INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

THERE HAVE BEEN REPORTS over the past few decades of gender-related differences in energy metabolism during exercise (3, 9, 15, 16, 24, 25). In particular, whole body fat oxidation has been observed to be greater in the exercising woman than in the man (3, 9, 24). These studies report that female subjects oxidize proportionally more fat during submaximal exercise than their male counterparts, but the metabolic mechanisms responsible for this gender-based difference in fuel oxidation are largely unknown (9, 25). In contrast with these studies on fat metabolism, the literature on gender effects on amino acid metabolism is controversial. One study found no difference in leucine kinetics between men and women exercising for 2 h at a moderate intensity (4). This study (4) contradicts the findings of two other reports that showed a higher rate of leucine oxidation in men during rest and 90 min of moderate-intensity exercise than in women (15, 18).

The purpose of the present study was to examine the effects of gender on leucine kinetics during rest, exercise, and recovery in an attempt to reconcile the differences between these reports (4, 15, 18). Because there are no previous gender reports and to allow for a comparison between amino acid kinetics, we also studied lysine metabolism. Lysine has a metabolic fate different from that of leucine, and it cannot be degraded by skeletal muscle.

This experiment was designed to pay special attention to other experimental factors that are known to influence exercise metabolism and may have altered the gender-based findings of the previous studies (4, 15, 18). First, gender pairs were matched according to age as well exercise training habits. Second, menstrual cycle phase was directly determined with hormonal indicators. Finally, the preexperimental dietary intake of men and women was controlled and standardized. Thus both of our groups ate similar amounts of protein and were in a state of energy balance for 1 wk before the study.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects. We recruited 14 individuals for this experiment (7 women and 7 men). A health and physical examination was performed on all subjects to confirm that there was no medical reason for their exclusion from the study. All subjects had normal electrocardiograms. All participants were nonsmokers and did not have a family or personal history of diabetes mellitus. The menstrual cycle is known to alter protein catabolism and leucine kinetics in women (11, 13). Therefore, we determined the menstrual cycle phase of all our female subjects. 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). Six of the women were studied while in the follicular phase of their menstrual cycle. Our male subjects were chosen to match the seven female subjects on the basis of their exercise training habits and age. Our pool of subjects consisted of seven matched pairs. Five of these seven matched pairs exercised regularly, and the remaining two pairs were sedentary. Of the five regularly exercising matched pairs, two subjects in each group were moderately active recreational joggers and bikers who exercised for 1 h 3-5 days/wk. The remaining three pairs were very active, highly trained marathon runners or triathletes. The physical characteristics of both groups can be found in Table 1. The Investigational Review Board approved the study, and a written informed consent was obtained from each subject before participation.

Experimental protocol. Using a dietary exchange method (7), a registered dietitian designed a weekly meal plan for each subject. These meal plans were designed to be weight maintaining and employed the equation of Harris and Benedict (8) to determine daily caloric needs. Any increase in daily energy expenditure due to exercise training was computed for those subjects who regularly exercised. Exercise energy expenditures were determined with metabolic equations (2), and the subsequent value was added to the resting energy requirements to compute daily caloric needs. Thus the caloric intake averaged 1,893 ± 113.3 and 2,442 ± 85.7 kcal/day for the women and men, respectively (P < 0.003). The dietary composition for the men and women was controlled and consisted of 58-60% carbohydrate, 30% fat, and 10-12% protein. Therefore, the women and men received a similar amount of protein [1.0 ± 0.1 and 1.0 ± 0.3 g protein/kg body wt, respectively, P = not significant (NS)]. The subjects who regularly exercised were instructed to refrain from physical activity for 2 days before the tracer study to avoid an acute exercise recovery effect on leucine and lysine kinetics. Tracer infusions of the stable isotopes of leucine and lysine were performed after 7 days of equilibration to the controlled diet.

Tracer infusion studies. Subjects reported to the Clinical Research Center in a postabsorptive state (~15 h) on the morning of day 7. Two intravenous cannulas were placed into superficial veins, one in each hand. One cannula was used for the tracer infusions of L-[1-¹³C]leucine (99 atom %excess of ¹³C), L-[-¹⁵N]lysine (99 atom %excess of ¹⁵N), and NaH¹³CO₃ (99 atom %excess of ¹³C). All these isotopes were purchased from Merck (Dorval, PQ, Canada). We weighed and dissolved the tracers in normal saline and then sterilized the solution by micropore filtration (0.22 µm). The tracers were tested for sterility and pyrogenicity before their infusion. The second intravenous cannula was used for collecting blood samples and was kept patent with isotonic saline (10 ml/h). To reach an early isotopic steady state, priming doses were administered as follows: 1.2 µmol/kg of NaH¹³CO₃, 4.0 µmol/kg of L-[1-¹³C]leucine, and 6.8 µmol/kg of L-[-¹⁵N]lysine. The priming doses were followed by a 6-h constant-rate infusion of L-[1-¹³C]leucine at 5.0 µmol · kg¹ · h¹ and L-[-¹⁵N]lysine at 7.0 µmol · kg¹ · h¹. We obtained a background sample of expired air and venous blood from each subject before the tracer infusion. Next a weighed amount of labeled water (H₂¹⁸O, 99 atom %excess of ¹⁸O; MSD Isotopes) was given orally to determine total body water (17).

Tracer infusion during rest. The first 3 h of the experiment were used to obtain an isotopic plateau for the determination of leucine and lysine kinetics during supine rest. During these 3 h, venous blood samples were withdrawn every 30 min. The blood samples were centrifuged immediately, and the plasma was stored at 70°C for later analyses. Breath samples were collected every 30 min using a Hans Rudolph one-way nonrebreathing valve that was 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. CO₂ production and O₂ were determined continuously throughout the 3 h of rest. The average isotopic enrichment for the 3rd h of the infusion was used to calculate leucine and lysine kinetics during rest.

Tracer infusion during exercise. After 3 h of supine rest, the subjects began to exercise at 50% of their predetermined O_2 max using a constant-load pan weight cycle ergometer (Monark, Varberg, Sweden). Blood samples were withdrawn at 0, 15, 30, 45, 50, 55, and 60 min of exercise. We continuously measured O₂ and CO₂ production using a Hans Rudolph adult facemask interfaced with the metabolic cart. In addition, aliquots of the breath samples were trapped in evacuated glass tubes at 0, 5, 13, 27, 43, 50, 55, and 57 min of exercise for the subsequent determination of ¹³CO₂ enrichment. The average isotopic enrichment value for the last 20 min of exercise was used to calculate leucine and lysine kinetics during exercise.

Tracer infusion during recovery from exercise. After 1 h of exercise, the subjects again rested in the supine position for 2 h. This time period was used to determine leucine and lysine kinetics during the initial recovery phase from exercise. Expired air was collected every 30 min to determine CO₂ enrichment. Respiratory gas and blood samples were also gathered every 30 min. Leucine and lysine kinetics were calculated with the average isotopic enrichment during the last 30 min of recovery.

Analytic methods. We used the urease reaction to determine plasma urea nitrogen concentration with a urea nitrogen analyzer (model 2, Beckman Instruments, Fullerton, CA). Plasma free fatty acid (FFA) levels were determined according to Laurell and Tebbling (14). Plasma glucose was determined using the glucose oxidase method on a glucose analyzer (Beckman Instruments). Total plasma protein concentration was measured with refractometry (model SPR-T2, Atago). The percent increase in plasma protein concentration above rest was used to correct the glucose, FFA, and urea nitrogen concentrations for fluid volume shifts that occur with exercise (20). Total urine volumes were collected on days 6 and 7. Total 24-h urinary urea nitrogen excretion was determined with a colorimetric assay (model 640A, Sigma Chemical).

Body composition. Total body water and fat-free mass (FFM) were calculated using labeled water. An H₂¹⁸O tracer dilution method was employed for body composition analyses (21). An isotopic plateau for expired C[¹⁸O₂] was achieved within 3 h (12, 17). Body composition was calculated using the assumption that water constitutes a fixed fraction (73.25%) of the FFM (21, 22).

Data analyses. Steady-state tracer kinetic equations were used to calculate leucine and lysine kinetics. The reciprocal pool model was used in the calculation of leucine kinetics. Repeated-measures ANOVA with Newman-Keuls post hoc tests were employed for these data analyses. The statistical power for the ANOVA of leucine oxidation at an -level of 0.05 was found to be 1.00. An analysis of covariance was performed on the RER data, with the resting RER being used as the covariate. Where appropriate, a nonparametric statistical test, the Wilcoxon-Mann-Whitney test for two independent groups, was also used for statistical analyses. Values are means ± SE. P < 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Descriptive data. Our male and female subjects were matched according to their exercise habits and O_2 max. There was no difference between groups in O_2 max whether expressed per kilogram of body weight or per kilogram of FFM (P = NS). However, the male subjects were heavier and leaner than their female counterparts. Not surprisingly, the women had a greater percentage of body fat than the men (Table 1; P < 0.001).

Isotopic steady state. Labeled CO₂, -KIC, and lysine exhibited an isotopic plateau between 2.5 and 3 h of supine rest. We observed isotopic plateaus for labeled CO₂, -KIC, and lysine between 40 and 60 min of exercise (Table 2).

Indirect calorimetry. The average exercise O₂ for both groups was 1.43 ± 1.1 l/min. Figure 1 demonstrates that there were gender differences in the nonprotein RER during rest and exercise (P < 0.001 by ANOVA). The women had a consistently lower RER during rest and at all exercise time points than did the men. When the resting RER was used as a covariate, there was no significant interaction effect between gender and exercise (P = NS). Table 3 indicates that there were gender differences in the percentage of fat, carbohydrate, and protein used for exercise energy needs when calculated from this indirect calorimetry data.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Nonprotein respiratory exchange ratios for male and female subjects during rest, exercise, and recovery. Values are means ± SE. *Significant difference between genders (P < 0.001 by ANOVA).

Plasma and urinary substrate concentrations. Table 4 indicates that there was no difference between genders in plasma urea nitrogen or FFA concentrations at any time point (interassay coefficient of variation = 7%). However, the plasma glucose did differ between genders (15 min, P < 0.05). During day 7, there was a greater urinary urea nitrogen excretion in the men than in the women (5.6 ± 0.48 and 12.5 ± 1.99 g/day for women and men, respectively, P < 0.01). When nitrogen balance was estimated from these data (28), there was a difference between genders (0.22 and 3.95 g/day for women and men, respectively, P < 0.05).

Exercise and recovery effects on leucine and lysine kinetics. Within-groups statistical analyses indicated that the leucine rate of appearance was not altered by exercise in the men or the women (Table 5). However, leucine rate of appearance was significantly decreased in both groups during recovery (Table 5; P < 0.05). Leucine oxidation was significantly greater in both groups when exercise was compared with rest (Fig. 2; P < 0.05). Also, in both men and women, there was a significant attenuation in the nonoxidative leucine disposal during exercise compared with rest (Fig. 2; P < 0.05). The lysine rate of appearance remained unchanged from rest to exercise and from rest to recovery in both groups (Table 5).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Leucine oxidation and nonoxidative leucine disposal (NOLD) during rest, exercise, and recovery in male and female subjects. FFM, fat-free mass. Values are means ± SE. *Significantly different from rest within each group (P < 0.05); **significant difference between genders (P < 0.05).

Gender effects on leucine and lysine kinetics. Between-groups statistical analysis indicated that there were no differences between genders in leucine rate of appearance or lysine rate of appearance during rest, exercise, or recovery (Table 5). However, leucine oxidation during exercise was greater in the men than in the women (Fig. 2; P < 0.05), but there was no gender difference in leucine oxidation during rest or recovery (P = NS). Furthermore, there was a gender effect on nonoxidative leucine disposal during exercise, with the female subjects having a larger nonoxidative leucine disposal than their male counterparts (Fig. 2; P < 0.05). There was no difference between genders in nonoxidative leucine disposal during rest or recovery (Fig. 2).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The purpose of this investigation was to examine the effects of gender on leucine and, for comparison purposes, lysine kinetics. We controlled for extraneous variables that have previously been shown to alter gender-related metabolism by 1) hormonally determining menstrual cycle phase (11, 13), 2) standardizing the preexperimental diet (4), and 3) matching gender pairs by age and exercise training habits (9, 24). The experimental limitations of this study are the use of an assumed bicarbonate retention factor and the assumption of no ¹³CO₂ drift during the exercise bout.

Within these limitations, the results of our experiment indicated a greater oxidation of leucine during exercise in men than in women. Also, men had a lower rate of nonoxidative leucine disposal during exercise. In contrast, in women, a greater proportion of whole body energy sources during moderate-intensity exercise came from fat, whereas in men, a greater proportion of energy needs came from carbohydrate. However, there were gender differences in the indirect calorimetry data before initiation of exercise. If the resting RER was used as a covariate, the resulting interaction effect between gender and exercise was not significant.

Similar to other researchers, we found that leucine kinetics changed during exercise regardless of the gender of our subjects (12, 19, 26, 27). Although the rate of appearance of leucine was not altered by exercise, leucine oxidation was increased, and the nonoxidative leucine disposal was decreased during exercise compared with rest. Others have reported no change in leucine rate of appearance during exercise compared with rest (26). The magnitude of change in oxidative and nonoxidative disposal of leucine during this exercise was similar to that of previous studies (4, 12). Another research group has reported a lack of change in lysine rate of appearance during exercise regardless of whether [1-¹³C]lysine or [-¹⁵N]lysine was infused as the tracer (27).

Our data confirm gender effects on leucine kinetics during moderate-intensity exercise. To date, this and two other studies (15, 18) report that male subjects oxidize more leucine during exercise than their female counterparts. The mechanism for this gender difference remains unknown but has not been attributed to differences in the percent activation of skeletal muscle branched-chain 2-oxoacid dehydrogenase (15). However, it has been speculated that there are gender differences in hepatic branched-chain 2-oxoacid dehydrogenase activation (15). Not only did our male subjects oxidize more leucine during exercise but they also catabolized more protein than their female counterparts. Our leucine kinetic data are contrary, however, to those of Bowtell et al. (4), who reported no difference between genders during exercise. The lack of a gender-based difference in leucine oxidation in this previous study (4) may be due to the subject selection procedure. For instance, there was no mention of the training habits of their female subjects, nor was their menstrual cycle status noted. Even more importantly, this previous study (4) had a small female sample size (n = 2), increasing the chances of making a type II error of statistical inference.

In contrast to previous research, we found differences between genders in nonoxidative leucine disposal during exercise. Our data indicate that men had a diminished nonoxidative leucine disposal and, hence, reduced protein synthesis rate during exercise compared with women. Again, the only reasonable explanation for the differences between nonoxidative leucine disposal in our study and the previous gender studies (4, 15, 18) is the experimental method. During our experiment and the study of Bowtell et al. (4), the isotopic infusions were performed while subjects were in the fasted state, whereas in two other studies (15, 18) the isotopic infusion procedures were performed in subjects after they were fed. Previous research indicates that the state of feeding or fasting impacts leucine metabolism (16).

There were no gender effects on leucine or lysine kinetics in these postabsorptive subjects when they were studied during the resting state. Thus our data indicate that amino acid kinetics were similar between genders when studied during resting conditions. A lack of difference between genders in leucine oxidation and nonoxidative disposal was also found during the initial few hours of exercise recovery. However, leucine rate of appearance decreased during exercise recovery in both genders. Another research group reported similar reductions in leucine rate of appearance (and oxidation) during exercise recovery (6).

When women and men exercise at the same relative intensity, there appears to be a distinct difference in the contribution of fat and carbohydrate to total energy needs. Our data and those of others (3, 9, 24) would indicate that fats contribute more to energy needs during moderate-intensity prolonged exercise in the women, whereas carbohydrates contribute more in the men. The metabolic mechanism(s) responsible for these gender differences in fat and carbohydrate preference is unknown (9, 25). Some have speculated that there may be a gender difference in substrate delivery, hormonal action, or the capacity for substrate use in exercising men and women (9). It should be underscored that the metabolic mechanism(s) responsible for this reduction in leucine oxidation and protein catabolism in the exercising woman also remains unknown. It could be speculated that women derive more of their exercise energy needs from fat, thereby using less carbohydrate, amino acid, and protein as fuel. These metabolic differences between men and women indicate that nutritional needs and optimal training methods may not be generalized from one gender to the other. Gender-specific guidelines for nutrition and training may be needed to allow the female athlete to maximize her endurance performance. Certainly these data indicate the need for more gender-specific research in exercise biochemistry.

A gender comparison of lysine kinetics has not previously been performed. Therefore, these lysine data extend previous observations on the effects of gender on leucine kinetics. Our lysine kinetic data indicate that there are no gender differences in lysine rate of appearance during rest, exercise, or recovery. Therefore, gender-related effects in one amino acid might not be generalized to occur for all amino acids.

In conclusion, the results of this study confirm that there are gender-related differences in leucine oxidation, nonoxidative leucine disposal, and protein catabolism during moderate-intensity exercise. However, these gender-related differences in leucine kinetics may not be generalized to occur for all amino acids. These data also indicate that women rely to a greater extent on fat and less on carbohydrate, amino acid, and protein as a fuel source for exercise. This study confirms that there is a need for more gender-specific research in exercise biochemistry and that this type of research must pay careful attention to subject selection, study design, and experimental methodologies.