular organ. It should be noted, however, that the use of stable isotopes in conjunction with muscle biopsies or arteriovenous difference measurements allows the researcher to evaluate organ-specific metabolic processes (such as protein synthesis specifically in muscle rather than whole body or fuel utilization across an exercising limb) (Essen et al., 1992; Tessari et al., 1995; see
Young et al., Chapter 10 in this volume). In addition, the use of stable isotopes to monitor metabolic processes is safe to the environment, requiring no special disposal arrangements.
Stable isotope methodology requires highly sophisticated equipment for preparation and analysis of samples taken from body pools, with highly trained and attentive staff to do the derivatizations and run the equipment, as well as trained investigators to evaluate the validity of the data produced. Although the cost of the mass spectrometers used to analyze the derivatized products has decreased in recent years (gas chromatography mass spectrometer [GC-MS] costs about $75,000), the initial outlay for setting up a facility is still expensive because more than one kind of spectrometer usually is required to analyze a variety of isotopic tracers in one experiment (GC-MS may be used for 15N or 2H, but an isotope ratio mass spectrometer [$200,000] is required if 13C or 18O are to be used). Isotope prices themselves are no longer prohibitive, although not trivial (approximately $200–$500 per test per subject) (Personal communication, G. E. Butterfield, Palo Alto Veterans Affairs Health Care System, Palo Alto, Calif., 1996).
The continued development of the field is demonstrated by Bier, Wolfe, and Young and coworkers (see Chapters 8, 9, and 10 in this volume). Bier describes the use of high-energy bombardment to ionize high molecular weight molecules (matrix-assisted laser desorption ionization) so that fuel precursors, such as glycogen and protein, can be studied using very little product.
Wolfe (see Chapter 9 in this volume) describes the process of monitoring the naturally occurring 13C to 12C ratio in expired air in individuals "primed" with 13C-glycogen to determine utilization of endogenous fuel stores (glycogen, fat, and protein), a methodology made possible by an increase in the sensitivity of mass spectrometers. Although this latter method may hold promise for field use because it requires only breath sampling, it still relies on indirect calorimetry for estimation of total fuel oxidation, necessitating the concomitant use of an additional technology for measurement of oxygen consumption and carbon dioxide production, according to Wolfe (see below, and also see DeLany, Chapter 12 in this volume) to give fruitful data.
Use of Positron Emission Tomography to Study Protein Metabolism
V. R. Young, Y-M. Yu, H. Hsu, J. W. Babich, N. Alpert, R. C. Tompkins, and A. J. Fischman (see Chapter 10 in this volume) describe the use of positron emission tomography (PET) as an emerging tool for the evaluation of protein metabolism. This method involves the use of readioactive atoms of carbon, nitrogen, oxygen, and fluorine that are made by bombarding naturally occurring
nuclei with particles generated by a cyclotron. These atoms can be incorporated into a variety of biological fuels (glucose, oxygen, and fatty acids), and the organ-or organelle-specific metabolism of these fuels then can be followed using the noninvasive technique of PET. The use of this technique in conjunction with stable isotopes is proposed by Young and coworkers to evaluate the contribution of individual organs and body areas to whole-body protein metabolic processes. They describe a preliminary study in which 11C-methionine was used to assess hind limb-muscle protein breakdown in dogs (Hsu et al., 1996), and good agreement with previously published values was found (Biolo et al., 1995). According to Young and coworkers (see Chapter 10 in this volume), the efficacy of this method in humans in the field is questionable due to the radioactive nature of the nuclei introduced, short half-life of those nuclei, and extreme cost of the facilities necessary to produce and monitor the nuclei (the approximate cost is $2,000,000 for a cyclotron facility, making this method purely a research tool at this time). Although the chapter focuses on the use of PET in evaluating protein metabolism, the technique can be useful for measuring the utilization of a range of metabolic components in a variety of organs.
Nuclear Magnetic Resonance as a Tool to Study Metabolism
Nuclear magnetic resonance (NMR), a measurement technology whose use in body composition assessment is discussed earlier (see page 10), also allows noninvasive investigation of intracellular processes. The theory behind the method is described by Gerald I. Schulman (see Chapter 11 in this volume), as well as Heymsfield and coworkers (see Chapter 5 in this volume).
NMR can be used to monitor water and fat in the whole body by activation of hydrogen nuclei (Jardetzky and Roberts, 1981) or can be used to evaluate more specific processes on an individual organ level using the naturally occurring 13C or 31P nuclei (Rothman et al., 1992). Because the natural abundance of these latter nuclei is so low, their measurement is less sensitive. However, because they are found in molecules of particular interest, such as glycogen and adenosine triphosphate (ATP), they are used to monitor, respectively, liver and muscle glycogen metabolism and muscle glycogen synthesis, as well as ATP production and utilization (Shulman et al., 1990). In addition, new techniques of editing signals, stronger magnets, and improved software recently have increased sensitivity (see Shulman, Chapter 11 in this volume).
The technique requires a large magnet into which a body or body part can be placed. Although mobile units are available and expand the utility of the method, other factors limit its applicability in the field. Extensive electronics, computer equipment, and software are required for analysis of signals produced; an electrical engineer or physicist is required as full-time attendant for maintenance and interpretation (Personal communication, J. Kent-Braun, San
Francisco Veterans Affairs Health Care System, San Francisco, Calif., 1996). Thus, the method is expensive (a good magnet may run $500,000 to $2,000,000).
Stable isotopes can be used to study turnover of protein, carbohydrates, and fats and thus allow monitoring of changes in energy expenditure, relative fuel utilization, and gluconeogenesis, as well as a wide variety of other aspects of metabolic substrate oxidation in response to various stimuli. The ability to adapt these techniques for field use depends upon the route of isotope administration and the pool to be sampled. A significant problem that must be overcome with the use of stable isotopes, particularly in the field, is the natural abundance of these isotopes, which differs from place to place. In addition, while the cost of analytical equipment limits the processing of samples to large, well-funded research facilities, the cost of the isotopes themselves limits to some extent the numbers of subjects who can be included in studies.
PET utilizes radioisotopes incorporated into biological fuels to study organspecific substrate metabolism noninvasively. Use of this technique in the field is proscribed by its reliance upon radioisotopes and the size and immobility of the PET scanner. In addition, the cost of the equipment limits its use to that of a purely research tool.
The use of NMR to study body composition (MRI) and whole-body, organspecific metabolism also is described. NMR is expensive, and its technical complexity makes it a technique that is of use primarily to the research laboratory. With the availability of more mobile units, this technique may offer a noninvasive way of measuring metabolic processes in real time.
AMBULATORY TECHNIQUES FOR MEASUREMENT OF ENERGY EXPENDITURE
The measurement of energy expenditure and fuel utilization during military operations is of considerable importance to the MND. Total energy expenditure, as well as preferential fuel utilization, has been measured during special operations under a variety of environmental conditions (see IOM, 1993a, 1996), as well as in garrison, with the aim of optimizing the nutrient contents of military rations, designing supplemental rations for work in extreme conditions, and providing nutritional advice to soldiers. The need remains for optimizing these measurements with the best techniques available, for more accurately measuring total body protein turnover, and for determining energy expenditure in populations of female soldiers, among other groups.
This section reviews the discussion by: James P. DeLany on doubly labeled water for energy expenditure, Reed W. Hoyt and Peter G. Weyand on foot
Doubly Labeled Water for Energy Expenditure
The use of the doubly labeled water (DLW) method for the determination of energy expenditure, as described by James P. DeLany in Chapter 12 in this volume, is based on many of the principles described above regarding stable isotopes. It employs the differential excretion of two forms of water labeled with stable isotopes (2H2O and H218O) as a means of estimating carbon dioxide production and inferring oxygen consumption (Lifson and McClintock, 1966; Lifson et al., 1975). Deuterated water (2H2O) leaves the body in the water compartment (measured via urine or saliva), and the rate of dilution of the ingested dose is a measure of the production of metabolic water, which is proportional to the energy expended and the dilution that occurs due to fluid intake. Water labeled with oxygen-18 leaves the body as both water and carbon dioxide (see DeLany, Chapter 12 in this volume). Thus, if the regression lines for the excretion in urine or saliva of the two isotopes are compared, the difference between the two lines represents the excretion in the breath of label as carbon dioxide. Knowing carbon dioxide excretion and the respiratory quotient (RQ = carbon dioxide produced/oxygen consumed), an estimate of oxygen consumption can be made and energy expenditure computed using the energy equivalent of oxygen at the RQ measured.
According to DeLany, the method has the advantage of being noninvasive because it requires only that subject drink the labeled water and collect urine or saliva at the beginning and end of the testing period. It also requires no special dietary considerations on the part of the subject, thus increasing compliance. Also according to DeLany, the incorporation of additional sampling times other than start and end points does not increase the accuracy of the method, with the sample-to-sample variation being greater than the increased reliability obtained, although others do not agree with this assessment (Cole and Coward, 1992; Edwards et al., 1991).
Analysis of the data, in contrast, requires a great deal of care and attention. Calculations must be corrected for background enrichment, which may vary with the normal water supply and may change if the water supply itself changes (Dansgaard, 1964). Corrections must be made for fractionation of the deuterium in the body water compartments, such that evaporative losses from lung and skin have a greater deuterium content (Lifson and McClintock, 1966); correction must be made for the differential distribution of the two isotopes in total body water (ND/TBW = 1.041; NO/TBW = 1.007) (Coward et al., 1994; Racette et al., 1994). Sample analysis is problematic in that the isotopes may fractionate during preparation, or the sample matrix may interfere with measurements (as with saliva) (Ritz et al., 1994). Finally, the method gives only
an integrated value for average daily energy expenditure over 4 to 21 days and cannot clearly differentiate energy requirements on individual days (Coward, 1990).
This method has been used successfully in several military field studies to determine energy expenditure and water turnover under a variety of circumstances (Moore et al., 1992). Situations of high water turnover have been found to flush out the isotope and require large initial doses or redosing on subsequent days to attain information over long study periods (such as 28 days) (DeLany et al., 1989).
The major disadvantage of the DLW method is its cost; according to DeLany, an isotope ratio spectrometer is required for analysis, and extensive laboratory facilities are required for preparation of samples for analysis. The isotopes are now easily obtained and cost about $400 per dose. These costs prohibit use in large-scale troop assessments, especially if repeated dosing is required due to the high rate of energy expenditure (DeLany et al., 1989).
Using Foot Contact Time to Estimate the Metabolic Cost of Locomotion
Determination of the metabolic costs of locomotion is of interest to the military in determining energy needs (and food supplies) for troops in the field. In Chapter 14 in this volume, Reed W. Hoyt and Peter G. Weyand describe a device and a microcontroller that can be inserted into the shoe of the soldier to record the time of foot contact on the ground with each step. This device, which is constructed of a sensor circuit and an analog-digital converter, is called a foot contact monitor (FCM). The information derived from such a device can be used in conjunction with body weight to determine the metabolic cost of locomotion. This device has been used in the laboratory setting to determine the validity of the measure of metabolic cost of locomotion from foot contact time and body weight, and has been found to produce data that vary less than 20 percent from measured cost (Hoyt et al., 1994).
The device has the ability to measure the energy cost of locomotion in free-living conditions and estimate absolute or relative fitness levels as they change with training (Blair et al., 1992). The authors further point out that the device could be used to clarify the relationship between walking or running and training injuries, and to provide a biofeedback tool (with a FCM attached to a display of real time energy expenditure) to weight control programs.
According to Hoyt and Weyand, the device has several advantages. It offers the potential to estimate accurately the expenditure of energy at a variety of activities of varying intensities without the need for indirect calorimetry and breath gas collection. Data are expressed in standard energy units, and there is no need for calibration beyond determination of body weight. In addition, the ease of use and lack of subject input make it suitable to large epidemiological and field studies. Finally, the cost is low, estimated to be $50 to $100 for each device.
The disadvantages of the device, according to the authors, include the failure to determine total energy expenditure or that for upper-body work; the need to incorporate body weight, which may change over time and while carrying heavy equipment; and the inability to account for up-and downhill movement.
Near-Infrared Spectroscopy for Measuring Plasma Metabolites
In Chapter 15 in this volume, Donald Bodenner discusses the development of techniques using NIR spectroscopy to measure the concentration of organic compounds in complex mixtures such as food and blood. These techniques could be used for the noninvasive measurement of plasma metabolite concentrations because most organic compounds exhibit unique absorption spectra when scanned over the range of NIR wavelengths. The wavelength at which peak height is proportional to concentration must be determined for the compound of interest. While concentrations of single compounds in solution traditionally are deduced from Beer's law, the use of multiple linear regression analysis to construct regression equations that correct for interfering peaks (Beebe and Kowalske, 1987; Haaland, 1992; Honigs et al., 1983) enables interfering substances to be taken into account. In addition, the use of complex mixtures with known concentrations of constituents as standards (sometimes the subjects' own blood) will increase accuracy.
In vitro, NIR spectroscopy has been used widely to measure food constituents, such as protein in flour and sugar in breakfast cereals (Baker and Norris, 1985; Hruschka and Norris, 1982). Of clinical relevance, NIR has been used for the measurement of serum cholesterol (Peuchant et al., 1987) and fecal fat (Peuchant et al., 1988). In vivo, NIR spectroscopy has been used as a noninvasive way to measure tissue oxygenation through fingertip monitoring blood flow (Jobsis, 1977). Other applications have included monitoring hemoglobin saturation in the liver (Kitai et al., 1993), changes in protein and lipid in the brain (Carney et al., 1993), and blood flow and oxygen consumption in fetal brain (Faris et al., 1994). Measurement of plasma metabolites in vivo using this noninvasive technique is a more formidable problem because the composition of blood and tissue is in constant flux. Some success has been achieved measuring plasma glucose in diabetic patients (Robinson et al., 1992); however, each patient's samples require an individual calibration curve (Meehan et al., 1992; Vallera et al., 1991). In addition, the effect of changes in other metabolites, such as urea, is unknown.
Two newer techniques are available to measure energy expenditure in ambulatory individuals. Doubly labeled water, the older of the two, utilizes
stable isotopes of water to measure average expenditure over a given time period. The technique is relatively expensive to employ, and analysis of the data requires considerable care. A newer technique utilizes a small device built into a shoe to estimate the metabolic cost of locomotion by measuring foot contact time. Although considerably less expensive, easier to interpret, and potentially able to monitor the effects of an imposed stimulus, this technique cannot as yet measure total energy expenditure or that resulting from upper body exertion. The technique of near-infrared spectroscopy has been used to monitor blood metabolites noninvasively. Its potential for field use awaits considerable technical improvement, according to the Bodenner (see Chapter 15 in this volume).
Molecular and Cellular Approaches to Nutrition
In Chapter 17 in this volume, Howard C. Towle provides an overview of the processes that contribute to gene expression and describes the techniques used to study the regulation of gene expression, drawing for examples upon genes involved in nutrient metabolism. In Chapter 16, Raymond K. Blanchard and Robert J. Cousins review what is known about genes whose expression is regulated or influenced by trace minerals, particularly iron and zinc. In Chapter 18, Donald B. McCormick describes the use of isolated cell techniques to study the cellular uptake and metabolism of naturally occurring glucosides of two water-soluble vitamins, riboflavin and pyridoxine. Guy Miller (see Chapter 19 in this volume) discusses the use of isolated cell systems to study the impact of hypoxia and oxidative stress on cellular function.
Regulation of Gene Expression by Nutrients and Metabolites
Gene expression is a multistep process involving transcription of DNA to RNA (ribosomal, transfer, and messenger), mRNA processing (capping, splicing, and polyadenylation), transport of the processed message to the cytoplasm, and attachment to ribosomes and translation of the message to protein. Control of gene expression can be exerted at many different sites in the process, but transcription is the most common site for regulation.
Howard C. Towle (Chapter 17 in this volume) cites several examples of metabolic pathways in which the role of nutrient-or metabolite-mediated regulation at the transcriptional level has been examined and transcription factors identified. One such pathway is that of biosynthesis of cholesterol in which the factors that mediate the cholesterol-sensitive regulation of transcription of the biosynthetic enzymes have been identified (Goldstein and Brown, 1990; Hua et al., 1993; Yang et al., 1994). Another example is the synthesis of triglyceride that is induced by feeding a diet high in carbohydrates; the genes for the enzymes whose transcription is induced share a common
sequence in their promoter regions (Lefrancois-Martinez et al., 1994; Shih et al., 1995). In neither of these two examples are the exact mechanisms understood by which a nutrient or metabolite alters the binding of the transcription factors to the promoter regions. In contrast, Towle describes some members of the steroid receptor family, all of which are themselves transcription factors that are induced by dietary components or metabolites (Forman et al., 1995; O'Malley and Conneely, 1992).
Raymond K. Blanchard and Robert J. Cousins (Chapter 16 in this volume) describe the roles played by trace metals, such as zinc, in the control of gene expression. Three mechanisms have been identified by which metals may influence gene expression (Cousins, 1995). First, the metal may play a structural role, facilitating interaction among binding groups to alter their conformation. Alternatively, the metal may catalyze an enzymatic reaction required for gene expression. Finally, the metal may bind to a specific factor involved in the initiation of transcription. These zinc-containing transcription factors, known as zinc-finger proteins, exhibit what are called zinc-finger domains, which contain zinc and may assume one of four different conformations (Dawid et al., 1995; Klug and Schwabe, 1995; Vallee et al., 1991).
The metalloprotein-binding consensus sequences found in the promoters of metal-sensitive genes are termed metal-responsive elements. These may overlap or about other sites, are orientation-independent, and can function in heterologous promoters (a property that has been exploited extensively, as described below) (Cousins, 1994; Stuart et al., 1984).
Applications for Techniques Used to Study Gene Expression
The ability of nutrients and their metabolites to influence metabolic processes by altering gene expression has been recognized for at least 20 years. While the techniques described both by Blanchard and Cousins and by Towle (see Chapters 16 and 17 in this volume) have permitted further elucidation of the mechanisms by which this occurs, understanding of the control of gene expression is still very basic and rudimentary and of limited immediate practical application.
Blanchard and Cousins describe several applications of the knowledge that has been gained from studying mechanisms of control of gene expression. Among them are the introduction of metal-responsive elements into heterologous genes of interest to produce metal-responsive transgenes, as exemplified by the creation of zinc-inducible growth hormone genes in mice and livestock (Palmiter et al., 1982). Another application involves synthesis of small peptides containing zinc-finger domains; because of their DNA sequence-specific binding capacity, they can inhibit the binding of actual zinc finger-containing transcription factors to promoter regions, thus preventing these factors from exerting their transcription-promoting effects (Rebar and Pabo, 1994; Wu et al., 1995). This is analogous to the use that has been proposed for antisense DNA
(the noncoding or nonsense strand of DNA that is complementary to a region encoding a gene or its control sequence).
It is generally recognized that the synthesis of a protein may be influenced at a number of different stages, all of which may be roughly categorized as either transcriptional, translational, or posttranslational in nature. As Towle points out in Chapter 17 in this volume, the impact of nutrients and metabolites on the transcriptional control of gene expression, in terms of adaptation, is likely to be a long-term one, with the effects being observed over a matter of hours to days, rather than being a short-term adaptive response, such as the response of ferritin mRNA translation to increasing serum concentrations of iron. The implications of long-term adaptive responses presumably include allowing the organism to operate more efficiently in the face of changing nutritional status. Although the significance of the results of techniques such as ''differential display" (that is, techniques that show which genes are being expressed under a specific set of conditions) (Kendall and Christensen, 1995; Liang et al., 1993; Orr et al., 1994) may be difficult to interpret, they may permit the identification of genes that would be beneficial to control during stressful situations. Blanchard and Cousins and Towle also raise the future possibility of combining nutritional therapy with gene modification, which might permit enhancement, for example, of the body's ability to counteract exposure to ionizing radiation, toxins, or oxidative conditions. It has been shown that high-level zinc supplementation provides protection against the cellular damage caused by exposure to ionizing radiation as well as a number of chemical toxins. It is believed that the mechanism responsible involves the prevention of free radical formation, although at what level(s) the zinc exerts control is unknown (Willson, 1989).
Isolated Cell Approaches
Isolated cell systems are being used increasingly to investigate physiological phenomena at the cellular level. In their two chapters, Donald B. McCormick (see Chapter 18 in this volume) and Guy Miller (see Chapter 19 in this volume) provides examples of current studies utilizing this approach. In addition to the more research-oriented applications described in Chapters 18 and 19, a number of potential clinical applications for isolated cell techniques are being evaluated. These include in vitro toxicity and allergy testing and drug-efficacy screening (Purcell and Atterwill, 1994).
According to Miller, the growing popularity of this approach stems from its relative ease of use (once requisite training is obtained and adequate facilities are in place) and the ability it confers on the investigator to isolate the experimental system, control variables rigidly, process a large number of samples within a short time frame, and generate data rapidly. While these are important advantages, it must also be verified that the in vitro test is a proper model for the in vivo question being asked. Miller enumerates the issues
involved in the use of these techniques in general and the choice of cell types for investigations of particular questions. He emphasizes that cellular responses to stimuli, in his case to the stimulus of hypoxic stress, are dependent on the cell type, the nature of the stress, and the environment (cellular, exocrine, and endocrine) of the cells so that, if the purpose of using an isolated cell system is to model a more complex in vivo counterpart (which is almost always the case), the choice of cell system becomes critical. As a general rule, to maximize the utility of a particular cell model, the cell type chosen must display a pattern of response and sensitivity to the stimulus similar to the tissue or system of interest. An important challenge to the workers in this field will be to present convincing evidence that results obtained from such cell-based systems (in both the clinical and the research laboratory) are indeed applicable to intact organ systems.
Increased understanding of the mechanisms of gene expression has led to the development of techniques such as differential display that permit the identification of genes whose transcription markedly changes in response to a given stimulus. Such knowledge ultimately may lead to the development of preventive or enhancing treatments that work at the level of promoters or suppressers of transcription. The use of isolated cell systems enables researchers to examine the effects of stimuli at the organelle or cellular level where variables can be controlled that would not be possible to control in vivo. Great care must be taken to choose cell culture models that are as similar in response as possible to the entire organism and situation in question. Nevertheless, both gene expression and isolated cell approaches to nutritional problems are limited in their application and are largely restricted to basic research at this point.
ASSESSMENT OF IMMUNE FUNCTION
The role of nutrition in immune function has been of considerable interest to the military since it was established that the proinflammatory hormones secreted in response to infection were responsible for the anorexia, weight loss, redistribution of trace minerals, and shift in hepatic protein synthesis toward production of acute-phase proteins that are characteristic of this state (Beisel, 1980). Subsequent research has established that the hormones in question are the cytokines tumor necrosis factor-alpha (TNF-a), interleukin-1 (IL-1), and IL-6 (Dinarello, 1994). Earlier reports of the CMNR (IOM, 1992, 1993b) helped to identify the possible part played by acute undernutrition in the immune compromise suffered by Special Forces troops and helped pave the way for initiatives in optimization and assessment of immune function in the field. In addition, the military maintains a special interest in the development of
improved methods of immunization, particularly oral vaccines directed against gastrointestinal and respiratory pathogens. In this section, Lyle L. Moldawer and Gabriel Virella, Candace Enockson, and Mariano La Via review techniques for assessment of abnormal immune function (see Chapters 20 and 21, respectively, in this volume), while COL Arthur O. Anderson focuses on the development of vaccines targeted toward mucosal immune function (see Chapter 22 in this volume).
New Approaches to the Study of Abnormal Immune Function
In Chapters 20 and 21 in this volume, Lyle L. Moldawer and Gabriel Virella and coworkers identify parameters of abnormal immune function that are valid and can be measured reproducibly in the field. This identification is complicated by the complexity of the immune system and by uncertainty as to how much assays of circulating factors or immune cells truly reflect the physiological state of organs responsible for the initiation and sustainment of immune responses.
Evaluation of Humoral Immunity (Production of Antibodies)
While humoral immune responses are easy to measure, the concentrations of circulating or secreted antibodies at any given time correlate poorly with the ability to mount an immune response to antigen (Virella, 1993a, b). In addition, wide variability exists in both primary and secondary humoral responses to an antigen challenge. For example, Virella showed that administration of fish oil over an 18-wk period to healthy subjects resulted in a decrease in circulating immunoglobulins by the sixth week relative to subjects receiving olive oil; while levels of IgA and IgM returned to normal by the eighteenth week, IgG remained suppressed. The considerable intersubject variability of the data nearly obliterated the effect of the treatment; however, longitudinal measurements demonstrated significant effects.
Two additional measures of in vivo humoral immune response are described by Virella and coworkers. The measurement of a primary response, that is the response to an initial vaccination, can be confounded by prior exposure to the antigen. The alternative is to measure the secondary response to a booster vaccination. When subjects who were receiving supplements of fish or olive oil were challenged with tetanus toxoid booster, no difference was observed between the two groups in their ability to mount a response, although this type of immune assessment has been used in the past to evaluate the influence of nutritional status.
In vitro tests of humoral immune function (using isolated cells), in contrast, may not be sufficiently sensitive, require viable cells, and demand skilled personnel to perform them (Virella et al., 1991). Finally, the use of whole blood
for immunoassays adds confounding factors, such as inhibitors (Virella et al., 1988).
Evaluation of Cell-Mediated Immune Functions
Monoclonal antibody and flow cytometry technology permit the identification of a large number of subpopulations of lymphocytes in blood, based on membrane marker phenotype (Del Prete et al., 1995; Elson et al., 1994). However, due to the lack of strong correlations between membrane markers and cell function (Rahelu et al., 1993; Smyth and Ortaldo, 1993) and to the variability in the subpopulations (Hughes et al., 1994), the significance of changes in the distribution of lymphocyte subpopulations in peripheral blood is unclear. Furthermore, it is likely that the most significant changes occur not in blood but in lymphoid tissues, so that sampling peripheral blood may be irrelevant. Finally, changes in immunocompetence have not been related clearly to any documented change in lymphocyte profile (Virella et al., 1991).
Assays of Circulating Cytokines and Soluble Receptors. Thus far, at least 15 interleukins, a larger number of immunoreactive cytokines, and 3 types of interferon have been characterized and can be measured by enzyme-linked immunoabsorption assay. Of these, TNF-a, IL-1, and IL-6 have been the focus of much attention, primarily because they appear to be produced initially in response to inflammatory challenge and in turn induce secondary inflammatory mediators, such as IL-8 and some of the macrophage inflammatory proteins (see Moldawer, Chapter 20 in this volume). Hence, they have been viewed as the most proximal mediators of inflammation. Secondly, as cytokines, TNF-a, IL-1, and IL-6 are pluripotent. They were first identified (in the 1970s) as the activity called leukocyte endogenous mediator, induced after inflammatory challenge. In addition, they are the primary mediators responsible for the induction of the proinflammatory response to infection (Dinarello, 1994; Fong et al., 1990b; Powanda and Beisel, 1982).
A problem with discussing these cytokines is their dual nature: they can have both beneficial and detrimental effects (Beisel, 1975; Dinarello and Wolff, 1993). Thus, the challenge, according to Moldawer (see Chapter 20 in this volume), is how to interpret measurements of these cytokines when it is not clear whether they are playing a beneficial or an adverse role. The central questions are: (1) What is the diagnostic value of blood or urinary measurements of these cytokines for identifying the presence and magnitude of an inflammatory response? and (2) Can blood or urinary measurements be used to make decisions regarding therapy or performance in a field setting?
Under experimental conditions, cytokines are induced in response to the injection of certain bacteria (Fong et al., 1990a; Michie et al., 1988). In contrast, changes in the plasma concentrations of these cytokines are often not seen in
patients with most types of acute inflammation, that is, in those exhibiting an acute-phase response (Waage et al., 1989). A poor correlation has been observed between TNF-a levels in blood and burn-induced sepsis, and there is not a strong correlation with survival or mortality. The likelihood of detecting TNF-a in the blood of septic burn patients is only slightly greater than that in healthy controls, with a tendency for values to be higher in those who died (Marano et al., 1990). The relationship was even less strong for IL-1 (Marano et al., 1990). In contrast, IL-6 values were correlated highly with the presence of and mortality from systemic inflammatory response syndrome (Moscovitz et al., 1994). Thus, IL-6 may be a reliable indicator of the presence of infection, while IL-1 and TNF-a clearly are not.
The failure to observe infection-associated elevations in blood IL-1 and TNF-a levels is attributable to a number of factors. First, both IL-1 and TNF-a are secreted episodically (Beutler et al., 1986). Second, they both have short half-lives. These two characteristics alone would decrease the likelihood of reliably observing increases in the circulating plasma concentrations of the cytokines following infection (Beutler et al., 1986).
The third reason for failing to observe infection-associated increases in TNF-a and IL-1 is that there are factors in the blood that bind, inactivate, and inhibit the assay of these cytokines (Engelberts et al., 1991). These binding factors consist primarily of receptors that are proteolytically cleaved from the cell membrane during inflammation but which bind to the TNF-a and IL-1 in circulation, preventing them from binding to functional receptors or to antibodies (Engelberts et al., 1991; Moldawer, 1994). Concentrations of these shed receptors as well as that of another circulating inhibitor, IL-1 receptor antagonist, may provide an indirect estimate of the presence and intensity of inflammation (Moldawer, 1994; Van Zee et al., 1992).
Finally, the primarily paracrine production of these cytokines (that is, the synthesis and secretion of these locally acting substances into the intercellular spaces of adjacent cells) (Suter et al., 1992) can account for the inability to observe a correlation between circulating TNF-a and IL-1 and the degree of infection since any of the cytokine that appears in the blood probably represents spillover from some paracrine compartment or direct production in plasma by immune cells in the blood (Suter et al., 1992). Data from several groups (Suter et al., 1992; Personal communication, G. Schultz and L. M. Moldawer, University of Florida, Gainesville, 1995) support the role of compartmentalization in controlling circulating levels of TNF-a. Concentrations of TNF-a in wound sites of patients with nonhealing wounds and in bronchoalveolar lavage of patients with acute respiratory distress syndrome were elevated significantly over values observed in healing wounds or in the circulation, respectively. The same pattern was observed for IL-1 (Colotta et al., 1993).
Field Measurements of Cytokines. The difficulty of collecting blood samples in the field necessitates validating the use of urinary cytokine values as a reflection of those in blood (or tissue). Because many of the cytokine inhibitors were originally identified and purified from urine, it is clear that they are present in urine (Prieur et al., 1987; Seckinger et al., 1987a, b, 1988), although urinary levels tend to be lower than those in blood (see Virella et al., Chapter 21 in this volume). As for the cytokines themselves, increases in both circulating concentrations and urinary production of IL-6 and TNF-a have been observed in conditioned athletes after a 20-km run, consistent with a nonspecific inflammatory response (Sprenger et al., 1992). The increases observed in urinary excretion were markedly greater than those in blood (Sprenger et al., 1992). In contrast to the conditioned athletes, normal volunteers and unconditioned athletes had no IL-6 excreted in their urine. Hence, urinary IL-6 excretion appears to respond to an inflammatory challenge, suggesting that elevated urinary IL-6 measurements could be detected in samples collected in the field. Consideration must be given, however, to any factors that might alter urinary output and concentration (renal function and hydration status), and data must be expressed relative to osmolarity or some other parameter (such as creatinine) that reflects changes in urinary volume. Furthermore, the questions that remain to be answered regarding the correlation between levels of circulating cytokines and immune status and the relevance of circulating lymphokines relative to their concentration in lymphoid tissues must be answered for urinary cytokine levels as well.
One application of cytokine measurements that is of potential interest to the military has been an attempt to determine if weight loss during spaceflight is related to a nonspecific inflammatory response. Data suggest that the psychological stress of spaceflight and adjustment to the decrease in gravitational force may induce a proinflammatory cytokine response, which is followed by a secondary (postflight) response during readjustment to normal gravitation (Stein and Schluter, 1994).
In Vitro Assays of Lymphocyte Function
In vitro assays of lymphocyte function can be performed in a variety of ways (for example, by measurement of incorporation of labeled DNA precursors or release of cytokines) and may provide a better reflection of immune status. The disadvantage of in vitro assays, according to Virella and coworkers (see Chapter 21 in this volume), is their requirement for freshly isolated cells.
Mitogenic assays (in vitro), in which the proliferation of lymphocytes in response to a known irritant is measured, are the classic test of lymphocyte responsiveness (see Virella et al., Chapter 21 in this volume). These assays are typically performed on monocytes and have the disadvantage of requiring the use of radioactivity (3H-thymidine) or other expensive techniques. Furthermore, the assays are difficult to reproduce and are relatively insensitive. Recently, the
trend has shifted to looking at more physiologically relevant end points, such as the release of one of the interleukins, IL-2. Because IL-2 release is often decreased even when mitogenic indices are normal or elevated, IL-2 release may be a sensitive index of regulatory abnormalities affecting initial stages of T-helper cell proliferation (Virella et al., 1993). IL-2 receptor (CD25) expression in response to mitogenic stimulation also is considered a useful end point in studies of the effect of stress on the immune system (La Via et al., 1996). However, the method requires access to a large number of flow cytometers to handle large numbers of samples in a short time.
Functional assays for helper or suppressor T-cell activity and cytotoxicity assays are complex, difficult to standardize, and rarely performed, according to Virella (Personal communication, Medical University of South Carolina, Charleston, 1997). Such techniques have given way to cytokine production assays (for example, assay of IL-10 as an indication of suppressor activity) on cells sorted by flow cytometry. The measurement of natural killer cell activity is not performed as its functional significance is unclear at this time.
Phagocytic Cell Assays
Phagocytic cell assays are usually carried out with polymorphonuclear leukocytes, but functional abnormalities involving phagocytes are rare compared to granulocytopenias in patients with less dramatic problems than severe trauma, such as burns and severe malnutrition (Virella, 1993b). These assays also require trained personnel, sophisticated equipment, and fresh cells and are difficult to perform on large numbers of individuals.
New Techniques for Producing Immunity via Oral Immunization
In Chapter 22, COL Arthur O. Anderson discusses several technical advances that have begun to influence the production of vaccines, in particular those targeted at enteric diseases, such as that produced by the biological warfare agent Vibrio cholerae. Because the effects of exposure to infectious agents and toxins can significantly impair the function of the military, safe and effective immunization becomes a major prerequisite of readiness.
The ability to immunize troops against the types of agents that they are likely to contact is influenced by many factors. The route of entry into the body for many pathogens is the gastrointestinal (GI) tract, respiratory tract, or epithelial lining of the reproductive tract. The mucosal immune system provides the first line of defense against most pathogens and the only one against agents such as cholera.
Stimulation of the mucosal immune system leads to the production of tetrameric secretory immunoglobulin A (Ma et al., 1995). In mammals, 75
percent of synthesized immunoglobulin is IgA, and two different types of cells are required for its synthesis and assembly (Mostov, 1994).
Whereas parenterally administered (injected) vaccines stimulate peripheral immunity but have very little effect on the mucosal immune system, oral vaccines tend to be relatively ineffective in stimulating peripheral immunity (a phenomenon referred to by Anderson as cross-regulation) (Koster and Pierce, 1983). Efforts to develop and administer oral vaccines against enteric agents have been hampered by the apparent need to use particulate antigens, such as live or killed intact microorganisms, and the ability of the GI tract to inactivate polypeptide antigens or to induce an immunologic tolerance (analogous to the process by which the GI tract is prevented from mounting an immunologic response to the antigens in food) (Chen et al., 1995). As outlined below, several lines of research have sought to overcome these problems by using different approaches.
Production of Recombinant Bacterial Antigen for Oral Immunization
Cholera bacteria (encountered as a biological warfare agent) and enterotoxigenic E. coli multiply in the small intestine, producing enterotoxins that bind to a glycoprotein, GM1 ganglioside, on the surface of the epithelial cells that form the lining of the GI tract, thus exerting a pharmacological (ADP-ribosylation) effect that results in massive hypersecretion of intracellular fluid (Sack, 1980; Sixma et al., 1991). Both cholera toxin (CT) and the heat-labile enterotoxin (LT) of E. coli consist of six subunits, one nonbinding but toxic A subunit and a pentamer of GM1-binding but nontoxic B subunits (Sixma et al., 1991). Attempts to develop vaccines against these pathogens have utilized the B subunit because antibodies against the B subunit block the ability of the toxin to bind to cells (Clemens et al., 1990; Peltola et al., 1991). Utilizing an Agrobacterium-mediated plant transfection system, Arntzen and coworkers (Haq et al., 1995) constructed expression vectors containing the gene for the LT-B subunit and transfected tobacco and potato plants. Mice administered extracts of soluble tobacco leaf protein or potato tuber protein by gavage developed both serum and gut mucosal LT-B antibodies that inactivated the toxin in vitro. Oral immunity was conferred upon mice fed fresh transgenic potato tubers that expressed the B subunit.
Thus, the plant material provides both the expression and delivery systems, eliminating the need for trained medical personnel to administer the vaccine (Haq et al., 1995; see Anderson, Chapter 22 in this volume). Because the vaccine can be fed as part of a meal, deployment need not be delayed to allow for immunization of troops. Finally, the use of plant systems for the expression of heterologous genes (those not native to the species) permits almost limitless production of vaccine and may represent an economically feasible alternative use for the tobacco that is now grown for cigarettes (Haq et al., 1995).
Use of Enterotoxins as Vaccine Carriers and Adjuvants
As has been described, the binding subunits of enterotoxins such as CT and LT are potent antigens for both mucosal IgA production and peripheral IgG production, regardless of route of administration. Coadministration of a relatively weak antigen of interest with CT, or conjugation of the antigen with CT subunit B (CTB), enables the CT to function in an adjuvant capacity to promote the formation of antibodies to the weaker antigen (Elson and Dertzbaugh, 1994). Because conjugation of antigen to CTB can damage the tertiary structure, decreasing its adjuvant effect, efforts have been under way to utilize recombinant techniques to fuse antigenic constructs to the A subunit (Dickinson and Clements, 1995; Hajishengallis et al., 1995).
Use of Transgenic Plants to Generate Antibodies for Passive Immunotherapy
The ability to produce unlimited quantities of antigen-specific secretory antibodies would make it possible to induce passive mucosal immunity to a variety of organisms. Whereas two types of cells are required to synthesize and assemble secretory IgAs in mammals, researchers have demonstrated the ability to generate assembled, functional antibodies to a cell surface adhesion molecule of Streptococcus mutans (the bacteria responsible for tooth decay) in single cells of transgenic plants (Ma et al., 1995). By introducing the genes encoding each subunit of the immunoglobulin molecule into separate transgenic tobacco plants and performing the appropriate Mendelian crosses, plants synthesizing fully assembled, secretory IgA-G hybrid antibodies have been obtained. A product containing these antibodies in a toothpaste is undergoing testing.
Use of Polymerase Chain Reaction for Identification and Cloning of Antibodies
Unfortunately, the desire to produce an antibody in large quantities does not always have the advantage of a preexisting monoclonal antibody. The antibody molecule is composed of two pairs of polypeptide chains: one pair of light (low molecular weight) chains and one pair of heavy chains, all linked by disulfide bonds. The amino acid sequences of both light and heavy chains consist of alternating regions of variable and constant sequences. According to Anderson, the specificity of an antibody for its antigen is conferred by an area of the variable region known as the complementarity determining region (CDR). Following in vivo exposure to an antigen, clones of B-cells expressing specific antibodies undergo a process of "affinity maturation" in which single base pair mutations occur in the DNA of the CDRs, resulting in higher affinity of the antibodies for their antigens. Immunoelectrophoresis on frozen tissue slices and polymerase chain reaction amplification of the CDRs in cells that display high-affinity binding permits the identification and cloning of sequences responsible
for that binding (Jacob et al., 1991, 1993; Marks et al., 1991). Following insertion of the sequences into heavy-chain variable regions, large-scale antibody production can occur the recombinant plant system.
Microencapsulation of Antigens and Antibodies
Microencapsulation technology, first developed for delivery of pharmaceutical agents, has now been applied to the delivery of vaccines (Michalek et al., 1994). Administration of microencapsulated vaccines using polymers of lactic and glycolic acids confers several advantages, including the ability to protect the antigen from degradation in the GI tract (resulting in the need for less antigen); the ability to administer antigen in a timed-release manner; the adjuvant effect of the microencapsulation material itself, which boosts antibody production; and the ability of microparticles, when administered in a range of sizes, to undergo size-based migration to both mucosal and peripheral immune tissues and to induce both mucosal and peripheral immunity (Eldridge et al., 1990).
Assessment of Immune Function
A convincing rationale must exist to support the study of any given parameter of immune function. The choice of adequate end points is difficult because of the complexity of the immune response. Furthermore, the choice must be based on the availability and reproducibility of techniques.
Longitudinal studies of serum IgG concentrations, measurement of humoral responses to vaccines or boosters, and the determination of serum or urinary concentrations of select cytokines involved in inflammatory and immunoregulatory processes are parameters that are relatively easy to obtain and whose measurement can be supported by currently available basic and clinical research data. According to Moldawer (see Chapter 20 in this volume), plasma and urine levels of TNF-a and IL-1 are not reliable indicators of inflammation. Increased concentrations of the shed TNF-receptors and IL-1 receptor antagonists, however, are generally indicative of a local inflammatory response and may be used with caution as indicators of local TNF-a and IL-1 production.
Plasma and urinary IL-6 levels are elevated in a large number of inflammatory processes and seem to correlate with physiological parameters. Plasma and urinary measures of IL-6 receptor antagonist and shed TNF-receptors can be used to detect the presence of inflammation and metabolic stress.
Several of the in vitro functional assays, such as IL-2 release, IL-2 receptor (CD25) synthesis, or immunoglobulin synthesis in response to mitogenic stimulation appear to have the potential to reveal immunoregulatory abnormalities when other parameters (particularly cytokine concentrations in blood)
appear normal. While such assays require freshly isolated cells, precluding collection of samples in the field, experiments could be designed around training exercises.
Techniques were described that have been applied successfully to assess the influence on immune response of factors such as dietary alteration, heavy exercise, emotional stress, surgical trauma, and other physical trauma, all of which are encountered in the military situation. The ability to obtain useful information is dependent on careful experimental design, elimination of as many variables as possible, taking into consideration the types of samples that can be obtained in the field, and, if possible, designing assessments around field exercises so that a wider variety of sample types can be obtained in a laboratory setting.
Techniques for Oral Vaccine Production
The gastrointestinal mucosa is the target for a significant number of pathogens that are encountered in the military situation and are directly or indirectly responsible for altered nutritional status. An increased understanding of mucosal immunology combined with progress in the fields of biotechnology and molecular genetics have led to a better understanding of how to optimize vaccine administration as well as significant advances in the production of antibodies and antigens for use as oral vaccines. These include the use of recombinant techniques to produce bacterial antigens for use as vaccines and antibodies for passive immunotherapy, both of which can be delivered in edible form. In addition, the polymerase chain reaction technique permits identification and amplification of antibodies. New encapsulation technologies permit the delivery of reduced doses of oral vaccines by protecting them from destruction in the GI tract and enable targeting of the vaccines to the mucosal or peripheral immune system.
FUNCTIONAL AND BEHAVIORAL MEASURES OF NUTRITIONAL STATUS
In this section, James S. Hayes describes the use of involuntary muscle contraction to assess nutrition status, while Mary Z. Mays as well as Harris R. Lieberman and Bryan P. Coffey focus on cognitive assessment in the military (see Chapters 23, 24, and 25 in this volume). Summaries of the presentation by David F. Dinges on sleep and circadian rhythms and chapter by Ginger S. Watson and Yiannis E. Papelis on the use of simulators follow (see Chapter 26 in this volume).
Involuntary Muscle Contraction to Assess Nutritional Status
Skeletal muscle function has been studied by measuring both handgrip strength (voluntary contraction) and the response to electrical stimulation of the branch of the ulnar nerve that innervates the adductor pollicis (thumb) muscle (involuntary contraction). The use of the latter technique, referred to as muscle function analysis (MFA), to assess nutritional status is described by James S. Hayes (see Chapter 23 in this volume). According to Hayes, MFA has the advantage of being less susceptible to subject motivation than the measurement of voluntary contraction and, therefore, may be more sensitive and reliable. The stimulus is applied at various frequencies, and the pattern of involuntary contractions is measured and recorded. Although the basic technique was developed in 1954 (Merton, 1954), computerized data collection and analysis methods recently have been coupled to the MFA technique, making it much easier to apply.
The MFA response is thought to be associated with the nutritional status of the individual who is being assessed. There is some correlation between MFA results and total-body protein measurement; as the latter decreases, MFA relaxation rates also decrease (Brough et al., 1986; Russell et al., 1983a, b). The developers, therefore, contend that lower MFA relaxation rates indicate malnutrition or some other abnormality.
The advantages of MFA, according to Hayes, are its noninvasiveness and apparent sensitivity to early changes in muscle function due to malnutrition or to refeeding, the immediate availability of results, and the low cost of both the equipment and test administration.
The disadvantages, according to Hayes, include the difficulty of locating the ulnar nerve; discomfort; the size of the equipment; and the other mechanisms, such as voluntary muscle control and sodium potassium pump activity, that may influence the results. Therefore, the validity of the concept needs further testing.
In Chapter 23 of this report, Hayes describes studies currently under way on MFA. These include examining the ability of MFA to detect and monitor the nutritional status of HIV patients during nutritional intervention and throughout the course of the illness, evaluating the optimum type of protein supplement for trauma patients and the ability of nutritional support to improve the outcome and survival of renal dialysis patients, and determining the ability of MFA to assist in monitoring malnourished hospital inpatients. Because the results of such studies are as yet unavailable, the validity of the technique cannot be assessed.
Application of Cognitive Performance Assessment Technology to Military Nutrition Research
The field of military nutrition is based on the premise that readiness, hence physical and cognitive performance, are dependent, in part, on the nutritional state of soldiers. While it is known that cognitive performance in the military setting is influenced by nutrition (Consolazio et al., 1967, 1968; Johnson and Sauberlich, 1982; Johnson et al., 1971), the extent of this influence is not well understood. Traditionally, according to Mary Z. Mays (Chapter 24 in this volume), the Army's interest in the influence of nutrition on cognitive function has focused on three specific issues: food deprivation in the field, underconsumption of rations, and identification of performance-optimizing ration components. The lack of information concerning the role of nutrition in cognitive function is, in large part, the result of limitations in assessment technology. Assessments of cognitive performance attempt to identify and quantify measurable end points of complex intellectual behaviors. According to Mays, cognitive assessment has been accomplished largely through the use of one of three methods: observation of behavior, paper-and-pencil tests (or their computer counterpart), and tests of manual dexterity or hand-eye coordination (as part of a battery of tests). Increasingly, such tests have been replaced by assessment technologies that make use of computer games or performance tracking devices built into actual or simulated vehicles or other machinery.
As reviewed by Harris R. Lieberman and Bryan P. Coffey (see Chapter 25 in this volume), the measurement of cognitive performance in humans is associated with many problems. First, data collected using formal laboratory behavior tests may be difficult to relate to real-world performance. Second, circadian variation in performance occurs (Moore-Ede et al., 1982) and may overwhelm any differences caused by the nutritional alterations. Third, the underlying behavioral function that the performance test actually is assessing may be unclear, so it cannot be assumed that the limiting or critical factor being assessed can always be accurately defined. Finally, it may be difficult to identify the optimal test to evaluate the behavior of interest, so as to avoid the possibility of failing to detect treatment effects, especially when the effects of the manipulations are expected to be modest (which is the case for nutritional interventions).
The military's assessment of cognitive performance is of great importance, given the complexity of weaponry and the high level of performance and vigilance that is required under difficult working conditions. This assessment is further complicated, however, by a number of factors unique to the military situation. First, the desire to conduct testing in field settings limits the types of tests that are feasible. Field testing usually requires that subjects stop their ongoing activities to participate in the task, which is a particular problem if subjects are engaged in training exercises or actual operations. Studies performed in the field must be designed to be minimally intrusive on soldiers'
time (see Lieberman and Coffey, Chapter 25 in this volume), and the tests used must be reliable under field conditions. Second, cognitive assessments of military personnel must employ tests that are psychometrically valid substitutions for critical military functions or tasks (see Mays, Chapter 24 in this volume). Third, because the primary nutritional deficits soldiers experience are likely to be the acute lack of energy intake, cognitive assessment must be extremely sensitive to any subtle performance decrement that may result. Fourth, the effects of battle stress, as well as boredom and frustration with the assessment task, can result in a decrease in the reliability of data collected. Finally, the process of identifying nutrients that will enhance performance in the field requires carefully controlled, clinical, dose-response evaluation that is best performed in a laboratory setting.
Technical Advances in Cognitive Performance Assessment
Computers and Miniaturization
Mays suggests that while the basic methods of cognitive assessment are unlikely to change significantly in the next 20 years, advances in computer software and hardware along with miniaturization should increase the ability of researchers to assess cognitive function in the field. The use of handheld computerized devices would decrease the invasiveness of performance assessment by permitting remote data collection and would allow repeated measures.
Lieberman and Coffey describe two electronic activity-monitoring devices designed to be worn on the wrist. The Motionlogger Actigraph, a commercially designed device, monitors sleeping and waking activity using a piezoelectric motion detector. Data collected by the monitor are downloaded to a computer that uses an algorithm validated to calculate sleeping and waking time. While the Actigraph does not provide as much information as polysomnography and cannot assess performance per se, it provides continuous assessment of physical activity behavior, which can be related to the physical and mental state (Lieberman et al., 1989; Tryon, 1991).
The Vigilance Monitor, developed at USARIEM, combines the measurement capabilities and characteristics of the Actigraph with vigilance assessment and intervention capability. Vigilance reflects the ability to process relevant information and to respond in a timely fashion (Koelega, 1989). Vigilance has been found to be sensitive to the effect of diet (for example, caloric restriction and caffeine intake), as well as a number of other factors (Clubley et al., 1979; Green et al., 1994; Lieberman, 1992). According to Lieberman and Coffey, the Vigilance Monitor functions by presenting an audible tone to the wearer and measuring response time. By repeating the stimulus presentation randomly and intermittently, vigilance is monitored on a more or less continuous basis. The Vigilance Monitor also is configured to collect data about environmental
variables, such as air temperature, sound, and duration and amplitude of light exposure of the subject so that an attempt can be made to correlate the level of vigilance with these variables. The Vigilance Monitor has a number of advantages over traditional methods of cognitive assessment. These include its unobtrusiveness (it enables the wearer to continue with most activities while being monitored), its ability to be programmed to provide data on several variables concurrently, and its ability to monitor a number of subjects simultaneously over the course of many days. The device also has the novel attribute, although not yet validated in the field, of being able to maintain alertness in the wearer by functioning as an alarm. Finally, the cost of the monitor is relatively low, once the appropriate computer capabilities have been developed and implemented.
A similar method of psychomotor vigilance testing that utilizes a handheld device (the ''psychomotor vigilance task") was described in a presentation by David F. Dinges (see Kribbs and Dinges, 1994). To perform this test, the subject is instructed to push a button on the device in response to the appearance of a light in the display window. The advantages of the test include its ability to test subjects of all intelligence levels on a basic cognitive skill (alertness or attention) that is required for almost all other cognitive functions, the absence of a learning curve for the task (which is known to confound much of the published data on cognitive performance [Dinges and Kribbs, 1991]), and high validity and reliability. The test utilizes an instrument that is portable and completely programmable with respect to interstimulus interval, auditory and visual feedback, and signal load rate. the use of a visual stimulus rather than sound is based on the finding that while auditory response time is faster than visual, visual response time is affected more quickly by fatigue (Dinges, 1992). The performance parameters (types of errors) that can be measured with the vigilance task include false starts, lapses (a long reaction time or a sudden period of nonresponse), decline in the optimal response capacity (response slowing), false response, and accelerated habituation (increase in the number of lapses with length of time on the task) (Dinges and Powell, 1985). Fatigue produces an increase in lapses and a decrease in the minimal ("best") response time, consistent with observations from traditional cognitive performance tests that, given the opportunity, people slow down to maintain accuracy (Dinges, 1992).
Improvement in Interface Technology
According to Mays, the design of natural interfaces will improve in the near future, permitting the use of identical assessment tools in the laboratory and in the field with no special training. One example of this may be the Iowa Driving Simulator (IDS), described in Chapter 26 in this volume by Ginger S. Watson and Yiannis E. Papelis, which also takes advantage of advances in computational methods to create a high-fidelity computational vehicle model set in a
fully interactive, virtual environment. The IDS provides the subject with realistic motion and visual, auditory, and force feedback cues to simulate a wide range of driving conditions and scenarios (Kuhl et al., 1995). Thus far, military applications of the IDS have been limited to the development of a "virtual proving ground" that closely matches test courses at the Aberdeen Proving Ground, Maryland, for the design and testing of new Army vehicle prototypes (one simulator has been made to resemble the internal appearance and behavior of a high mobility multipurpose vehicle). Use of the IDS for research purposes is somewhat limited by the need for different degrees and types of fidelity for different performance measures (Alessi and Watson, 1994). In order for data to be valid, subjects must perceive that the experience is real and respond in a real way, and their perception must persist over time, with the novelty factor being eliminated or overcome. Research that has utilized the IDS has tended, thus far, to measure the effects of factors such as age, gender, and visual impairment on reaction time (Romano and Watson, 1994). To date, no studies have examined the effects of nutritional variables, and only one student-run study has examined the influence of sleep deprivation.
While there is evidence that cognitive performance is influenced by nutritional status, there are many problems associated with trying to evaluate this influence, particularly in the field. Two types of portable monitors have been described for the assessment of vigilance, one aspect of cognitive performance that is highly relevant to field situations. A third device, the IDS, was described that measures the multiple cognitive functions associated with driving. Each of these techniques can be used to assess the influence of some nutritional stimulus or change in status, but all await further validation.
The CMNR's overview and summary of emerging technologies for nutrition research set the stage for responding to the questions posed by the Army. The committee's responses, as well as its conclusions and recommendations, are presented in the next chapter.
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