Nitrogen retention and protein balance are maintained or become more positive during periods of energy balance, and more negative during energy deficit (2,5,25). Negative energy balance increases amino acid oxidation for energy, possibly limiting protein and amino acid availability to support other physiological functions, such as immune response, neurotransmitter synthesis, and accretion of lean body mass (18). For example, short-term (≤ 4 d) fasting increases leucine flux, leucine oxidation, and proteolysis during rest and exercise (11,12,19,26). Thus, countermeasures to mitigate the effects of energy deficit on protein and amino acid metabolism have been studied.
For a given dietary energy deficit, nitrogen balance is less negative when the protein content of the diet is higher (3,8). Additionally, the total dietary energy required for an individual to maintain nitrogen balance (zero net loss) appears to decrease as the dietary protein content increases (21). These observations suggest that a countermeasure likely to be effective in maintaining the integrity of protein metabolism during energy deficit is an increased dietary protein intake. High-protein diets result in the preservation of lean body mass during weight loss (13,14). However, in all of these previous investigations, the negative energy balance was induced via a decrease (relative to the normal or accustomed level) in caloric intake, rather than by increasing energy expenditure. While that model may be appropriate for understanding physiological responses of obesity treatment interventions for sedentary persons or those whose physical activity level is low, it is probably not useful for studying the physiological responses of persons who experience energy deficit attributable to prolonged or repeated periods of high-energy expenditures without adequately increasing (again, relative to normal or accustomed) energy intakes-for instance, certain athletes and deployed military personnel. Understanding how negative energy balance impacts protein metabolism, under conditions of high energy expenditure (high metabolic rates) is necessary to provide a scientific basis for development of countermeasures to minimize health effects attributable to limited protein and/or amino acid availability arising from those conditions.
Controlled experimental studies investigating whole-body protein turnover and dietary protein requirements of physically fit individuals' abruptly increasing total daily energy expenditure have not been conducted. In 1999 and 2006, the Institute of Medicine recommended that research was needed to better quantify effects of an energy deficit, especially energy deficits arising under high-energy expenditure conditions, on protein requirements (9,10). Therefore, this investigation sought to determine the adequacy of current recommendations regarding optimal dietary protein content for persons such as athletes or deployed military personnel who experience periods of high energy expenditures without an adequate increase in energy intakes. The working hypothesis was that nitrogen balance and protein synthesis would decrease while protein turnover and breakdown would increase in individuals who sustained an unaccustomed increase in total daily energy expenditure without a compensatory increase in total energy intake. Conversely, nitrogen balance, protein synthesis, and protein breakdown would remain unchanged in those who increased energy intake sufficiently to match the increase in energy expenditure. Additionally, it was hypothesized that the decrease in nitrogen balance and protein synthesis coupled with an increase in protein breakdown, associated with an unaccustomed increase in total daily energy expenditure unmatched by increased energy intake, would be mitigated if the diet contained higher amounts of protein compared with diets providing the same total energy but with lower protein content.
This study was approved by the appropriate institutional review boards. Twenty-two healthy men gave informed, written consent to participate in this investigation following an oral and written explanation of all study procedures and risks. All subjects completed an initial screening form and were medically cleared for participation in accordance with United States Army Research Institute of Environmental Medicine guidelines for human use. Subjects were required to be weight stable (± 2.2 kg) for 2 months prior to the start of the study, have a 6-month endurance training history (≥ 5 d·wk−1, 30 min·d−1), and a V˙O2peak ≥ 54 or 52 mL·kg−1·min−1 for ages 18-29 and 30-35 yr, respectively. Individuals who used tobacco products, had a disease or took medications known to impact macronutrient metabolism, or possessed any cardiovascular or musculoskeletal conditions that prohibited strenuous exercise were excluded.
The investigators have adhered to the policies for protection of human subjects as prescribed in Army Regulation 70-25, and the research was conducted in adherence with the provisions of 32 CFR part 219. Human subjects participated in this study after giving their free and informed voluntary consent. Investigators adhered to AR 70-25 and USAMRMC Regulation 70-25 on the use of volunteers in research.
Volunteers resided in a research dormitory for the duration of the experimental phase of the study, allowing for strict control and monitoring of energy intake and energy expenditure (Fig. 1). Subjects were divided into three groups: balance (BAL), deficit (DEF), and deficit high protein (DEF-HP). During the first 4 d of the experimental phase, all groups were in energy balance. Energy intake and expenditure was matched to subjects' normally accustomed levels. The normal accustomed level was assessed by diet and physical activity records subjects maintained for 3 d before beginning the experimental phase of the study. Beginning on day 5, all volunteers increased their energy expenditure by 1000 kcal·d−1 by exercising at intensities corresponding to 50-65% V˙O2peak. In the BAL group, energy intake was increased to match this increase in expenditure, whereas the DEF and DEF-HP groups did not increase their energy intake, thereby creating a 1000-kcal energy deficit. Nitrogen balance was assessed daily, and whole-body protein turnover, derived from phenylalanine and tyrosine kinetics, was assessed in the fasted state at rest on days 4, 7, and 12, using a priming dose of L-[ring-15N]tyrosine and a 4-h primed, continuous infusions of L-[15N]phenylalanine and L-[ring-2H4]tyrosine. Baseline testing included assessment of aerobic capacity (peak oxygen uptake (V˙O2peak)), anthropometry (height and weight), body composition (DEXA), 3-d diet records, and 3-d physical activity records. Descriptions of these measures are given below.
Peak oxygen uptake.
Peak oxygen uptake (V˙O2peak) was determined by analysis of expired gases during a cycle ergometer test using indirect open-circuit spirometry (True Max 2400, Parvomedics, Sandy, UT). The protocol was progressive in intensity, continuous in nature, and performed indoors at a temperature of 20-22°C and 30-80% humidity. Volunteers first pedaled for 3 min at 100 W. Then, intensity was increased by 30 W every 2 min until the volunteer was unable to maintain a pedaling rate that maintained or increased O2 consumption.
Vertical height was measured in duplicate to the nearest 0.1 cm. Subjects were measured in stocking feet and standing on a flat surface, feet together, knees straight, and the head, shoulder blades, buttocks, and heels in contact with a vertical wall. Body weight was measured at baseline and twice daily (prior to morning meal, and following the evening meal) using a calibrated electronic battery-powered scale accurate to 0.1 kg. Body composition was determined by dual-energy x-ray absorptiometry (DEXA). DEXA measurements were done at baseline and on day 11 of the intervention.
Baseline dietary records.
Three-day dietary records were collected from each volunteer before they began the experimental phase of the study, to assess their usual nutrient intake for energy, carbohydrates, protein, and fat. All dietary records were analyzed by computer-based nutrient analysis software, Food Processor v. 8.5.0 (ESHA Research, Salem, OR). The average daily energy intake determined from these records was used in combination with the average daily energy expenditure determined from the physical activity records to establish the energy intake and expenditure levels for the first 4 d of the experimental phase of the study.
Baseline physical activity records.
Three-day physical activity records were maintained by each volunteer before beginning the experimental phase of the study, to assess their usual energy expenditure. Each subject's normal daily activities were grouped into different categories by duration and corresponding metabolic equivalent (MET). The average daily energy expenditure determined from these records was used in combination with the average daily energy intake determined from the dietary records to establish the energy intake and expenditure levels for the first 4 d of the experimental phase of the study.
Three to five days prior to starting the controlled diet, subjects were asked to consume a diet that contained the same amount of protein as the study diet in order to habituate hepatic enzymes to a specific dietary protein level. Subjects were instructed on how to do this during a prescreening evaluation of their 3-d diet record. Subjects consumed a controlled diet throughout the study. The diet consisted of whole foods and liquid supplements provided to each subject in individual amounts. All meals were prepared in the USARIEM metabolic kitchen by a registered dietitian. Throughout the study, the dietary protein source was consistent across groups while protein content was held constant at 0.9 g (BAL, DEF) or 1.8 g (DEF-HP) of protein per kilogram of body weight per day. Total energy intake for study days 1-4 was individualized to match each volunteer's usual energy intake and expenditure in order to maintain energy balance. The BAL group increased their energy intake by 1000 kcal on days 5-11 to match their increase in energy expenditure, while DEF and DEF-HP maintained the same energy intake during the period of increased energy expenditure as during the baseline period (days 1-4). Total energy content was adjusted by adding or subtracting fat- and carbohydrate-containing foods so that the caloric ratio of these nutrients in the diet remained approximately 1:2. The diet provided approximately 8-12.6% protein, 32-37% fat, and 55% carbohydrate. Meals were served according to a fixed schedule, but water and sugar-free noncaffeinated drinks were taken ad libitum.
Usual/normal exercise period: During study days 1-4, each subject's normal daily activities were grouped into different categories by duration and corresponding MET level. The type, intensity, and duration of all activities were tightly controlled by dividing each 24-h period into prescribed 15-min blocks at a specific MET level in order to duplicate the subjects' normal daily caloric expenditure.
Increased exercise period: During study days 5-11, volunteers increased their energy expenditure by 1000 kcal each day by exercising between 50 and 65% of their V˙O2peak. For subject comfort and to avoid injuries, exercise was divided between various modalities (bike, treadmill, elliptical) and split into 15-min intervals throughout the day. Volunteers were allowed to self-select how they wanted to distribute these 15-min blocks throughout the day, as long as they completed the prescribed energy expenditure.
Determination of total daily energy expenditure.
Total daily energy expenditure was determined periodically throughout the study, using indirect open-circuit spirometry (True Max 2400, Parvomedics, Sandy, UT). Total daily energy expenditure (EEtotal) was calculated using the following equation:
EEsleep was estimated via measurement of basal metabolic rate on days 1, 2, 5, and 8. EEphysical activity was measured during each prescribed exercise activity on days 1, 5, and 8. EEmiscellaneous was measured during nonexercise activities (i.e., watching TV, playing video games, reading) on days 1, 5, and 8.
Determination of nitrogen balance.
Nitrogen balance was determined daily using the following equation:
Nin was determined by computer-based nutrient analysis Food Processor, v. 8.5.0 (ESHA Research, Salem, OR) of each volunteer's daily dietary intake. Total N content of daily urine, feces, and sweat collections was determined using the micro-Kjeldahl technique. Accuracy of the Nurine was assessed using 24-h creatinine excretion. Daily fecal samples were homogenized and pooled into three separate collection periods according to the appearance of fecal markers (carmine red and charcoal). Total body sweat loss (Nsweat) was estimated via a modification of the regional sweat-collection method described by Lemon et al. (15). Briefly, volunteers affixed a sweat-collection pad to each thigh using an adhesive dressing prior to beginning the first exercise bout of the day. These pads were removed, the urea was extracted, and the extract was frozen for later analysis of total nitrogen following the last exercise session of the day. Total nitrogen loss was estimated by calculating the surface area of the collection pad, determining the nitrogen collected from this surface area, and extrapolating this amount to the surface area of the subject's body. Miscellaneous nitrogen losses (Nmiscellaneous, skin, hair, secretions, sweat while at rest) were estimated as 5.0 mg N·kg−1 body weight·d−1 (3,4). Daily nitrogen balance was calculated (mg N·kg−1 FFM·d−1), and results were then pooled and means were determined for three separate phases: baseline days 3-4, days 5-8 (EX1) and days 9-11 (EX2).
Whole-body protein turnover was derived from phenylalanine (Phe) and tyrosine (Tyr) kinetics assessed in the fasted state at rest on days 4, 7, and 12. Subjects began fasting at 1800 h on the evening before each protein-turnover assessment, and they were transported to the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, where they remained overnight in the Metabolic Research Unit under the supervision of nursing staff. Subjects were awakened the following morning at about 0450 h. A venous catheter was inserted into an antecubital vein for isotope infusion. Another catheter was placed in the contralateral hand vein, and the hand was heated with a heating pad, for the sampling of "arterialized" blood (1). At 0600 h, subjects were infused with a priming dose of L-[15N]phenylalanine (3.9 μmol·kg−1), L-[ring-2H4]tyrosine (2.9 μmol·kg−1), and L-[ring-15N]tyrosine (1.4 μmol·kg−1), followed by a 4-h continuous infusion (50 mL·h−1) of L-[15N]phenylalanine (3.9 μmol·kg−1·h−1) and L-[ring-2H4]tyrosine (2.9 μmol·kg−1·h−1). "Arterialized" venous blood samples were drawn immediately before the priming doses were administered, at 60-min intervals during the first 2 h of the infusion, and at 15-min intervals during the final 2 h of the infusion for subsequent analyses of plasma isotope enrichment. Resting metabolic rate and CO2 production rate were measured before the priming infusions begin, and for the last 5 min of each hour of the infusion. At the completion of the infusion period, catheters were removed, the subjects' arms were bandaged, and the volunteers were transported back to USARIEM to resume diet and exercise regimes at approximately 1200 h.
Phe and Tyr kinetics were calculated using the equations described by Thompson et al. (24). Phe and Tyr fluxes (Q; μmol·kg−1·h−1) were obtained from isotope dilution by the following equation:
where i is the rate of infusion of the tracer (μmol·kg−1·h−1), and Ei and Ep are the enrichments of the infusate and the plasma amino acids (Phe or Tyr), respectively. The conversion rate of Phe to Tyr (Qpt; μmol·kg−1·h−1) was calculated from the following equation:
where Qt and Qp are the flux rates for Tyr and Phe estimated independently by the primed constant infusions of [2H4] Tyr and [15N] Phe, respectively; Ep and Et are the respective enrichments of [15N] Phe and [15N] Tyr in plasma; and ip is the infusion rate of [15N] Phe.
Whole-body protein turnover.
Whole-body protein kinetics were estimated from Phe and Tyr kinetics based on the following equation:
where Sp is the loss of Phe from the free amino acid pool to protein synthesis, and Bp is the rate of entry of Phe into the free amino acid pool from protein breakdown. Because Phe is not synthesized endogenously, its flux in the fasted state is an index of whole-body protein breakdown, because it represents Phe derived exclusively from whole-body proteolysis. Similarly, the conversion rate of Phe to Tyr or Phe hydroxylation can serve as an index of net protein oxidation. Lastly, assuming that most Phe not oxidized is used for protein synthesis, the disposal rate of nonoxidative Phe, derived by subtracting the conversion rate of Phe to Tyr from Phe flux, can be considered an index of whole-body protein synthesis.
Results are presented as means (± SEM). Statistical analysis was completed using the SPSS statistical package version 13.0 (SPSS Inc., Chicago, IL). Students t-test was used to compare baseline and pre- and poststudy anthropometric data (i.e., body weight, body fat, and FFM), and repeated-measures ANOVA was used to compare NBAL and whole-body protein turnover over time between and within groups. Significant main or interaction effects were analyzed using the Student-Newman-Keuls post hoc test. A P value of ≤ 0.05 was considered statistically significant.
Descriptive characteristics of the test subjects are presented in Table 1. Baseline weight (kg) and FFM (kg) were significantly higher for DEF-HP compared with BAL and DEF. Groups were matched according to V˙O2peak (mL·kg−1·min−1).
Actual dietary intakes for the BAL, DEF, and DEF-HP groups are presented in Table 2. Protein intake in the BAL and DEF groups was slightly higher than prescribed (~1.0 g·kg−1·d−1), whereas the DEF-HP met the prescribed level during BL and EX (~1.8 g·kg−1·d−1). Mean energy deficits (kcal·d−1) for study days 5-11 were 40 ± 20, 868 ± 41, and 1006 ± 33 for BAL, DEF, and DEF-HP, respectively. There was no difference in energy deficit between DEF and DEF-HP.
Changes in body weight and body composition after the 11-d intervention are presented in Table 3. DEF and DEF-HP experienced significant decreases in body weight (kg), body fat (%), and fat-free mass (kg), whereas BAL experienced a significant decrease in fat-free mass only. There were no differences between groups for these outcome measures.
Pooled nitrogen-balance data from study days 3-4 (BL), 5-8 (EX 1), and 9-11 (EX 2) are presented in Figure 2. DEF experienced a significant decrease in nitrogen balance from BL (10.7 ± 1.0 mg N·kg−1 FFM·d−1) to EX 1 (−28.4 ± 0.7 mg N·kg−1 FFM·d−1) that was maintained in EX 2 (−25.0 ± 1.0 mg N·kg−1 FFM·d−1). The BAL group did not experience any significant changes in nitrogen balance over time (−21.3 ± 0.3, −0.9 ± 0.7, and 0.3 ± 0.7 g mg N·kg−1 FFM·d−1 for BL, EX 1, and EX 2, respectively). Similarly, there was no difference in nitrogen balance for DEF-HP across all three time points (−24.4 ± 12.0, −10.9 ± 11.0, and −4.1 ± 15.0 g N·d−1 for BL, EX 1, and EX 2, respectively).
Phenylalanine and tyrosine kinetics.
Data for Phe and Tyr kinetics are presented in Figures 3-5. No within- or between-group differences were found over time for Phe flux (Qp), conversion rate of Phe to Tyr (Qpt), or the derived protein synthesis value (Sp).
This investigation examined the impact of a 1000-kcal·d−1 exercise-induced energy deficit on nitrogen balance and whole-body protein turnover in a group of healthy, fit males, consuming either 0.9 or 1.8 g of protein per kilogram per day. The central aim of the study was to investigate whether the effects of negative energy balance on protein metabolism would be modulated by varying the protein content of the energy-deficient diet. This study used a unique approach by examining the effect of a negative energy balance achieved by increased energy expenditure, as opposed to decreased energy intake, on protein metabolism in a tightly controlled environment where both energy intake and energy expenditure were monitored daily during the 11-d intervention.
The primary finding of this investigation was that feeding a high-protein diet prevented a decrease in nitrogen balance during a 7-d negative energy-balance period. This effect is likely reflective of postprandial responses to habitual feeding of higher dietary protein, as no differences were observed between or within groups for whole-body protein synthesis, whole-body breakdown, or net protein oxidation measured during the fasted state.
The link between energy intake and nitrogen balance is well established. Nitrogen balance becomes negative in the face of a negative energy balance caused by a decreased energy intake (3,8,25,27). This relationship is confirmed and extended by results of the present investigation in which an exercise-induced negative energy balance shifted nitrogen balance from positive to negative throughout the 7-d energy-deficit period in the DEF group. In contrast, nitrogen balance did not become more negative during the energy-deficit period in the DEF-HP group. Therefore, the higher-protein diet acted to preserve nitrogen balance in the face of a 1000-kcal·d−1 energy deficit produced by increased energy expenditure. This finding is consistent with previous observations that, for a given level of caloric restriction, nitrogen balance is less negative when the protein content of the diet is higher (3,8,28). Thus, in combination with those studies involving restricted energy intake, our investigation illustrates that the effect of negative energy balance on nitrogen balance can be mitigated by increased dietary protein, regardless of how the energy deficit is induced.
The benefit of the high-protein diet observed in nitrogen-balance data was not evident in the whole-body protein-turnover measurements made during fasting conditions. There were no differences between DEF and DEF-HP in protein synthesis, protein breakdown, or net protein oxidation during either the energy balance or energy-deficit period. Furthermore, energy deficit also had no impact on fasting whole-body protein turnover at rest, as there were no within-group differences noted over time. This lack of change in whole-body protein turnover was unexpected and could have resulted from one of, or a combination of, the following factors: the energy deficit experienced by the subjects in our investigation was not severe enough to impact fasting whole-body protein turnover at rest; the effects of dietary protein are manifested during postprandial but not fasting conditions; the effect of the exercise-induced energy deficit may only become evident during or following an exercise bout (not at rest); and whole-body protein turnover responds differently to an exercise-induced energy deficit compared with a diet-induced energy deficit.
Previous work examining the effect of a negative energy balance on whole-body protein turnover varies depending on the degree and length of the energy-deficit period. Short-term (≤ 4 d) fasting in nonobese volunteers has been shown to increase leucine flux, leucine oxidation, and proteolysis during both rest and exercise (11,12,19,26). However, as the length of the deficit increases, and the severity of the deficit decreases, there appears to be a reversal of this response. Yang et al. found no change in leucine flux at rest following 13 d of 25% (~700 kcal·d−1) caloric restriction (30). Similarly, Friedlander documented no change in leucine flux or oxidation at rest following 21 d of 40% (~1300 kcal·d−1) caloric restriction (6). Our whole-body protein-turnover data are in agreement with these investigations, in that resting whole-body protein turnover remained unchanged when a 1000-kcal·d−1) energy deficit was induced by exercise during 7 d. Interestingly, Friedlander did note a decrease in leucine flux and oxidation during exercise, suggesting that the adaptive response of protein metabolism to an energy deficit may only be evident under more stressful conditions (6). A limitation of our investigation was the lack of an assessment of whole-body protein metabolism during or immediately following an exercise bout.
In sum, the two methods used to assess changes in protein metabolism in this investigation seem to yield conflicting results. The nitrogen-balance data appear to indicate a benefit of higher-protein feeding during an exercise-induced energy deficit, whereas the whole-body protein-turnover data do not. Similar disagreement between these two methodologies has been shown previously (8). An interpretation of these apparently discrepant observations must consider when the measurements were taken, which highlights some of the advantages and disadvantages of these two methods for studying human protein metabolism. During the course of a day, there are acute variations in protein balance, which shifts from negative to positive with the normal cycle of feeding and fasting (20). Nitrogen-balance measurements reflect net effects of events occurring over a much longer period. Thus, nitrogen balance, although less sensitive compared with stable isotope methodology, provides an overall view of protein balance for a complete 24-h period, thereby accounting for the normal swings in protein balance following feeding/fasting. Although stable isotopes provide more sensitive and specific quantification of individual contributions of protein synthesis, breakdown, and oxidation to overall protein balance, this methodology only provides a relatively short "snapshot" period of time. In the present study, protein-turnover measurements reflect 4 h in the morning, following an overnight fast. Therefore, protein turnover was in a basal state during this fasting period, and any potential benefit of higher protein feedings was not detected.
We speculate that the benefit of these higher-protein meals actually occurred after each feeding, presumably resulting in a slightly higher positive protein balance in comparison with the adequate protein diet. Ultimately, these small increases in protein balance with each feeding may account for the more positive nitrogen balance we observed during a 24-h period.
This study is not without its limitations, which must be considered. The negative nitrogen balance observed during the baseline period for the BAL and DEF-HP groups was not expected. It is possible that for the BAL group, the 0.9 g of protein per kilogram per day was insufficient to maintain nitrogen balance, even at energy balance. Data from Tarnopolsky et al. (7,23) support this conclusion. However, the 1.8 g of protein per kilogram per day consumed by the HP group should have been sufficient enough protein to keep these subjects in nitrogen balance during the baseline period. The 3- to 5-d adaptation period used in this investigation may not have been long enough to achieve a true adaptation to the "in-study" protein level. A World Health Organization (1985) report concluded that the major initial changes in nitrogen excretion occur within approximately 5-7 d in adults (16). Additionally, the level of dietary control during the adaptation period could have impacted our baseline nitrogen-balance values. Volunteers were still free-living during this adaptation period, and food was not provided; therefore, it is possible that they may not have adhered to the run-in diet closely enough.
It is also important to acknowledge the choice of tracer when interpreting these results. Whereas phenylalanine is commonly used because it is not synthesized endogenously or oxidized by muscle, this model is not without its weaknesses, which have been pointed out previously (17,22). As previously discussed by Wolfe et al. (29), more than one tracer should be employed to most accurately assess whole-body protein turnover, because the metabolism of one particular amino acid may not be representative of all the amino acids in the body. Future studies should consider the use of multiple tracers in order to best validate the results observed.
Despite these potential limitations, our data show that a higher-protein diet was beneficial in attenuating the effects of negative energy balance, because of an increase in energy expenditure, on protein metabolism. Although we observed no differences in whole-body protein-turnover measures during the fasted state, the benefit of additional protein was demonstrated by a maintenance of nitrogen balance during the 7-d energy-deficit period, which is likely reflective of postprandial responses to habitual feeding of higher dietary protein. Future studies should be conducted that assess the impact of variations in habitual dietary protein intake in the fed state to better elucidate the acute variations in protein metabolism to nutrient provision.
The opinions or assertions contained herein are the private views of the author(s) and are not to be construed as official or reflecting the views of the Army or the Department of Defense or ACSM. Any citations of commercial organizations and trade names in this report do not constitute an official Department of the Army or ACSM endorsement of approval of the products or services of these organizations.
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