Committee on Military Nutrition Research: Activity Report 1994-1999 (1999)

As stated in Chapter 1 of this report, the Committee on Military Nutrition Research was asked to review the state of knowledge concerning the impact of nutritional status on immune function. Below are the committee's answers to the five questions posed by the Army regarding nutrition and sustainment of immune function in the field, followed by the committees conclusions and recommendations. Recommendations for areas of future development for the U.S. Army nutrition research programs are also included.

1. What are the significant military hazards or operational settings most likely to compromise immune function in soldiers?

As described previously and outlined below, many conditions or stressors have been associated with compromised immune function during Ranger training and basic combat training, as well as during arctic training and in deployments to locations such as Somalia, Haiti, Panama, and the Persian Gulf.

• Reduced ration consumption. Intakes less than 60 percent of the total energy needed, particularly during exposure to harsh environments and/or dehydration, were shown to be a significant stressor. In U.S. Ranger II, the increase in energy intake from that in Ranger I (2,780 to 3,250 calories or approximately 470 kcal/d), which tempered weight loss to only 12.8 percent of initial body weight, appeared to minimize the adverse effects on immune

• function. Thus, weight loss, particularly that involving lean body mass, appears to be a major factor in inducing immune system dysfunction.
  
  

  The effects of dehydration on immune function are not reviewed in this report. However, weight losses of as little as 3–5 percent in 24–48 h, which are primarily due to dehydration, have a significant impact on performance. Weight losses of 6–10 percent in a similar period may affect health adversely. Thus, the effects of dehydration must be separated from those of underconsumption of rations (see IOM, 1995).

• Prolonged moderate-to-heavy physical activity. The week-long Norwegian Ranger training studies with heavy exercise and limited sleep did not demonstrate significant weight loss or alterations in immune function, whereas the U.S. Ranger I study of 8-to 9-week duration demonstrated a greater weight loss (14 percent of body weight) and an altered immune response. Low- to moderate-intensity exercise (<60 percent Vo_{2 max} ), such as that performed in most troop activity of a duration of 60 minutes or less, appears to exert less stress on the immune system than activity that is more strenuous (>>60 percent Vo_{2 max} ) performed for longer than 1 h. Repeated bouts of strenuous activity may increase the risk of infection, particularly of the upper respiratory tract.
• Limited, interrupted or nonrestful sleep. Limited or nonrestful sleep over a prolonged period (as little as 3 hours or less was noted in the Norwegian Ranger studies), particularly when coupled with stressful physical activity, may result in some compromise of the immune system. Short periods of severe caloric and sleep deprivation appear to have less adverse effect on immune function than a more prolonged period with greater weight loss (caloric deficit).
• Increased infection and injury. This category includes infections associated with trauma and burns, such as cellulitis, osteomyelitis, wound abscesses, and sepsis, as well as naturally occurring infections and diseases such as conjunctivitis, otitis, upper and lower respiratory tract infections, urinary tract infections, and gastroenteritis. Diarrhea is commonly experienced by soldiers in military operations, most likely due to exposure to infectious organisms from strange environments (dust, water, local foods). Influenza also is common, and in some environments, other diseases occur that are rare in the United States.
• Increased exposure to extremes of temperature and humidity. Increased exposures in areas such as the tropics or desert, as well as with operations in the arctic areas of North America or northern Europe during winter conditions, can adversely affect food intake and sleep. Heavy activity or environmental extremes may increase energy requirements by as much as 15 percent after acclimatization without compensatory ration intake. For example, hypohydration may lead to temporary anorexia and a worsening cycle of lowered water and food intake. The factors that influence ration consumption may be even more significant for operations in the cold and at high altitudes.
• Increased psychological stresses. Stresses such as those imposed by deployment, separation from family, imminence of combat, threat of biological agents, and long periods of vigilance with interrupted sleep and inadequate rest,

• may also be significant and often result in field training- or combat-induced anorexia. All of these factors may impinge on immunological health.
• Prolonged exposure during training or battlefield combat to environmental assaults. Environmental exposure (for example, to smoke or fumes from fuels or chemicals, dust, dirt, and blast overpressure) may induce oxidative stress on protective systems.

2. What methods for assessment of immune function are most appropriate in military nutrition laboratory research, and what methods are most appropriate for field research?

It is important first to identify a number of methodologic issues that must be considered when assessing immune function.

In addition to the choice of assay, a large number of issues must be considered in the design of studies to assess immune function. The first consideration in a study of immune challenge is the choice of antigen, described previously for tests of primary and secondary antibody response (Cunningham-Rundles, 1999; Straight et al., 1994).

The second consideration is the timing of sample collection. As described by Erhard Haus (Chapter 20), the immune system is significantly influenced by biological rhythms; thus, samples must be drawn on an established schedule (Straight et al., 1994). Additionally, it is important to standardize collections in relation to physical activity because differential cell counts can change acutely during and immediately after exercise (DeRijk et al., 1996, 1997).

The third, and possibly most critical consideration, is the protocol for storage and transportation of samples. According to G. Sonnenfeld (University of Kentucky, Louisville, personal communication, 1997), human blood samples must be shipped at room temperature in Styrofoam containers, and for most status indicators, must be assayed within 24 h. If necessary, some preparatory steps, such as harvesting cells from blood, may be performed in rudimentary makeshift labs and the samples sent under controlled conditions to a central facility for completion of analysis.

A fourth but related consideration is the choice of laboratory for sample analysis. The Agency for Toxic Substances and Disease Registry (ATSDR) of the Department of Health and Human Services recommends the use of a central or core reference facility for all analyses to avoid small differences in protocols and solutions used. Some methods, such as the measurement of mitogen-induced lymphocyte proliferation by [³H]thymidine incorporation are extremely

sensitive to such factors (Cunningham-Rundles, Chapter 9). Thymidine incorporation is also a variable, relatively nonspecific measure not easily standardized and not well applicable to field studies. Because some procedures must be performed within a short period of time, the number of samples that can be processed is thus limited.

Finally, the use of controls is extremely critical both in the collection and assessment of samples and in the interpretation of data. It is recommended that each time samples are drawn and an assay is performed, a standard is drawn and included for the assay, consisting of the blood (or cells) of one individual, to correct for intraindividual and interassay variability. Whenever possible, subjects should be used as their own baselines, and longitudinal studies should be performed (Straight et al., 1994). Of major concern are the lack of population-based normative reference ranges for most immune function parameters and the need to obtain complete health histories (including such factors as smoking, use of other drugs, and pregnancy) from subjects to rule out possible confounding factors.

Immunologic function can be related to nutritional status by utilizing two distinct methodological approaches. First, under controlled conditions, normal healthy individuals can be studied; after an appropriate baseline period, a nutritional perturbation can be imposed and the changes in immune responses from baseline determined. This approach allows single nutrient or environmental perturbations to be studied while many other factors that also cause immune dysfunction are controlled. In addition, appropriate controls (with adequate sample size) can be included, and a period of refeeding (or second control period) can be included at the end of the experiment.

Second, in field studies, the conditions are quite different, and other variables, in addition to altered nutritional intake, affect individuals. Immune dysfunction due to both nutritional and other operational stressors may be present. Under these conditions, it is possible to study the incidence of infection using epidemiologic techniques, while food intake and nutritional status are determined. Appropriate ambulatory tests of immunologic function can be validated and compared to results obtained in more controlled settings.

A longitudinal study of immune function in simulated combat conditions in the field could be performed that would have the ability to detect accurately over time the subjects' nutritional state and the incidence of infection. When clinical signs are clearly defined and documented, and symptoms indicate the occurrence of infectious illnesses, studies to determine etiology and therapy can be initiated, along with serial studies of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), acute-phase reactants, whole blood, plasma, cytokines, and their receptors. The longitudinal course of illness can then be correlated with nutritional parameters.

Prior to pursuing field investigations, researchers must undertake appropriate studies in a controlled clinical setting to answer some of the more basic questions about the impact of altered nutritional status on immune function. These studies must precede those that attempt to confer a state of enhanced immune function or to study the response to a specific nutrient. For example, limited studies of subjects placed under conditions of reduced caloric intake could be undertaken. Attempts could also be made to see if supplementation with one or more essential single nutrients could maintain normal immunological competence in the face of generalized dietary deprivation. Before extensive field evaluations of the influence of nutrition on immune response are undertaken, carefully controlled laboratory studies should be performed and data collected from more fundamental research studies. To hypothesize which of the nutrients may enhance immune response, it may be helpful first to determine under controlled conditions which nutrients, by their deficiency or exclusion from the diet, impact the immune response negatively; however, this will not provide a complete picture.

The CMNR confirms the need to determine appropriate field measures for monitoring the immune response, particularly for determining the presence and magnitude of an acute-phase reaction, which may be adversely influenced by nutritional status in stressed individuals. Based on standardized test panels recommended by government agencies or private-sector scientists, the committee suggests the following. If clinical signs of infection are present or there has been significant weight loss induced by nutritional stress, a simple-to-use basic screening panel of immune function tests such as CRP protein, ESR, a baseline battery (testing six or more antibody titers for several previously administered military vaccines, immunoglobulins G, A, and M; and complete blood count with CD4 lymphocyte count and CD4:CD8 ratio should be employed initially. In the event that these basic tests of immune response indicate the existence of immune compromise of an unusual nature or unusually great incidence, the CMNR suggests a second tier of immune function tests. These would include natural killer (NK) cell numbers and activities; lymphocyte mitogenesis assays; thymosin measurements; and estimations of phagocytic cell chemotaxis and microbicidal activities (for example, Listeria monocytogenes-killing assay). However, these tests must first be validated for field use. A standardized battery of delayed dermal hypersensitivity tests may be employed at baseline and again if stress-induced weight loss exceeds 10 percent.

If validated, these tests would be valuable in research studies for rapid field assessment of immune status and might suggest steps that could be taken to improve resistance to potential exposures and thus improve unit effectiveness. As previously noted by the CMNR (IOM, 1997), tests based on cytokine assays, especially of the proinflarnmatory cytokines and related molecules excreted in urine and whole-blood cytokine production assays, have great potential for adding important new diagnostic measures at a relatively low cost-benefit ratio.

Differential changes in production patterns of specific cytokines (that is., shifts from T-helper 1 [Th1] to Th2-type patterns) may be the most sensitive way to determine whether changes in immune responses are stress related. Such tests currently are being evaluated in many civilian research studies and may have very real potential value for suggesting the presence of cytokine-induced malnutrition in military personnel who are being exposed to the stresses of rigorous training exercises or ongoing operational missions. Additionally, in the development and validation of more precise readout measures, attention should be paid to the development of microassays that can be applied in field settings to minimize stress and blood loss during sampling.

3. The proinflammatory cytokines have been proposed to decrease lean body mass, mediate thermoregulatory mechanisms, and increase resistance to infectious disease by reducing metabolic activity in a way that is similar to the reduction seen in malnutrition and other catabolic conditions. Interventions to sustain immune function can after the actions, nutritional costs, and potential changes in the levels of proinflammatory cytokines. What are the benefits and risks to soldiers of such interventions?

One of the most fundamental needs is to sustain the functional competence of the immune system in military personnel who must experience the stresses of rigorous training and operational assignments and who face the risks of infectious illnesses as well as diverse forms of trauma. Cytokine effects in the body can be influenced by a variety of factors as outlined below.

• Nutritional interventions. It is well known that in the course of infection, proinflammatory cytokines mediate the loss of specific nutrients, which must be repleted or redistributed. In turn, growing evidence suggests that a number of nutrients may influence immune function by affecting synthesis of specific cytokines, their soluble receptors, or inhibitory factors. For example, research on the antioxidant vitamins A, E, and C, as well as certain polyunsaturated fatty acids (PUFAs) and amino acids (AAs), has shown that their apparent ability to modulate immune status may be mediated by their effects on cytokines, at least under some conditions, but many questions remain regarding the efficacy of these nutrients in amounts that exceed Military Recommended Dietary Allowance (MRDA) levels. Research is also needed on whether nutritional intervention during stress is effective or whether it must be combined with agents that suppress inflammation.
• Pharmacological interventions (including immunizations). Research has demonstrated that a number of pharmacological agents including aspirin, ibuprofen, and glucocorticoids modulate the effects of cytokines and can be used to minimize signs and symptoms of cytokine-induced acute-phase reactions and the nutrient losses that accompany them. Glucocorticoids can

• block fever and reduce many of the metabolic consequences of acute-phase responses caused when proinflammatory cytokines are released by cells, but the adverse consequences of prolonged systemic administration of glucocorticoids have long been recognized. On the other hand, drugs such as aspirin and ibuprofen can block the intracellular formation of many of the eicosanoids (prostaglandins, prostacyclins, leukotrienes, thromboxanes) and thereby reduce the fevers, myalgias, and headaches that accompany cytokine-induced acute-phase reactions. Because losses of body nutrients during these reactions are often proportional to the magnitude and duration of fevers, the use of such generally safe and effective anti-inflammatory drugs serves indirectly to maintain the body's nutritional status and immune system functions. Further, the use of anti-inflammatory drugs for the management of minor traumas or infections (for example, upper respiratory tract infections) is well recognized and provides for sustained military performance during severe training exercises and operational missions. The immune system can be ''educated'' in advance by the prophylactic administration of immunizations against all possible foreign agents. Such immunization procedures do carry some risks, depending on the vaccine being administered, but the ultimate military benefits of such immunization practices far outweigh the risks. Furthermore, the risks of immunization can be reduced and the benefits increased (that is, improved vaccine effectiveness) by the use of oral vaccines, whose development by the military was recommended in an earlier CMNR report (IOM, 1997).
• Administration of products of biotechnology. Biotechnological methods have allowed the production of many individual cytokines and their receptors. At the present time, their use is limited to the administration of granulocyte macrophage colony stimulating factor for the treatment of bone marrow recipients and those undergoing a limited number of other experimental procedures, and their effectiveness has not been demonstrated in healthy subjects or in clinical trials. A recent review by Mackowiak and colleagues (1997) discusses the therapeutic use of pyrogenic cytokines and the use of their inhibitors. The authors comment on the failure, or even the harm, associated with their therapeutic use in humans (in contrast to rodents). The administration of exogenous cytokines and the modulation of cytokines in vivo are areas of active research in the civilian sector; the use of cytokines to enhance resistance to infections, however, should be carefully studied in animals before application to clinical situations.

4. What are the important safety and regulatory considerations in the testing and use of nutrients or dietary supplements to sustain immune function under field conditions?

The basic considerations in the testing and fielding of nutrients or dietary supplements to sustain immune function are to ensure, first, that the nutrients are in fact safe under the conditions of intended use and, second, that they are effective. Since the levels of some of the nutrients that must be fed to achieve potential effects are much higher than levels usually ingested in foods, further safety testing is warranted. Such testing involves attempts to delineate the upper limits of safety.

For any substance, there are a number of major considerations relevant to the question posed. These include the following:

• the intake levels that are suggested and referenced by the Recommended Dietary Allowance (RDA)/MRDA;
• the customary range of intake;
• the tolerable upper level;
• the safety and efficacy of the substance at the level of intended use; and
• special groups or circumstances that deserve attention.

Generally accepted tolerable upper intake limit values have not yet been established for individual nutrients, but the Food and Nutrition Board's (FNB's) Subcommittee on Upper Reference Levels of Nutrients is now considering these levels. For purposes of planning further military research on individual nutrients, there is already evidence that safety problems associated with excess consumption are much more likely for some nutrients than for others. In clinical practice, the general rule of thumb is that it is generally unwise to exceed three to four times the traditional RDA for most fat-soluble vitamins; however, margins of safety may be lower for vitamins A and D in some groups. In general, water-soluble vitamins tend to be less toxic and can be consumed in larger multiples of the traditional RDA than can fat-soluble vitamins.

Trace minerals are difficult to discuss in general terms. It is important to remember that supplements of a single nutrient cannot be considered in isolation. The World Health Organization (WHO, 1996) Expert Consultation examined upper safe levels for trace minerals and concluded that the toxicity and the potential for nutrient—nutrient interactions must be considered individually. Risks of pathology resulting from such interactions are higher when intakes of other essential nutrients with which they interact are low or marginal, accentuating the nutrient imbalance. Therefore, conservatism is warranted in the consumption of trace minerals in excess of traditional RDA or suggested safe and adequate levels. However, requirements may change during an episode of illness, and requirements for some minerals may substantially increase (for example, zinc during diarrhea). The FNB's Dietary Reference

Intakes (DRIs) Subcommittee on Upper Safe Levels is now considering the issue more fully.

Dietary deficiencies of a variety of nutritionally essential trace elements (zinc, copper, selenium) have been demonstrated to have an adverse impact on immune function in laboratory animals and elderly humans, and deficiencies of zinc and copper have resulted in increased susceptibility to certain infections in humans. Excessive intakes of some trace elements have led to immuno-suppressive effects. Therefore, care must be exercised in the use of single-nutrient supplements until the optimal range of intakes for these trace elements is determined.

Iron. Both iron deficiency and iron excess appear to have the potential to increase susceptibility to infection. In a military situation, it is likely that the potential reduction in immune function due to iron deficiency is of more significance than any effects of iron overload. Because of their higher iron requirement and lower intake of operational rations, the iron intake of female soldiers may be lower than recommended in the MRDA, increasing their risk for iron deficiency anemia. Utilizing as the criterion for iron deficiency a serum ferritin concentration of less than 12 μg/L, and a combination of low serum ferritin and a hemoglobin of less than 120 g/L as the criteria for iron deficiency anemia, it was shown that 17 percent of new female recruits entering basic combat training (BCT) fit these criteria for iron deficiency, while 8 percent could be classified as having iron deficiency anemias. A survey of a similar (but not the same) population of women at the end of BCT showed that by the end of training, 33 percent were iron deficient and 26 percent were anemic (Westphal et al., 1994, 1995).

Iron deficiency anemia can be expected to have adverse effects on the military performance of both men and women depending in part on its severity. Performance deficits in both men and women due to compromised iron status have been demonstrated most clearly during exercise of prolonged duration, such as long-distance running (Newhouse and Clement, 1988). Iron deficiency anemia may also have an adverse impact on recovery from serious wounds or injuries, especially those that involve large amounts of blood loss. However, data to support deficits in physical performance in iron-compromised individuals have not been systematically collected by the military. Some preliminary evidence suggests that iron supplementation of nonanemic women can improve aerobic capacity (J. Haas, Cornell University, personal communication, 1977). Male soldiers consuming operational rations appear to meet iron needs, as judged from current levels in the MRDA.

Glutamine. Glutamine is an amino acid that constitutes approximately 5 percent of most proteins. The CMNR recognizes that glutamine is a potential candidate for addition to operational rations to optimize immunity. It has

demonstrated potential for promoting immune cell proliferation and improving immune function, especially under the stress of surgery, infection, or bowel disease. However, before it would be appropriate to consider providing supplemental glutamine to soldiers in training or deployment situations, it will first be necessary to demonstrate in a healthy population the benefits of providing glutamine at levels significantly greater than those normally obtained in the diet. The results of one military study presented at the workshop showed no beneficial effects of glutamine supplementation on immune function parameters. The CMNR recently hosted a workshop (The Role of Protein and Amino Acids in Sustaining and Enhancing Performance) that addressed more fully the safety and efficacy issues for this and other amino acids.

Vitamin A and Antioxidants. Vitamin A intakes beyond the MRDA do not appear to be beneficial; in fact, excess intakes can be toxic. Healthy adult men and women of military age represent the lowest-risk group for the development of vitamin A deficiency; however, under certain conditions, such as chronic infection or prolonged dietary deprivation, the risk of vitamin A deficiency and associated immune abnormalities may be significant (Semba, Chapter 12). Carotenoids as supplied from fruits and vegetables may be important as modulators or stimulators of immune function.

Vitamins C and E are immunopotentiating agents most likely because of their function as antioxidants. Both of these vitamins are relatively nontoxic. However, it has not been demonstrated whether there is a functional benefit of increased intakes in protection against cancer, pathogenic viruses, or bacteria. Investigation of the role of these vitamins in protecting against or modulating the effects of infection is an active area of research. Toxicities of high-dose vitamin C supplements also have been difficult to demonstrate. Some evidence suggests that doses of 500 mg or more may result in increased excretion of oxalate (a precursor to one form of renal stone), but this observation has been limited to individuals who have an increased risk of forming stones (Urivetsky et al., 1992). A primary cause for concern among military personnel, who may be deployed on short notice, has been the risk of rebound scurvy due to sudden vitamin C withdrawal (Schrauzer and Rhead, 1973); however, clear evidence for this phenomenon is lacking. Likewise, the potential value of consumption from food sources rather than single-nutrient supplement intake requires greater study. However, the most prudent approach seems to be to increase fruit and vegetable consumption in the diet, thereby maximizing the potential benefits of antioxidant nutrients.

A factor that must be considered in recommending an increase in vitamin E intake is the level of PUFAs concomitantly being consumed in the diet. Increasing amounts of polyunsaturated fatty acids increase the vitamin E requirement because of the propensity of PUFAs to undergo lipid peroxidation. Approximately 0.4 mg of α-tocopherol equivalent for each gram of PUFA consumed has been suggested to be adequate in adult humans (Sokol, 1996).

Fatty Acids. Limited data suggest that moderate reductions in total fat calories (that is, 26 percent versus 30 percent) may have some beneficial effects in enhancing immune function. Increasing or decreasing the consumption of n-6 or n-3 PUFAs, or altering their intake ratios, may impact on immunological function. Although increased consumption of fish oils that supply eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may reduce the risk of heart disease and be beneficial in treating autoimmune diseases, their increased intake may reduce immune function, raise the dietary requirement for vitamin E, and affect blood clotting mechanisms (especially n-3 fatty acids).

Final Cautionary Notes. It is important to recognize that although modification of operational rations could potentially benefit the immune function of a large segment of the military population, a small but significant portion of the population could be harmed by such modifications because of genetic predisposition or other unknown factors. Also, it is possible that an elevated intake of a nutrient would result in a modification of immune function that is safe for a limited period but would diminish in safety or efficacy with prolonged use. Such a situation will necessitate a risk—benefit decision or the identification of a means by which to provide the supplemental nutrients in an additional ration component. Despite claims made by industry, some athletes, and sports coaches, most of the nutrients discussed in this report have failed thus far to demonstrate both safety and efficacy in modifying immune function, and further research is needed. Systematic studies should assess the extent to which subjects self-medicate with over-the-counter dietary food supplements, and such products should be evaluated carefully before their use is recommended.

5. Are there areas of investigation for the military nutrition research program that are likely to be fruitful in the sustainment of immune function in stressful conditions? Specifically, is there likely to be enough value added to justify adding to operational rations or including an additional component?

It is important to conduct research aimed at defining more specific nutrientimmune system interactions in order to elucidate the levels of key nutrients that are necessary to maintain proper immune function. Since these data also would be important for the general population, it is not necessary that this research be supported solely by the military; it could be conducted by other agencies. However, special studies of unique groups or circumstances (such as Ranger training) applying chiefly to the military might be warranted.

For some aspects of immune function, both beneficial and adverse effects related to nutrient intake may be encountered (some examples are vitamin A and

iron). With this in mind, the CMNR suggests that the following areas are worthy of further investigation by the military nutrition research program:

• Supplement use. The military needs to gain a better understanding of supplement use by its personnel. Little information is available regarding the real benefits or potential toxic effects of nutritional supplements in supranormal amounts to warrant their further study for widespread use in the military at present. Indeed, some data indicate frank adverse effects of consuming one or more of these nutrients (such as copper and zinc) in pharmacologic amounts.
  
  

  Although there seems to be relatively little risk associated with the use of vitamins C and E, and there may be relatively little risk associated with the use of β-carotene, major differences in potential benefits may exist between dietary exposure to such antioxidants and the use of supplements. Excessive supplementation with vitamin A, zinc, or selenium could prove toxic. The basis for this discrepancy and its impact on how such supplements are used should be addressed. In addition, the use of botanical and herbal supplements may be associated with risks that require further study.

• Cytokines as an index of immune function. As previously recommended by the CMNR (IOM, 1997), further research will be needed to determine if stress-related changes in cytokines can be detected reliably in spot urine samples collected during military field operations. Proinflammatory cytokines and their receptors and antagonists are all excreted in the urine. The magnitude of stress-related increases in the production of proinflammatory cytokines can be determined in whole-blood stimulation assays and possibly in 24-h urinary samples obtained during periods of stress, infectious illness, and/or trauma; however, the practicality and validity of urinary cytokine measures for field research studies must be determined.
  
  

  Studies are necessary to determine if cytokine-related measurements have greater value, greater sensitivity, or greater stimulus-related specificity than the standard measurements of red blood cell sedimentation rates and CRP protein as indicators of systemic disease and/or as models of stress-induced release or suppression of proinflammatory cytokines.

• Disease conditions and long-term host defense. Ensuring prompt etiologic diagnosis of infectious illness and early therapy with effective antimicrobial agents will spare the loss of body nutrients by minimizing disease severity and duration. The severity and duration of fever are in proportion to measurable losses of nutrients from the body and/or their accelerated consumption. Accordingly, the control of high fevers (but not necessarily their total elimination) by specific drugs (ibuprofen, and to a lesser extent, aspirin) that prevent the conversion of arachidonic acid to fever-related eicosanoids (for example, the prostaglandins) will conserve body weight and nutrient stores. During protracted infections, nutritional supplements (multivitamin and/or multimineral pills, antioxidants, and amino acids such as glutamine and arginine) may provide valuable immunological support. The potential value of

• similar combinations of supplements, given as a possible prophylactic measure during periods of severe military stress, is currently unknown but warrants future study. Further, the consumption of high-quality diets should be encouraged early in convalescence to restore body nutrient pools and lost weight.
  
  

  One important disease condition as yet unstudied is diarrhea. This condition should be examined to evaluate its effect on immune status indicators, both as a single variable and in combination with other important variables such as immunization, exercise, and reduced food intake. The major losses during diarrhea are those of water, sodium, potassium, and bicarbonate. None of these are known to have a direct effect on immune system functions; nonetheless, the resultant acidosis affects a variety of cell functions.

  A key question involving the immune status of Special Forces troops is how acute nutrient deprivation during training may influence host defense on a long-term basis, and whether temporary nutritional and immune deficits incurred during training may produce long-term vulnerability (see recommendation in the CMNR's report of Ranger I studies [IOM, 1992]). Research also will be needed to determine if cytokine-induced losses of essential body nutrients are important concerns in military personnel exposed to other nonnutritional stresses.

• Immune function in women. Most studies to date have focused largely on male soldiers. Therefore, there is a paucity of information about the immune response of energy-and sleep-deprived female personnel who participate in training activities. Research is needed to evaluate the interrelationships among sleep, nutrition, physical activity, female sex hormone responses, menstrual cycle, and immune function in women in the military. An emerging area of interest is the evaluation of the effects of endogenous and exogenous (phyto- and xeno-) estrogens on immune function. Of particular importance are the deficiency of iron in many military women and the immunological consequences of iron deficiency.

The study of the interaction of nutrition and immune function is an exceptionally active area of research in both the military and the civilian (academic and commercial) sectors.

A considerable number of conditions encountered by the military act as immune stressors. These stressors include operationally induced undernutrition

and dehydration; alterations in biological rhythms; atmospheric conditions such as temperature, humidity, and altitude; and environmental pollutants such as dust, smoke, and chemical fumes, as well as injuries and infectious agents themselves. As a result, studies of immune function in field situations contain many uncontrollable variables, and it is often difficult to attribute observed effects to one variable such as nutritional status.

The military's use of prophylactic immunization provides sufficient benefit beyond risk to warrant continued development. Recommendations concerning research on militarily relevant vaccines are contained in an earlier CMNR report (IOM, 1997). This is supported by a recent decision of the Secretary of Defense to begin systematic immunization of all U.S. military personnel against the biological warfare agent anthrax.

Pharmacologic agents such as aspirin, ibuprofen, and glucocorticoids modulate the effects of cytokines and can be used to minimize signs and symptoms of cytokine-induced acute-phase reactions and the nutrient losses that accompany them. Their use in military operations for the management of minor traumas and infections is well recognized and has been shown to sustain military performance during severe training exercises and operational missions.

Evidence to suggest that the administration of recombinant cytokines can modulate immune function in a desirable manner is limited at the present time to a small number of disease states. Their effectiveness has not been demonstrated in healthy subjects.

Field studies must be based on the results of prior experiments conducted in controlled laboratory and clinical settings. Experimental designs and methods must be validated by pilot tests prior to use. Because of the effects of circadian rhythms on immune function, samples must be collected at precisely defined times. In addition, because of the sensitivity and low levels of the molecules of interest, biological samples must be handled, transported, and stored according to recommendations for the materials in question, and appropriate controls must be included.

Total energy intake appears to play the greatest role in nutritional modulation of immune function. Since it has been demonstrated that prolonged energy deficits resulting in significant weight loss have an adverse effect on immune function, emphasis should be placed on the importance of adequate ration intake during military operations to minimize weight loss. Weight loss in the range of 10 percent in operations extending over 4 weeks raises the concern of reduced physical and cognitive performance and has potential health consequences for some individuals (IOM, 1995).

The nutritional status of soldiers should be optimized prior to deployment or engagement in any exercise or training course or even brief encounters with anything that would present a potential immune challenge

(disease, toxic agent, or environmental stress). When consumed as recommended, operational rations provide adequate energy and macronutrients.

In addition to energy intake, nutrients that appear to play a role in immune function include protein, iron, zinc, copper, and selenium; the antioxidants β-carotene and vitamins C and E; vitamin A and the B-group vitamins, especially B₆ , B₁₂ , and folate; the amino acids glutamine and arginine; and the polyunsaturated fatty acids (PUFAs). It is difficult to consider the role of one nutrient in isolation. Evidence for a role for vitamin C in immunomodulation remains controversial, and the role of vitamin E has been demonstrated chiefly in the elderly. Available data also suggest that altered dietary intakes of essential polyunsaturated fatty acids (PUFAs), either the n-6 or the n-3 PUFAs, may influence immune functions. Iron deficiency impairs immune system competence and depresses the bactericidal functions of phagocytic cells. Excess iron as well as iron deficiency may also compromise immune status. Selenium deficiency is associated with increased susceptibility to particular infectious pathogens and may modify the virulence of a coxsackie virus that causes heart muscle damage. The latter observation may explain the apparent prevalence of Keshan disease, an endemic juvenile cardiomyopathy thought to be caused by a coxsackie virus, in areas of China experiencing periodic selenium deficiency. Glutamine has demonstrated potential for improving immune function in critical illness, and parenteral and enteral administration of glutamine has been observed to improve recovery following gastrointestinal surgery, but its usefulness in healthy populations has not been determined. Studies to evaluate the effects of supplemental glutamine on the immune function of soldiers have shown no demonstrable effects. The amounts of vitamins and trace elements (including zinc, copper, and selenium), contained in operational rations, meet all MRDAs (Military Recommended Dietary Allowances) if the diet is fully consumed. However, varying combinations of military stresses may increase the need for certain essential nutrients to values greater than the MRDA to maintain immunological competence.

The effects of providing supplements of vitamins A, C and E, as well as certain polyunsaturated fatty acids and amino acids, prior to, during, or following infections are virtually unknown in young, healthy adult men. Many questions remain regarding the efficacy of these nutrients in amounts that exceed Military Recommended Dietary Allowance (MRDA) levels. However, during protracted infections, nutritional supplements (multivitamin and/or multimineral pills, antioxidants, and amino acids such as glutamine and arginine) may provide valuable immunological support. Further, the consumption of high-quality diets should be encouraged early in convalescence

to restore body nutrient pools and lost weight. The most prudent approach seems to be one of increasing fruit and vegetable consumption in the diet, thus maximizing the potential benefits of antioxidant nutrients.

Safety problems associated with excess consumption of supplements are much more likely for some nutrients than for others. Toxicity and the potential for nutrient-nutrient interactions must be considered individually. Excess intakes of vitamin A may be toxic, whereas vitamins C and E are relatively nontoxic and have been shown to enhance the immune response. Trace minerals are particularly problematic because requirements may be altered during periods of illness (increased), while at the same time, excessive intakes of some trace elements may be immunosuppressive.

Excess iron as well as iron deficiency may compromise immune status. The problem of compromised iron status in female personnel is a matter of concern because it may impact immune function, physical performance, and cognitive function. It is important to maintain adequate iron status in female soldiers and to do so without causing excess iron intake by males.

Glutamine has demonstrated potential for improving immune function in critical illness, but its usefulness in healthy populations is unknown. Parenteral and enteral administration of glutamine has been observed to improve recovery following gastrointestinal surgery. Thus far, the effect of glutamine has been observed only in supraphysiological amounts and only in patients undergoing bone marrow transplantation or major operations and those who sustain life-threatening sepsis. Studies to evaluate the effects of supplemental glutamine on the immune function of soldiers have shown no demonstrable effects. An effect of glutamine deficiency also has not been demonstrated.

Although none of the major body nutrients lost during severe diarrheal episodes (sodium, potassium, and bicarbonate) are known to influence immune function, rehydration strategies (and in some situations, supplementation with glutamine) may be of use in the treatment of diarrhea.

Finally, it must be emphasized that the results of studies performed in deficient animals or individuals are different from those done on adequately nourished ones and that, in many cases, an "overdose" of a nutrient, as well as a deficiency, leads to negative consequences.

• The CMNR recommends the use of medically appropriate and directed prophylactic medications and procedures to minimize the adverse effects of infectious agents. However, the CMNR sees no potential value at this time administering cytokines or anti-cytokines to healthy military personnel.

It is generally assumed that the body's production of endogenous cytokines during stressful situations is beneficial to the host. However, if endogenous proinflammatory cytokines accumulate in large excesses or are given in large doses, they may have noxious or even dangerous consequences. The military should remain cognizant of the very active civilian-sector research concerning cytokines, their complex control mechanisms, and their functions, and should apply any pertinent new findings to the management of militarily relevant infectious diseases, trauma, or other stresses. The military should also keep apprised of advances (in the form of proven treatments) that emerge from this research.

• In light of the importance of military immunization programs for achieving and maintaining immune status at optimal levels, the CMNR reiterates its previous recommendations (IOM, 1997) that vigorous research efforts be undertaken to create and evaluate militarily relevant oral vaccines.

These should include optimization of administration schedules and elucidation of the influence of nutritional status on vaccine efficacy. Immunological responses to vaccines may be altered by the stresses of mobilization and/or overseas deployments. Antibody responses to vaccines are known to be depressed by protein-energy malnutrition. The potential problem of reduced responsiveness to military vaccines given during periods of mobilization and deployment stresses (in comparison to normal responses, as measured in control studies) also deserves future study.

• It is recommended that soldiers maintain good physical fitness via a regular, moderate exercise program as a means of sustaining optimum immune function. Since the intensity and duration of physical activity can affect immune function, training regimens that achieve high levels of physical fitness without adverse effects on immune status should be established.

• Additionally, the CMNR recommends the use of methods to minimize psychological stresses, including training, conditioning, and structured briefing and debriefing.

• In view of the compromised immune function noted in studies of Ranger trainees, the CMNR recommends that, where possible, individuals who have lost significant lean body mass should not be redeployed until this lean mass is regained.

Although data showing an effect of weight loss on immune function may be limited, it is reasonable to suggest that the maintenance of body weight within 10 percent of ideal weight should increase the likelihood that adequate immune function will be maintained. Thus, the committee recommends that soldiers be

advised to achieve an energy intake sufficient to maintain normal weight. The energy intakes required to maintain body weight will vary with the intensity and duration of physical activity; therefore, the best field guide for individual soldiers and commanders is to monitor body weight changes and to emphasize, through a ''field-feeding doctrine,'' the importance of ration intake as the fuel for the soldier to maintain health and performance.

• The CMNR recommends that nutritional anemia be treated prior to deployment and that individuals classified as anemic¹ and requiring iron supplements not be deployed.

With the reduced personnel in today's Army and the potential for frequent deployment, it is important that soldiers be in good nutritional health at the time of deployment and that an effort be made to correct any compromise in status that may have resulted from previous deployment. Some scientists believe that iron supplements, if given during the course of bacterial or parasitic infections, may increase the severity of these illnesses. Because this topic is a controversial one, it requires further investigation. Nevertheless, it is recommended that if additional iron is required (for prophylactic purposes), it should be in the form of an optional ration supplement, and the iron content of operational rations themselves should not exceed MRDA levels.

• As a means of reducing the number of stresses encountered by military personnel, the committee encourages the development and implementation of nutrition education programs targeted at high-risk military groups, such as Special Forces troops and female soldiers.

The military should increase efforts to communicate information regarding healthy eating habits and supplement use to all personnel. Since dehydration and energy deficit have a great potential for compromising immune function, soldiers should also be educated regarding compliance with the "water doctrine."

Supplementation with certain nutrients may be of value for sustaining host defense mechanisms (including those conferred by the immune system) at normal levels during periods of extreme physiological and physical stress. Carefully controlled pilot and more extensive field studies will be necessary to investigate this possibility. It is unlikely, however, that nutritional supplements can produce a state of superimmunity in normal subjects or military personnel.

• At this time, the CMNR cannot recommend general supplementation of military rations above the MRDAs for the purpose of enhancing immune function.

There are no definitive studies that demonstrate positive benefits to young, healthy, active individuals of nutrient supplements at levels significantly in excess of those recommended by the MRDAs and commonly provided by foods. Encouraging ration intake to sustain nutrient levels as described in the MRDAs appears to be the best recommendation until further research clearly can define the likely benefits of specific nutrient supplementation under defined operational conditions. Soldiers should be cautioned regarding the indiscriminate use of individual supplements and the potential effects of inadequate nutrient intake, as well as the use of single or combined supplements, since their effects on immune status are not known.

• The CMNR recommends that, when needed, the preferred method of providing supplemental nutrients is through a ration component.

This would reduce both the potential for excessive intake by those individuals who do not need the nutrient and the potential misuse that exists when supplemental nutrients are provided in individual nutrient form. Because energy is one nutrient that has been identified as playing a role in immune function, provision of supplemental energy in the form of a food bar would allow soldiers to increase their nutrient intake as needed according to activity levels.

• The CMNR recommends that the military gain a better understanding of supplement use as well as supplement abuse by personnel and make strong recommendations for the appropriate use or nonuse of nutritional supplements.

The emphasis should be on education and wise choices. In the past, the CMNR has suggested the development of a "field-feeding doctrine" (IOM, 1995), with the guiding principle that the energy intakes of military personnel during training and combat operations should be adequate to meet their energy expenditures and to maintain body weight and lean body mass. This field-feeding doctrine would accompany the successful "water doctrine" that resulted from a recommendation in the report Fluid Replacement in Heat Stress, (IOM, 1991, 1993). The guiding principle of the water doctrine was to ensure that adequate fluid intake is maintained to avoid dehydration and subsequent decreased food intake. As more information is gained on supplement use and misuse and on the risks and benefits of supplements, the Army may want to consider formulating a ''supplement doctrine" similar to the water and food doctrines to address these concerns and add a component to nutrition education programs. A better understanding of supplement use will provide information on the prevalence and frequency of use, its impact on an individual's nutritional status, and the likelihood of reckless or dangerous nutrition practices. Such information will help provide for the delivery of targeted and focused nutritional education messages. The committee is aware that some information on supplement use will be obtained by the Army Food and Nutrition Survey and suggests that additional information on supplement use can best be obtained by

including appropriate questions in ongoing military health surveys, such as the Survey of Health-Related Behaviors Among Military Personnel.

The CMNR strongly encourages the military to keep apprised of relevant civilian research and consider the application of selected findings and protocols to the military situation.

• The CMNR recommends that research be conducted to determine the appropriate field measures for monitoring nutritionally induced immune responses, particularly for determining the presence of acute-phase reactions and changes in immune function of the type and degree that are likely to occur as a result of the nutritional insults suffered by soldiers in typical deployment situations.

This will require basing field studies on appropriate clinical investigations, piloting experimental designs, and using a simple panel of standard tests that have been validated for the field. Particular attention must be paid to the timing of sample collection; the conditions under which samples are transported, stored, and handled; and the use of proper controls.

A rapid assessment of immune functions for use in the field includes clinical evaluations of local lesions, sites of inflammation, and signs and symptoms of generalized infectious illness. The C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and white cell counts are the most rapid and least expensive lab tests. Skin tests are highly valuable markers of cell-mediated immunity but require 48 hours before they can be read. Other tests can be valuable if time and facilities permit. On the other hand, preliminary clinical trials may employ additional kinds of sophisticated immunological studies, along with those listed for field investigations.

• In addition, the CMNR recommends careful design of research protocols.

Efforts should be directed towards ensuring the control of as many environmental, behavioral, and treatment variables as possible so that the effects attributed to a deficiency of a particular nutrient are not in fact the result of some other operational stress. The military nutrition research program should attempt to differentiate between nutrition-induced immune dysfunction and that caused by other forms of operational stress.

• The CMNR strongly encourages the military to increase its awareness of and consider the military applications of the findings within the civilian research community regarding nutrition and immune function. The advice of civilian and military immunologists should be sought to identify the testing methods that have proven to be most useful and field applicable for monitoring immune status and function.

Very little is yet known about the immunological effects of short-term food deprivation when accompanied by varying combinations of other military stresses. Future investigations into the changing immunological status of troops in the field must obviously be based upon available current knowledge about the immunological impact of individual stresses. However, because multiple stresses (including food deprivation) are to be expected, these will have to be studied using experimental designs and methods that have been validated by pilot studies prior to their use in large field studies.

• The CMNR reiterates its previous recommendations (IOM, 1997) that laboratory-based studies be performed to determine if an interleukin-6 (IL-6)-creatinine ratio (or some comparable measure) can be measured in single "spot" urine samples as an index of the 24-h excretion of IL-6 and if 24-h IL-6 excretion is, in turn, a reliable indicator of acute stress response.

Such determinations should be made before urinary IL-6 measurements are used in field studies, where 24-h urinary collections are virtually impossible to obtain.

• The CMNR recommends the development and field testing of appropriate measurements of cytokines or their various markers in urine and blood that are reflective of ongoing acute-phase reactions and of changes in immune status in multistress environments.

Developmental efforts should focus on one or two measurements that could be standardized with sufficient accuracy to serve as marker replacements for an entire (and complex) cytokine battery and would have some clinical correlate in immune function, such as skin test response and peak titer following vaccination. These may be useful in studies of the effects of nutritional status on immune function. Civilian research efforts in this area should be followed carefully, and collaborative relationships should be formed.

• The CMNR recommends that if research is conducted on the ability of nutrients to influence immune status, priority at this time should be placed on the antioxidants β-carotene and vitamins C and E.

The committee acknowledges that insufficient data are available to identify any specific nutrient or combination of nutrients as having adequately demonstrated the ability to enhance immune function under the military operational conditions investigated. This would include vitamins C and E, as well as the amino acids glutamine and arginine.

• The influence of iron status on the risk of infection requires further investigation. This is also an area of interest to the civilian medical community.

• It is recommended that the military keep apprised of research being conducted in the civilian sector on immune function in physically active

women and consider conducting studies on military women in situations of deployment to augment the findings of civilian studies.

At present, there are very few studies on the immune function of healthy women or women in high stress situations.

The Committee on Military Nutrition Research is pleased to participate with the Military Nutrition Division, U.S. Army Research Institute of Environmental Medicine, and the U.S. Army Medical Research and Materiel Command in progress relating to the nutrition, performance, and health of U.S. military personnel.

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