FIGURE 1--Study design. Each subject was studied on one occasion after an overnight fast. The subjects were divided either in a resistance exercise group (exercise group, EG) or a rest group (control group, CG). Familiarization period was 14 d, and the fasting period was started 10 h before experiment. Infusion of ring-2H5-phenylalanine was started at 0 min. Blood samples (B) were taken at 55 min from an antecubital vein (AV), at 135,145,155,165 min (period 1) and at 270, 280, 290, 300 min (period 2) from femoral artery (FA) and femoral vein (FV). The muscle biopsies were taken at 165 and 300 min from vastus lateralis muscle. The leg blood flow was measured at 125-165 min (period 1) and at 260-300 min (period 2) during a continuous infusion of indocyanine green (ICG).
Data collection protocol.
The protocol was planned to study only one subject (either EG or CG) per day. The subjects fasted overnight and reported to the laboratory in the morning, where their body mass and height were measured. An 18-gauge polyethylene catheter was inserted into a left antecubital vein (AV) for basal blood samples. Thereafter, a primed, continuous infusion of L-[ring-2H5] phenylalanine was started and maintained throughout the measurements. The prime was 2 μmol·kg-1 and the infusion rate was 0.05 μmol·kg-1·min-1. At 125 min, polyethylene catheters (20-gauge) were inserted into the right femoral artery (FA) and femoral vein (FV), as well as an 18-gauge catheter was inserted into a right AV for drawing blood samples. The arterial catheter was used for the infusion of indocyanine green (ICG) for measuring blood flow (Fig. 1).
Collection of blood and muscle samples and blood flow.
The ICG infusion (0.5 mg·mL-1; 60 mL·h-1) was started 10 min before the first blood sample during periods 1 and 2. In period 1, the blood samples for isotopic measurements and blood flow were drawn at 135, 145, 155, and 165 min (30, 40, 50, and 60 min after RES) after the initiation of L-[ring-2H5] phenylalanine infusion. In period 2, the blood samples for isotopic measurements were taken 270, 280, 290, and 300 min (165, 175, 185, and 195 min after RES) after the initiation of infusion. For serum free amino acid concentration analysis blood samples were drawn 135, 165, 270, and 300 min after the initiation of infusion and analyzed by high performance liquid chromatography (HPLC). Blood flow samples were simultaneously drawn from an FV and a right AV. The ICG infusion was briefly halted to allow sampling from the FA for isotopic measurements. Blood for amino acid concentrations and enrichments was placed into preweighed tubes containing 2-mL sulfosalicylic acid and known amount of internal standard (13C6 phenylalanine; 50 μmol·L-1). Samples were mixed carefully and stored in ice. Blood flow samples were stored at room temperature until analysis. After the last blood sample (165 min), a muscle biopsy was taken for isotopic measurements and for analysis of free amino acid concentration from the VL muscle under local anesthesia. With the use of sterile technique, the skin and subcutaneous tissue were anesthetized, and a 6- to 7-mm incision was made. A 4-mm biopsy needle was advanced 3–5 cm into the muscle with the closed cutting window. The cutting cylinder was opened and closed two to four times and a sample of 30–50 mg was obtained. Visible fat and connective tissue were removed and the samples were rinsed with ice-cold saline before storing into tubes in liquid nitrogen. The second muscle biopsy was taken after the last blood sample (˜300 min after the initiation of L-[ring-2H5] phenylalanine infusion).
Analysis of enrichment and concentration of phenylalanine.
Enrichment and concentration of phenylalanine in whole blood were measured by gas chromatography-mass spectrometry (GC/MS; Hewlett Packard Agilent 5973N, GC 6890 Plus+, Palo Alto, CA). To determine the enrichment of infused amino acid in whole blood, the tertiary-butyl dimethylsilyl derivative was made. Isotopic enrichments were expressed as a tracer-to-tracee ratio. Concentration of phenylalanine was determined with an internal standard (13C6 phenylalanine; Cambridge Isotope Laboratories, Andover, MA) solution as previously described (1–3). In addition, detected masses were m/z 336 (natural phenylalanine), m/z 341 (infused 2H5-phenylalanine), and m/z 342 (13C6 phenylalanine in internal standard). Because the tube weight and the amount of blood were known, the blood amino acid concentration was determined from the internal standard enrichment on the basis of the amount of blood and internal standard (200 μL of 50 μmol·L-113C6 phenylalanine) added. Appropriate corrections were made for any spectra that overlapped, contributing to the tracer-to-tracee ratio (30).
Analysis of muscle samples.
Muscle tissue samples were analyzed for intracellular amino acid enrichments. On thawing, the tissue was weighed and the protein was precipitated with 0.8 mL of 14% perchloroacetic acid. The tissue was then homogenized and centrifuged, and the supernatant was collected. This procedure was repeated once, and the collected supernatant was processed like blood samples.
Analysis of leg blood flow.
Leg blood flow was determined from blood samples collected during a continuous infusion of ICG (12). The blood flow was determined by spectrophotometrically measuring the ICG concentration in serum from the FV and the peripheral vein as described previously (1,12). Leg plasma flow was calculated from steady-state values of dye concentration and converted to blood flow with the hematocrit (1). Serum from the blood samples was analyzed in a spectrophotometer with absorbance set at 805 nm.
The three-compartment model of leg muscle amino acid kinetics has been described previously by Biolo et al. (1,2). The use of this model allows determining the rate of utilization of phenylalanine for muscle protein synthesis and appearance from breakdown because phenylalanine is neither oxidized nor synthesized in muscle. The blood values for blood flow, blood and muscle phenylalanine concentrations, and enrichments are the averages of the sampling periods (period 1: 135, 145, 155, 165 and period 2: 270, 280, 290, 300 min). Muscle net balance was determined by calculating the difference between muscle protein synthesis and muscle protein breakdown.
The calculation of intracellular phenylalanine utilization (protein synthesis) and appearance (protein breakdown) assumes that there is no de novo trace production or oxidation in the leg. Net balance and the muscle biopsy data assume that the muscle accounts for the leg metabolism of amino acids. It is also assumed that the tissue enrichment and amino acid concentrations are representative of the intracellular space and that the intracellular free amino acid pool is homogenous. It is also assumed that the free amino acid pool is the precursor for protein synthesis. The detailed model assumption has been described earlier (e.g., 1).
Analysis of serum free amino acid concentrations in blood.
Concentrations of free amino acids in serum were determined applying the procedure of Pfeifer et al. (16) by reversed phase high performance liquid chromatography (RPHPLC) (Waters 501 pumps, Waters 717 autosampler and Zorbax C18 column). The 18 amino acids and two internal standards ([beta]-Abc and Nor-Valine) were detected by Perkin Elmer LS-4 fluorescent detector using wavelengths 338 nm (exitation) and 455 nm (emission); 100 μL of internal standard solution was added to the serum sample (50 μL) and acetonitrile (100 μL) was used to precipitate the proteins; 750 μL of distilled deionized water was added, and the resulted sample was vortexed and allowed to stand on ice bath for 1 h. The 200 μL of the sample was transferred to the ultraspin centrifuge filter and centrifuged. The clear mixture was transferred to the HPLC vial, derivatized with orto-pthaldehyde derivatizing solution and analyzed by Waters HPLC system using gradient two-buffer elution.
Analysis of free amino acid concentrations in muscle.
Concentrations of free amino acids in muscle were analyzed using HPLC equipped with fluorescent detector similarly to blood samples (see above). Samples were prepared by taking at least 10 mg of muscle and adding 400 μL of 5% perchloric acid and 100 μL of internal standard solution. After standing on an ice bath for 1 h, the samples were ground with pestles and centrifuged. Supernatants were transferred to clean tubes, and pH of the supernatants were adjusted to 5–7 by using potassium hydroxide. After spinning, 200 μL of the mixtures was transferred to the ultraspin centrifuge filter and centrifuged. The residual matters were removed and the solutions were ready for the HPLC analysis.
Data are expressed as means ± SEM or SD. The main effect of group and the interactions were assessed by MANOVA. With MANOVA, it can be constructed a statistical model corresponding to arrangements of the experiment. The dependent variables are TAA, EAA, NEAA, BCAA, and single amino acids, whereas the independent variables are vessels (FA, FV), group, and sample (time period 1: 135–165 min and time period 2: 270–300 min). If differences were detected, then a Bonferroni post hoc test was used to determine pair-wise differences. Also, residual examinations were made by diagnostic methods to make sure that the assumptions of statistical analysis were valid. Relationships between blood and muscle amino acids were determined using Pearson correlation coefficients. In addition, an unpaired Student’s t-test was used to determine differences between groups for physical activity, nutrition, free amino acids, leg blood flow, protein synthesis, protein breakdown, and protein net balance between groups. Statistical significance was set at P < 0.05. All the analyses were done using the SPSS 10.1. for Windows software package.
Free time physical activity and nutrition.
There were no statistical differences between the EG and the CG during 2 wk before the study day either in number of free time physical activity (7.8 ± 5.6 and 8.7 ± 2.7 times, respectively) or in total hours of physical activity (9.5 ± 5.7 and 12.3 ± 5.8 h, respectively). The nutrient intake was similar in the groups during the 5-d period (Table 2).
Main effect of group and interactions in serum free amino acids.
The results revealed that compared with CG, EG had significantly (P < 0.05) higher concentration in aspartate in both vessels. On the other hand, the concentration in leucine was lower (P < 0.05) in EG than in CG, respectively (Table 3). Furthermore, the concentration of alanine was significantly higher (P < 0.01) in EG than in CG during period 1 in both vessels. In addition, the concentrations of asparagine (P < 0.001) and threonine (P < 0.05) were higher in FV than in FA in CG. However, methionine (P < 0.05) concentration was higher in FV than in FA in both groups.
Serum free amino acid concentrations in AV, FA, and FV.
There were no significant differences between the groups in fasting free amino acid concentrations in AV (55 min before study period;Table 4). The concentration of methionine was higher (P < 0.05) in EG than in CG at 30 min of recovery. At 195 min of recovery, the sum of free BCAA in FV was significantly (P < 0.01) lower in EG than in CG (Table 4). There was a significantly higher concentration of alanine at 30 min (P < 0.01) and 60 min of recovery (P < 0.05) in FA, and at 30 min of recovery (P < 0.01) in FV in EG than in CG (Table 5).
Main effect of group, interactions and free amino acid concentrations in muscle.
There was a significantly higher (P < 0.001) concentration of alanine in EG than in CG after RES at 60 min of recovery (Table 5). All results for alanine and for the sums of free amino acids are presented in Tables 4 and 5. In addition, a significant (P < 0.05) difference between 60 min and 195 min of recovery (interaction in group × sample: time effect) was observed for alanine.
Correlation coefficients between serum amino acid and muscle amino acid concentrations.
Only low and nonsignificant correlation coefficients were observed in serum amino acid concentrations between blood (mean values of four serum samples per period) and muscle.
Leg blood flow.
Mean blood flow in leg was significantly (P < 0.01) higher in EG during both periods (period 1: 6.10 ± 0.48 mL·min-1·100 mL-1 leg volume and period 2: 5.58 ± 0.18 mL·min-1·100 mL-1 leg volume) than in CG (period 1: 3.62 ± 0.17 mL·min-1·100 mL-1 leg volume and period 2: 3.96 ± 0.22 mL·min-1·100 mL-1 leg volume).
Protein synthesis, breakdown, and net balance.
The only significant difference was observed during period 2 when protein synthesis and protein breakdown were significantly (P < 0.05) greater in EG compared with CG (Fig. 2). Net balance was negative during both periods.
One of the main results of this study was the increase of muscle protein synthesis (21%) and protein breakdown (17%) at 195 min after RES but not yet at 60 min compared with resting conditions. In fasting conditions, however, net muscle protein balance was negative in both recovery phases. During the 195-min recovery, the serum concentration (mean value of 30, 60, 165, and 195 min) of aspartate was greater in EG than in CG in both FA and FV, but the serum concentration of leucine was greater in CG than in EG in both vessels. The BCAA concentration in FV was also greater in CG than in EG at 195 min during recovery. The concentration of alanine was greater in EG than in CG at 30 min after RES in both vessels but at 60 min only in FA. In muscle, the concentration of alanine was greater in EG than in CG at 60 min but not at 195 min after RES.
Muscle protein synthesis, breakdown, and net balance.
The most important finding of this study was that at 60 min during recovery after RES both protein synthesis and protein breakdown increased only slightly (insignificantly), but at 195 min a strong increase was observed in both parameters compared with the resting values. The increase in protein breakdown possibly occurred in order to provide amino acids in the fasting conditions for muscle protein synthesis. In the later recovery, the catabolic responses may take place in the most active muscle fibers that have not contributed to the mobilization of protein resources during intense exercise. Biolo et al. (2) have examined untrained men 3 h after a heavy resistance exercise and found an increase of 100% in muscle protein synthesis and an increase (50%) in muscle protein degradation after physical exercise. In the present study, protein synthesis increased significantly by 21% and the protein breakdown by 17% in the second period after the recovery of 195 min after RES. The mean values of protein synthesis and protein breakdown in our study may underestimate the real effect of exercise on these parameters, as the deviation in the amount of protein synthesis and breakdown was remarkable among the EG subjects especially in the second period, possibly due to different individual response to the exercise workload. This assumption is supported by the report of Phillips et al. (17), who have examined both trained and untrained subjects and found that resistance training attenuates the exercise induced increase in muscle protein turnover. In the present study, the observed increase in absolute protein synthesis was low, although RES must have provided challenge for the muscles of the subjects in EG. The training before experiment was performed at low intensity and the rate of repetitions were only half from those of the study day. Thus, in order to give challenge for the muscles, the exercise session during experiment included 50% more repetitions than during pretraining. Also, the intensity was greater because the subjects were told to lift the load as quickly as possible in the study. Because RES was performed very effectively, we must consider the possibility of the challenge for the muscles. According to studies in rats (8) and humans (6), muscle protein synthesis can be also inhibited by extreme exercise. After resistance exercise, protein synthesis has been reported to rebound for 48 h, whereas protein degradation remains slightly elevated, resulting in positive net balance only if amino acid availability is increased (21). These findings are in line with our results, in which net balance remained negative in the fasting conditions both in CG but also in EG when low availability of free amino acids was further diminished after exercise.
Free amino acid concentrations in serum.
The response of alanine to exercise was similar as expected according to the literature. EG showed higher concentrations of alanine (43%) in FA than CG during recovery of 30 min. The observed postexercise increase in alanine was associated with the lower venous concentration of asparagine in EG which may due to the role as a donor of the amino radical in the formation of alanine (7). After exercise, the splanchnic uptake of gluconeogenic substrates has been shown to increase (25), and because alanine is utilized in hepatic gluconeogenesis (9), the concentration of alanine decreases. Thus, the difference between EG and CG was almost disappeared at 60 min of recovery. The changes in the concentration of threonine were similar as in asparagine, as both of these amino acids had higher concentrations in FV than in FA and, moreover, insignificantly lower in EG than in CG. Threonine is one of the glucogenic amino acids that are released from muscles during fasting (20). The smaller increase in threonine concentration in EG than in CG in FV may result from the increased gluconeogenesis due to both fasting and exercise. Methionine is a glycogenic essential amino acid, which is released from muscle during fasting (20) and which has a limiting role in the maintenance of body protein and nitrogen balance under conditions of greater protein turnover (13). In this study, the concentration of methionine remained at the same level through the periods at rest, whereas the significantly higher arterial and venous levels seen postexercise decreased to values similar to CG at 195 min of recovery after RES. The base level of BCAA, measured from antecubital vein before exercise, was similar in EG and CG. The BCAA concentration remained lower in EG than in CG throughout the recovery, but the difference between the groups was only significant at 195 min of recovery after RES, when the concentration was 20% lower in EG than in CG in FV. The release from other tissues into blood may have been reduced and/or the blood free amino acids may have been utilized in energy metabolism after RES in the fasting conditions, which confirms the previous study (14) suggesting that increased muscle protein metabolism results in decrease of blood BCAA. The results also indicated that the concentration of BCAA leucine decreased, whereas that of aspartate increased in EG during recovery compared with CG. Aspartate plays an important role in purine nucleotide cycle in maintaining the pool of adenosine triphosphate (ATP) in muscle (27). The observed inverse relationship between these two amino acids may reflect the degradation of leucine and the consequent synthesis of aspartate. BCAA degradation in muscle results in formation of glutamate, which can further provide the amino group with oxaloacetate to synthesize aspartate (27). It seems that the changes in serum amino acid concentrations after an intensive RES may be associated also with the energy demands.
Free amino acid concentrations in muscle.
Alanine is one of the major carriers of nitrogen among body tissues and is constantly released from skeletal muscle into the bloodstream (1). Alanine efflux increases in fasting and during exercise (7), which was confirmed in this study. Our results indicated that there was a significantly higher concentration of muscle alanine in EG than in CG after RES. During recovery total amino acids remained high, whereas alanine decreased by 30%. When the muscle biopsies and blood samples of 60 and 195 min during recovery were compared, the decrease of alanine concentration was observed to occur more slowly in muscle. There was seen a difference between the groups in alanine, which showed a 40% higher concentration in EG than CG at 60 min of recovery but only 21% at 195 min, respectively. Our results indicate that the differences in the concentrations of free amino acids in muscle between CG and EG are scant, except in alanine, which increased significantly after RES. The fasting conditions (at least 12 h before the first samples) in both groups may partly explain the differences via the decreased availability of the free amino acids. In addition, no correlative relationships between muscle and blood amino acid concentrations were seen.
Leg blood flow.
As expected, according to the literature, the leg blood flow was increased in the exercise group during both periods of recovery. The basal mean values (period 1: 3.62 and period 2: 3.96 mL·min-1·100 mL-1 leg volume) in CG and the postexercise mean values in EG (period 1: 6.10 and period 2: and 5.58 mL·min-1·100 mL-1 leg volume) were in line with the results of Biolo et al. (2), who reported the basal leg blood flow to be at rest 3 mL·min-1·100 mL-1 leg volume and after the bout of exercise to increase to 5–6 mL·min-1·100 mL-1 leg volume in normal adults. Increased blood flow affects muscle protein metabolism by increasing transport of amino acids into cells and consequently stimulating protein synthesis (3). Amino acid availability has also been shown to be an important factor in the regulation of muscle protein metabolism (e.g., 3). In this study, the increased blood flow may have enhanced free amino acid availability for protein synthesis in EG. The leg blood flow does not alone explain the increased protein metabolism at the end of recovery because the flow slightly decreased during the second period. Other things such as increased amino acid availability through breakdown at the end of recovery or increased blood flow wash out effect on amino acids in the beginning of the recovery may have affected protein metabolism.
In summary, this study provides evidence that RES induces a significant increase in muscle protein synthesis and muscle protein breakdown at 195 min but not yet at 60 min during recovery in fasting conditions. However, negative protein net balance is not changed.
The authors wish to thank Robert R. Wolfe, Ph.D., for his assistance and guidance to learn a three-pool compartment model of leg muscle amino acid kinetics in Medical Branch and Shriners Burns Institute, Galveston, TX 77550. The authors thank also Risto Puurtinen for help in blood collection and Hannu Tuuri for valuable technical assistance in statistical analysis.
The study was supported by TEKES 40721/00.
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Keywords:© 2003 American College of Sports Medicine
FREE AMINO ACIDS; SYNTHESIS; BREAKDOWN; NET BALANCE; STABLE ISOTOPES; EXERCISE