INTRODUCTION

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ALTHOUGH STUDIES have been performed on laboratory animals (7, 13, 15); experiments on the effects of endurance training on amino acid kinetics in humans are limited (11). The need for research on humans is supported by the fact that there are large interspecies differences in the turnover rate of the free amino acid pool (11). There is one previous study that reported that differences in endurance-trained and sedentary humans in whole body leucine kinetics during rest are related to the size of the anatomic muscle mass (20). There are no comparisons of whole body amino acid kinetics during exercise or recovery in trained and sedentary individuals. Therefore, we studied whole body leucine and lysine kinetics in endurance-trained athletes and sedentary controls that were pair matched for body weight. Leucine was studied because it is an essential ketogenic, branched-chain amino acid that can be oxidized by skeletal muscle (10, 30). Lysine kinetics were simultaneously studied because, in contrast to leucine, lysine is an essential amino acid that cannot be degraded or transaminated by skeletal muscle (32). Although leucine turnover appears to be increased in the resting endurance-trained athlete, we postulated that this increase may be due to a difference in the size and not the function of the fat-free tissue mass (20). Therefore, the hypothesis tested in this experiment was that there will be no difference in whole body leucine kinetics during exercise or recovery in the endurance-trained athlete and pair-matched sedentary control.

	METHODS

Subjects. Fourteen healthy men (n = 8) and women (n = 6) were recruited for this experiment. Seven of these subjects (3 women and 4 men) were endurance-trained (Trn), and seven (3 women and 4 men) were nontrained, sedentary adults (Con). Five of the women were tested in the follicular phase of their menstrual cycle. Menstrual cycle phase was determined by counting days from the onset of menses and by using a monoclonal antibody self-test kit (Ovukit, Quidel San Diego, CA). The subjects were considered endurance trained if their cycle ergometry-obtained maximal oxygen consumption (O_{2 max}) was >50 ml · kg¹ · min¹. All athletes reported that they had a training frequency of five times a week or more and that their training sessions were at least 1 h/day. In addition, the trained subjects had been in training no less than 1 yr. These endurance-trained athletes were marathon runners, triathletes, or long-distance cyclists. All 14 participants had normal 12-lead electrocardiograms and were without a family or personal history of diabetes mellitus. This project was approved by the Investigational Review Board of the University Hospitals of Cleveland. A written informed consent was obtained from each subject before his or her participation in this experiment.

Experimental protocol. A 7-day weight-maintaining dietary-control period preceded the stable-isotope infusion experiment. The diets were formulated to meet daily caloric needs and consisted of ~58-60% carbohydrate, 30% fat, and 10-12% protein. Both groups received equal daily amounts of nitrogenous protein (Trn = 70.86 ± 5.30 g protein/day vs. Con = 70.50 ± 4.85 g protein/day). The equations of Harris and Benedict (12) were used to estimate each subject's resting energy needs. Increases in energy expenditure due to daily exercise training sessions were determined for each athlete by using standard metabolic equations (2). The energy expenditures for exercise training were added to the resting energy requirements to compute daily caloric needs for each athlete. A registered dietitian designed the individual meal plans for all subjects and used a dietary-exchange procedure to individually counsel each subject on his or her nutritional plans (9). The protein sources allowed on days 1-4 included meat proteins, and on days 5-7 they were switched to meat-free proteins. This dietary substitution was made to eliminate exogenous nutritional creatinine so that an accurate measure of anatomic muscle mass could be obtained. Total 24-h urine volumes (days 6 and 7) were collected for the determination of creatinine excretion. The subjects were instructed to refrain from physical exercise on days 5 and 6 to avoid an acute exercise recovery effect on amino acid kinetics during the experimental tracer infusion (day 7). Compliance with these dietary and experimental procedures were evaluated by reviewing the written dietary records and by interviewing each subject before the infusion experiment.

Tracer-infusion studies. All subjects reported to the Clinical Research Center after an overnight fast on the morning of day 7. Intravenous cannulas were placed into superficial hand veins of each arm. One cannula was used for the tracer infusions of L-[1-¹³C]leucine (99 atom %excess ¹³C), L-[-¹⁵N]lysine (99 atom %excess ¹⁵N), and sodium bicarbonate NaH[¹³C] O₃ (99 atom %excess ¹³C) (purchased from Merck, Dorval, Canada). The labeled tracers were weighed and dissolved in normal saline and sterilized by microfiltration (0.22-µm Millipore filter). Each labeled tracer was tested for sterility and pyrogenicity before the tracer infusions (16). The second intravenous cannula was used for blood sampling and was kept patent with an isotopic saline infusion (10 ml/h). A background sample of expired air and venous blood was obtained before the infusions were begun. Priming doses were administered to reach an early steady-state and were 1.2 µmol/kg of NaH¹³CO₃, 4 µmol/kg of L-[1-¹³C]leucine, and 6.8 µmol/kg of L-[-¹⁵N]lysine. This prime was followed by a 6-h constant-rate L-[1-¹³C]leucine (5 µmol · kg¹ · h¹) and L-[-¹⁵N]lysine (7 µmol · kg¹ · h¹) infusion. An accurately weighed amount of labeled water (H₂¹⁸O, 99 atom % excess ¹⁸O; MSD Isotopes) was given orally to determine total body water (22, 26, 27). The first 3 h of the primed infusion resulted in the acquisition of an isotopic plateau (18). During this 3-h period, venous blood samples were withdrawn every 30 min. These blood samples were immediately centrifuged, and the plasma was stored at 70°C for later analyses. Breath samples were collected every 30 min by using a Hans-Rudolph, one-way nonrebreathing valve connected to a 5-liter anesthesia bag. An aliquot of each breath sample was trapped in an evacuated glass tube for the subsequent analysis of ¹³CO₂ (16). Carbon dioxide production (CO₂) and O₂ were determined throughout the 3 h of rest. The average isotopic enrichment for the last hour of the infusion during resting conditions was used to calculate leucine and lysine kinetics.

Analytic methods. Plasma glucose was determined on a glucose analyzer (model 2, Beckman Instruments, Fullerton, CA) by using the glucose oxidase method. Plasma urea nitrogen was measured with a urea nitrogen analyzer by using the urease reaction (model 2, Beckman Instruments). Plasma free fatty acid (FFA) levels were determined according to Laurell and Tebbling (21). Total plasma protein concentration was measured by using refractometry (model SPR-T2, Atago). The percent increases in plasma protein concentration above resting levels were used to correct the FFA, glucose, and urea nitrogen concentrations for fluid-volume shifts that occurred with exercise (25). Twenty-fourhour urinary creatinine excretions were determined with a colorimetric assay (kit no. 555A, Sigma Diagnostics, St. Louis, MO).

Calculations and statistics. In a separate 7-h control experiment, we determined that our stable-isotope infusion procedures resulted in steady-state enrichments (data not shown). Enrichments for ¹³CO_2, [¹³C]KIC, and [¹⁵N]lysine are shown in Figs. 1, 2, and 3, respectively. Isotopic plateaus were observed for breath ¹³CO_2, plasma [¹³C]leucine, [¹³C]KIC, and plasma [¹⁵N]lysine after 2.5 and 3 h of rest. In addition, isotopic plateaus were observed for ¹³CO_2, plasma [¹³C]leucine, [¹³C]KIC, and plasma [¹⁵N]lysine in both the trained and control groups between minutes 40 and 60 of exercise. Therefore, steady-state tracer kinetic equations were used (20). Plasma KIC enrichments and the reciprocal pool model was used for the calculations of leucine kinetics. Leucine kinetics were corrected for bicarbonate retention. The bicarbonate retention factors employed were 83.1% for rest and 98.9% for exercise in the trained athletes and 83.1% for rest and 96.6% for exercise in the control subjects (4).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Breath 13CO2 enrichments during rest, exercise, and recovery in endurance-trained athletes and sedentary, nontrained controls. Values are means ± SE for rest and recovery. Time is in min.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Plasma [13C]-ketoisocaproate (KIC) enrichments during rest, exercise, and recovery in endurance-trained athletes and sedentary, nontrained controls. Values are means ± SE for rest and recovery. Time is in min.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Plasma [15N]lysine enrichments during rest, exercise, and recovery in endurance-trained athletes and sedentary, nontrained controls. Values are means ± SE for rest and recovery. Time is in min.

	RESULTS

Each endurance-trained subject was matched by age, gender, and body weight to a nontrained control subject (Table 1). As expected, the trained athletes had lower resting heart rates (Trn = 55.14 ± 6.2 vs. Con = 69.43 ± 6.2 beats/min; P < 0.05) and exercise heart rates (P < 0.05). Ratings of perceived exertion (Borg scale) were also lower throughout exercise in the trained compared with the nontrained subjects (P < 0.05). Although both groups exercised at the same percentage of O_{2 max}, the endurance-trained athletes had a higher mean O₂ during exercise (Trn = 1.63 ± 0.18 l/min vs. Con = 1.30 ± 1.4 l/min; P < 0.05). Plasma substrate and RER data are displayed in Table 2. The RER was significantly lower during rest and exercise in the trained group. Plasma FFA concentrations were lower and plasma urea nitrogen concentrations were greater during rest, exercise, and recovery in the trained group. Plasma glucose decreased with exercise, but the decrement was not significant and there were no differences between groups at any time points.

Leucine kinetics expressed relative to body weight. Leucine kinetics were calculated by using the reciprocal pool model (plasma KIC enrichments) and are shown in Table 3. The nontrained control subjects showed a significant decrease in leucine R_a during exercise and recovery in contrast with their resting R_a (P < 0.01). However, in the athletes there was no change in leucine R_a due to the stimulus of exercise (exercise vs. rest), but leucine R_a did decrease during recovery compared with rest (P < 0.01). A between-group comparison indicated that leucine R_a was greater during both exercise and recovery in the trained compared with the nontrained group (P < 0.05).

Leucine kinetics corrected for fat-free body mass and O₂. Leucine kinetics corrected for fat-free body mass are shown in Table 4. When leucine R_a, oxidation, and nonoxidative leucine disposal rates were corrected for fat-free tissue mass, the exercise and recovery effects were similar to those found when leucine kinetics rates were expressed relative to body weight. However, when leucine R_a and oxidation, and nonoxidative leucine disposal were corrected for fat-free mass, there were no differences between groups (P = NS; Table 4). Also, the correlation between exercise leucine oxidation and whole body fat-free mass was significant (R = 0.80; P < 0.001; n = 14). Therefore, there was a physiological relationship between whole body leucine kinetics and the size of the fat-free tissue mass.

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Lysine kinetics. There was no difference between groups in lysine R_a at any experimental time point. Similarly, there was no change in lysine R_a with exercise regardless of aerobic training. However, both groups showed a decrease in lysine R_a during the initial few hours of recovery from exercise (Trn rest = 104.92 ± 5.11 and Trn recovery = 88.36 ± 5.30 µmol · kg¹ · h¹; P < 0.01 vs. Con rest = 111.16 ± 7.47 and Con recovery = 94.49 ± 3.45 µmol · kg¹ · h¹; P < 0.007).

	DISCUSSION

The purpose of this investigation was to compare whole body leucine and lysine kinetics in endurance-trained and sedentary adults. Both groups were matched by gender, age, and body weight. Both groups also had similar body compositions, reflecting an equality in whole body protein mass. The similarity between groups in anatomic muscle and fat-free body mass is underscored because of a previous report that the protein pool size may alter the expression of whole body leucine turnover and oxidation in the resting human (20). The present data indicate that making comparisons between whole body amino acid kinetics in groups who differ in fat-free body mass may be problematic. With a few exceptions, most investigators express leucine kinetics relative to body weight and do not consider specific body compartments such as the skeletal muscle or fat-free tissue compartment. The present investigation supports the concept that correcting whole body leucine kinetics for fat-free tissue mass may be important when cross-sectional studies on humans are performed (20). It should be underscored that correcting leucine oxidation for fat-free mass equalized any between-group difference due to state of training. The elimination of an experimental effect when leucine oxidation was corrected for lean tissue mass (protein pool) may indicate that training effects on leucine metabolism are localized within skeletal muscle and are minimal when viewed at the whole body level.

An even more important finding may be that the rate of whole body leucine oxidation, when corrected for mean O₂ during exercise, was not different between these two groups. Isolated muscle studies of single hindlimbs of rats have demonstrated small reductions in leucine oxidation relative to O₂ in trained compared with nontrained animals (15). This animal study, however, employed a prospective experimental design and a 9-wk exercise training protocol. The present data are the first to indicate that there was no difference between whole body leucine oxidation when endurance-trained and sedentary humans are exercising at the same relative intensity and are studied in a cross-sectional manner.

In the present study, the endurance-trained athletes had an accelerated leucine R_a (whole body proteolysis) compared with their nontrained counterparts. Submaximal exercise in these athletes provided less perturbation of whole body proteolysis (expressed relative to body weight) when leucine was the amino acid marker used and must be reconciled with the fact that there were no between-group differences in lysine R_a. As others have indicated, the response of leucine and lysine kinetics during exercise may be disparate (18, 32). However, it seems unlikely that leucine was selectively hydrolyzed from protein pools due to the stimulus of exercise. The discrepant R_a for these two essential amino acids may be due to a greater proteolytic rate occurring in those proteins that contain relatively more leucine than lysine. Many different protein pools contribute to the increased proteolysis with exercise (3, 6, 17), and the proteolytic mechanisms involved are still unknown (17). One recent study indicates that nonmyofibrillar protein proteolysis may be caused by an enhanced flux rate through lysosomal pathways (17). However, studies of endurance-training effects on proteolytic enzyme activity are conflicting (23, 28).

Others have reported that the leucine R_a decreases or remains unchanged and that leucine oxidation increases with prolonged exercise (20, 24, 29, 30, 32). Previous studies also indicate that lysine R_a remains unchanged or decreases with submaximal exercise (20, 32). The present study extends those findings and suggests that the underlying state of aerobic conditioning may influence the response of leucine kinetics expressed relative to body weight during aerobic exercise. In fact, the disparity between experimental outcomes in past research may actually reflect the variability in aerobic capacity of the study participants.

The present data collected during exercise recovery are consistent with past research in this area. Whole body leucine oxidation, leucine R_a, and lysine R_a expressed relative to body weight have been reported to decrease during the initial few hours of exercise recovery (5, 18). However, these previous studies did not examine the effects of endurance-training on leucine kinetics during recovery. The present study indicates that the state of aerobic conditioning can effect the fate of leucine during the immediate hours after endurance exercise. Specifically, leucine R_a and oxidation (relative to body weight) are greater in the recovering endurance-trained athlete.

In conclusion, when leucine kinetics were expressed relative to body weight, prolonged submaximal exercise increased the rate of leucine oxidation while simultaneously decreasing nonoxidative leucine disposal in both endurance-trained athletes and sedentary humans. Also, when the data were expressed relative to total body weight, endurance training was associated with an increased leucine oxidation at all times and a heightened leucine R_a and hence proteolysis during exercise and recovery. However, when leucine kinetics were expressed relative to fat-free mass, these intergroup differences were eliminated. Therefore, whole body leucine kinetics are similar in trained and sedentary individuals when expressed relative to fat-free tissue mass. These data indicate that correcting whole body leucine kinetics for fat-free mass may be important when cross-sectional studies on humans are performed. Finally, whole body leucine oxidation relative to metabolic rate was found to be the same in exercising, endurance-trained athletes and sedentary controls. Therefore, it is concluded that there was no difference in whole body leucine kinetics between endurance-trained and sedentary humans when exercising at the same relative intensity.