Participants included in this study were healthy, active-duty Soldiers assigned to an elite U.S. Army Special Operations unit with full medical clearance, indicating they were free of disease or health conditions that would incur duty limitations or restrictions. Exclusion criteria included being deployed to a combat zone for ≥30 days within the previous four months, although no potential participants met this criterion for exclusion. All combat, combat support, and combat service support Soldiers assigned to the unit and eligible to deploy were invited to attend an informational study briefing. The briefing time was coordinated with unit leadership and scheduled to maximize the number of available Soldiers not otherwise actively engaged in training. Participants provided verbal informed consent after being recruited from the informational briefing with an ombudsman present. Approximately 90% of those that were briefed participated in the study. Pre-deployment baseline data on markers of nutritional status were collected between January and October of 2013 from 107 male participants 4–8 weeks before deployment. After excluding for early redeployment (n = 4), late deployment (n = 1), and missing background information at baseline (n = 7), post-deployment follow-up data on markers of nutritional status were collected from 50 participants during the reintegration period, within 10 days upon returning from deployment. The majority had measures obtained between 4 and 7 days (76%, 38/50) and fewer had measures obtained between 2 and 3 days (14%, 7/50) or later at 10 days (10%, 5/50). Loss to follow-up of the remaining participants occurred predominately due to changes in deployment schedules, such as that the participant did not deploy or did not return yet from deployment. One participant sustained a combat related injury and did not complete the deployment. The changing and often unpredictable nature of Soldier schedules and personnel assignments is common before, during, and after operational deployment and a similar pattern of loss to follow-up has been reported in previous deployment studies [7, 9]. Of the 50 participants with markers of nutritional status at baseline and post-deployment, all participants completed a food frequency questionnaire (FFQ) and background questionnaire at baseline and 33 also completed a FFQ and follow-up questionnaire upon return from deployment. Not all Soldiers were able to complete the FFQ and follow-up questionnaire upon return from deployment due to schedule time constraints during the reintegration period. A flow chart describing participant selection is provided in Fig. 1.
Deployment length was a uniform duration of time between 3 to 6 months (all Soldiers completed the same length of deployment). Eighty-six percent were deployed to Afghanistan and the remaining participants were deployed to other locations outside the continental U.S. All Soldiers had access to hot meals through cafeteria-style dining facilities for up to three meals per day, depending on individual schedules. Some Soldiers may have been required to be on duty during hours when dining facilities were closed, which occasionally (but not regularly) could have limited the number of meals they were able to obtain. Other shelf-stable food items were available to Soldiers through personal shipment, such as care packages, or supply by the unit. Meal, Ready-to-Eat (MRE) field rations were available if required by mission conditions. This study was approved by the Human Use Review Committee at the U.S. Army Research Institute of Environmental Medicine, Natick, MA. Investigators adhered to U.S. Army Regulation 70–25 and U.S. Army Medical Research and Material Command regulation 70–25 on the use of volunteers in research.
Dietary intake assessment and demographic characteristics
Dietary intake over the previous 3-months was assessed at baseline and upon return from deployment with a 101-item self-administered paper and pencil version of the 2005 Block FFQ (NutritionQuest, Berkeley, CA) [10, 11]. The FFQ was chosen as the dietary assessment method to reduce participant burden and because the potential for research efforts to distract from military operations precluded the use of other dietary assessment methods during the deployment. Healthy Eating Index-2010 (HEI-2010) scores were calculated according to maximum and minimum score standards for each component, which have been described in detail elsewhere . HEI-2010 is a measure of diet quality that assesses conformance with federal dietary guidelines [13,14,15]. The index is comprised of 12 components; 9 components assess compliance with adequate intakes (total vegetables, greens and beans, total fruit, whole fruit, whole grains, dairy, total protein foods, seafood and plant protein, and fatty acid ratio) and 3 components assess compliance with moderation (sodium, refined grains, and empty calories) . Higher scores for each component are reflective of greater compliance with federal guidelines. The sum of each component score together yields the total HEI-2010 score, which ranges from 0 to 100.
Daily consumption of foods and food groups, dietary intake of key micronutrients, as well as carbohydrate, fat, and protein (expressed in grams and percent of calories), total caloric intake, and percent of calories from sweets and desserts were derived from the frequency and quantity of reported food items on the FFQ and corresponding nutrient values provided in the USDA’s Food and Nutrient Databases , as calculated by NutritionQuest. Self-reported frequency of intake of food items was assessed by asking participants to select “How often in the past 3 months” each food item listed on the FFQ was consumed. Response options included: ‘never’, ‘once per month’, ‘2–3 times per month’, ‘once per week’, ‘2 times per week’, ‘3–4 times per week’, ‘5–6 times per week’, or ‘every day’. Quantity of reported food items was then assessed by asking participants to select “How much on those days”. Response options included portion sizes appropriate for each food item and respondents were provided with standardized portion size pictures.
Demographic information (age, marital status, education level, combat classification, grade, prior combat deployments, and smoking status) was determined from a standardized self-administered demographic/background questionnaire at baseline. MRE field ration use was determined from a follow-up questionnaire administered upon return from deployment. Participants were asked: “During the past 6 months, during the time when you were deployed only, did you consume any MRE (Meal, Ready-to-Eat) type of product listed below? If so, how often did you use these products?” Products included ‘Meal, Ready-to-Eat’, ‘First Strike Ration’, ‘First Strike Bar’, and ‘Ergo Drink’ and response options of frequency of use included ‘never’, ‘once a month’, ‘once a week’, ‘2–6 times per week’, ‘once a day’, ‘2 times per day’, or ‘3+ times per day’.
Markers of nutritional status
Blood samples were collected by median cubital venipuncture following an overnight fast at baseline and post-deployment. Immediately after collection, heparinized whole blood was used to measure hemoglobin and glucose using the iSTAT 1 handheld blood analyzer (Abbott Point of Care Inc., Princeton, NJ). Remaining samples were centrifuged, processed to isolate serum, frozen, and stored at −80 °C until the time of assay. Ferritin and PTH were measured using immunoassay with the Immulite 2000 XPi automated analyzer (Siemens Medical Solutions, Malvern, PA). Iron, total iron binding capacity (TIBC), total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), and triglycerides were assessed using a UniCel DxC 600 PRO clinical chemistry analyzer (Beckman Coulter, Brea, CA). Enzyme-linked immunosorbent assays (ELISA) were used to measure hepcidin (DRG Hepcidin ELISA, DRG International Inc., Springfield, NJ) and soluble transferrin receptor (sTfR) (Quantikine IVD Human Soluble Transferrin Receptor ELISA, R&D Systems Inc., Minneapolis, MN). Transferrin saturation (TS) was calculated as iron concentration × 100 / TIBC concentration. Levels of 25-hydroxy vitamin D (25-OH vitamin D) were assessed using radioimmunoassay (RIA; 25-Hydroxyvitamin D 125I RIA kit, DiaSorin Inc., Stillwater, MN).
SAS statistical software (version 9.3; SAS Institute, Cary, NC) was used to perform all analyses. Findings were considered statistically significant for all analyses at P ≤ 0.05. Change in means from baseline to post-deployment for HEI-2010 scores, foods and food group intakes, nutrient intakes, and markers of nutritional status were assessed with a paired t-test. Paired measurements are well established to increase power. The proportion with iron status markers below threshold cut-offs indicative of iron deficiency anemia (ferritin <12 ng/mL and hemoglobin <13.7 g/dL) was also determined.
Decreases in dietary calcium and vitamin D intake and increases in serum PTH over the deployment were concurrently observed, therefore post hoc correlation and regression analyses were performed to determine associations with post-deployment PTH. PTH concentrations were regressed onto post-deployment serum 25-OH vitamin D concentrations, as well as total (dietary + supplemental) vitamin D and calcium intake during deployment using the General Linear Model (GLM) procedure in SAS. One outlier (where serum 25-OH vitamin D > 200 nmol/L) was omitted prior to analysis. Models were not adjusted for demographics or overall diet quality (HEI-2010 total score) during deployment because there were no univariate associations with PTH (P > 0.05).