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Effect of vitamin B-6 supplementation on fuels, catecholamines, and amino acids during exercise in men

VIRK, RICKY S.; DUNTON, NANCY J.; YOUNG, JENNY C.; LEKLEM, JAMES E.

Medicine & Science in Sports & Exercise: March 1999 - Volume 31 - Issue 3 - p 400-408
Basic Sciences: Original Investigations

Effect of vitamin B-6 supplementation on fuels, catecholamines, and amino acids during exercise in men. Med. Sci. Sports Exerc., Vol. 31, No. 3, pp. 400-408, 1999.

Purpose: In two separate but identical studies, the effect of vitamin B-6 supplementation was examined on plasma energy substrates, catecholamines, and 13 amino acid concentrations during exercise.

Methods: Eleven trained men performed two separate exhaustive exercise tests at 71.0 ± 4.6% O2max during two separate 9-d controlled diet periods. Exercise test 1 (T1C) occurred following a control diet, and test 2 (T2B6) occurred following a vitamin B-6 supplemented diet (20 mg PN·d−1). Blood was drawn pre, during (60 min), post, and post-60 min of exercise, and plasma was analyzed for glucose, lactate, glycerol, free fatty acids (FFA), catecholamines (N = 5), and amino acids (N = 5).

Results: Mean FFA concentrations changed over time in both tests (P < 0.001) and were lower in T2B6 compared to T1C at pre (P = 0.03), during (P = 0.05), and post-60 min (P = 0.04) of exercise. Mean lactate, glycerol, and catecholamine concentrations only changed over time (P < 0.0001). The only significant changes in amino acid concentrations were for lower tyrosine (P = 0.007) and methionine (P = 0.03) concentrations in T2B6 relative to T1C at post-60 min of exercise and postexercise, respectively. No differences were observed in exercise times to exhaustion between T1C (108+32.6 min) and T2B6 (109+51.2 min).

Conclusions: These results indicate that vitamin B-6 supplementation can alter plasma FFA and amino acid concentrations during exhaustive endurance exercise without affecting endurance.

Department of Nutrition and Food Management, Oregon State University, Corvallis, OR 97331

Submitted for publication July 1997.

Accepted for publication June 1998.

This study was supported by the Oregon Agricultural experiment Station. We would like to thank Karin Hardin and Jim Ridlington for their help with this project.

Address correspondence to: James E. Leklem, Ph.D., Department of Nutrition and Food Management, Oregon State University, Corvallis, OR 97331.

There is limited research on vitamin B-6 and exercise. Manore (26) has written a recent review on physiologic aspects of vitamin B-6 metabolism during exercise and dietary vitamin B-6 intakes of athletes.

Vitamin B-6 (as pyridoxal 5′-phosphate; PLP) plays a critical role in energy production during exercise as a necessary coenzyme in the pathways of glycogenolysis and gluconeogenesis. PLP serves as a coenzyme for both glycogen phosphorylase in the initial step of glycogen degradation and for alanine aminotransferase in the gluconeogenic cori-alanine cycle. Studies in both animals (1,2) and humans (5) have found increases in muscle concentrations of vitamin B-6 following a period of pyridoxine supplementation. In animals, this increase resulted in a significant elevation in glycogen phosphorylase activity (1,2). Increases in plasma lactate concentrations (22) with concomitant decreases in plasma FFA concentrations (27) during exercise after a period of vitamin B-6 supplementation have been observed, suggesting an increased rate of carbohydrate utilization.

Studies in animals have shown that acute doses of vitamin B-6 can increase both the rate of synthesis (35) and the rate of release (21) of catecholamines from the adrenal medulla, resulting in an elevated concentration of blood glucose via the immediate mobilization of liver glycogen. Thus, vitamin B-6 can potentially alter substrate utilization through its effect on the concentrations of circulating hormones.

Several research groups have shown unequivocally that plasma concentrations of PLP increase during exercise (7,18,23). However, there is no agreement on the reason for this physiologic response. Crozier et al. (7) have postulated this finding is due to temporary shifts in protein concentrations from the interstitial fluid to the plasma at the start of exercise. An adaptive response directed toward the production of hepatic glucose via the process of gluconeogenesis also has been suggested as a possible reason (24). Provided this second hypothesis is correct, one might expect a change in plasma concentrations of selected amino acids involved in the gluconeogenic/transamination process to synthesize glucose. To our knowledge no investigators have examined the effects of vitamin B-6 supplementation on plasma amino acid levels during exercise. However, several research groups have investigated the effects of either depletion (9,16,32) or supplementation (20) of vitamin B-6 on plasma amino acid concentrations at rest in humans.

It was the intent of this study: 1) to determine if supplemental vitamin B-6 would enhance the rate of carbohydrate metabolism during submaximal exhaustive exercise and if this possible effect would result in a change in endurance performance, and 2) to determine whether supplemental vitamin B-6 and submaximal exercise to exhaustion would have an effect on the catecholamines epinephrine and norepinephrine and on selected plasma amino acid concentrations, thus providing a more complete perspective of fuel utilization.

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METHODS

Experimental design. Two similar but separate studies were conducted within a 2-yr span to examine the effects of vitamin B-6 supplementation on fuel utilization, catecholamines, and plasma amino acids during exhaustive endurance exercise in men. Following the completion of study 1 (N = 6), a freezer malfunction resulted in the unfortunate loss of samples to be used in several planned assays. Therefore, study 2 was performed as a repeat of study 1. Study 2 was slightly modified to strengthen the study design yet allow for the combination of data from both studies to increase the statistical power. See Figure 1 for a description of the studies. Because of the level of vitamin B-6 supplementation and the time required for vitamin B-6 status to return to baseline following supplementation, both studies were nonrandomized in design. Aerobically trained male subjects exercised to exhaustion on a cycle ergometer at 75% of a predetermined O2max. The first exercise test in each study occurred in a control state (T1C) and the second test in a vitamin B-6 supplemented state (T2B6). The tests were separated by approximately 2 wk. There was a 9-d dietary period (6 d before and 3 d after each of the testing sessions) in which the subjects were fed a controlled diet that was nutritionally adequate. To determine any changes in fuel utilization, blood was collected and respiratory gases were analyzed. Plasma was analyzed for glucose, lactate, glycerol, free fatty acids (FFA), epinephrine (study 2 only), norepinephrine (study 2 only), and amino acids (study 2 only). The appropriate data from both studies was combined after checking for normality using the univariate procedure and analyzed as described in the Statistics section.

Figure 1

Figure 1

Subjects. Eleven, healthy, trained, male subjects were recruited to participate in the study from the Oregon State University (OSU) campus and from the local community by a flier and by word of mouth (one subject participated in both studies). The study was approved by the OSU Human Subjects Committee, and written informed consent was obtained from each subject. For the subject selection in this study, training was defined as 180 min of aerobic activity (i.e., running, biking, swimming) per week, with a minimum of 3 d·wk−1. All subjects selected actually exercised between 200 and 400 min·wk−1 for 5-7 d·wk−1. They had maintained this exercise standard for at least 1 yr. In addition, each individual was requested to maintain his fitness regimen throughout the investigation to minimize a training effect. A prestudy questionnaire and a brief interview were used to evaluate training and health status. None of the subjects had used vitamin supplements for at least 4 wk before the start of the study and none had smoked or used other drugs known to interfere with vitamin B-6 metabolism or methodology. All subjects kept a daily log to help assure compliance with the study and to monitor activity levels. Body composition was determined by using hydrodensitometry. Residual volume was determined by the oxygen dilution method (39), and body density (13) and body fat percentages (37) were computed by standard equations.

Exercise testing. All exercise testing was performed on a cycle ergometer (Monark, Quinton Instruments, Seattle, WA). A total of four exercise tests per subject were conducted. These consisted of a O2max test in the week preceding the start of the study, an orientation ride within 2 d of starting the study (study 2 only), and two endurance rides during each of the dietary phases. The O2max test protocol consisted of increasing the workload in 30-W increments until each subject showed a plateau in oxygen consumption along with an R-value of greater than 1.1 and a maximum heart rate (HR) of greater than 90% of the predicted maximum or until each subject requested to stop the test. An average of the three highest oxygen consumption values obtained during the max test was used to set subsequent workloads during the orientation ride. In this orientation session, which lasted about 20 min, the workload which corresponded to 75 ± 1% O2max was determined. On the morning of the 7th d of each dietary period, the aerobically trained male subjects exercised to exhaustion at 75% of their maximum aerobic capacity following a 12-14 h fast. A brief warm-up consisting of 5 min allowed subjects to loosen up. During the test, HR was monitored by an ECG using three-limb leads (Quinton Instruments, Model 630 A). Respiratory gases were collected for 3 min at 10-min intervals to calculate R-values and measure oxygen consumption (Sensor Medics Metabolic Cart Model 2900, Yorba Linda, CA). One hundred mL of water was given to subjects while cycling (at 60 min into exercise). After the postexercise blood draw, an additional 100 mL of water was given to the subjects. Even though the lab temperature and humidity were comfortable for exercise (i.e., 20-24°C, < 60%, respectively), a fan was provided for evaporative cooling. Body weights were recorded pre and posttesting. By using the 20-point Borg scale, ratings of perceived exertion (RPE) were asked of subjects every 10 min to help identify progressive fatigue and subsequent exhaustion. Exhaustion was defined as the inability to maintain within 5 rpm of the initial cadence (i.e., 80 rpm) for a total of 20 s. During both exercise tests subjects were not aware of cycling times (i.e., clocks/watches were concealed).

Diet. Two 9-d feeding periods involved serving meals in a metabolic kitchen. Both dietary phases were identical in food (i.e., macronutrient) content, and composed of 60% carbohydrate, 23% fat, and 17% protein (approximately 3600 total kcal). Before the study, 3-d diet records were used to determine the energy needs of the subjects. Certain foods were allowed ad libitum. These foods were used primarily to adjust for each individual's variation in energy need, as determined by appetite and daily weight monitoring. Foodstuffs provided 2.30 mg·d−1 of vitamin B-6 in study 1 and 1.9 mg·d−1 in study 2 and did not change between each of the respective feeding periods during either of the studies. Identical foods and amounts were provided in both studies. Despite this, the difference in vitamin B-6 content between the two studies was due primarily to a difference in vitamin B-6 content of the animal foods purchased and subsequently analyzed. We have observed this in other metabolic studies. The vitamin B-6 content was determined by microbiological assay of aliquots of food composites (29). Phase 1 (T1C) was supplemented with a placebo capsule, while phase 2 (T2B6) was supplemented with an additional 20 mg pyridoxine-hydrochloride (PN-HCl)·d−1 in similar capsule form as the placebo. Based on previous studies in our lab, it was estimated that 20 mg would be necessary to raise plasma PLP concentrations high enough to observe a possible effect. A single blind design was chosen. All other micronutrients met 100% of their recommended dietary allowances. When possible, all foods were purchased from the same lot. Careful preparation by means of weighing foods to ± 0.1 g assured consistency within and between the diets of all subjects. Alcoholic beverages were prohibited throughout the study, and caffeine was not allowed on the day before, the day of, or the day after exercise testing.

Blood collections. Blood was drawn by a registered medical technologist in the week before the start of the experiment, on the two exercise test days (day 7 and 30), and once during the vitamin B-6 supplementation phase midway between the two dietary phases (days 16-19). The mid-study nonexercising blood sample provided plasma PLP blood data on subject compliance of supplementation. Based on these data, capsule counts, and urinary vitamin B-6 metabolite excretion (15), all subjects were taking the supplement. On exercise test days, the first blood sample was taken 30-40 min before exercise after the subject had rested for 10-15 min. The second sample was taken 5-10 min before starting exercise. The third sample was taken 60 min into exercise. During this time the workload was decreased slightly to allow for a safe blood draw. The fourth and fifth samples occurred in the exercise recovery phase at immediately postexercise and post-60 min of exercise, respectively. Heparinized blood tubes were used for collection, and all samples were kept on ice approximately 5-10 min until centrifuged. After extraction of the plasma portion, the separately aliquoted samples were frozen at either −40°C for fuel analysis or −80°C for hormone and amino acid analysis. Plasma for amino acid analysis was deproteinized with sulfosalicylic acid before freezing at −80°C.

Analyses. All samples were done in duplicate unless otherwise specified. Plasma glucose was measured by the glucose oxidase method (38) using a Technicon Autoanalyzer System II (Alpkem Corp., Clackamas, OR).

Plasma lactic acid was measured spectrophotometrically (Sigma Chemical Co., procedure no. 726-UV/826-UV, St. Louis, MO) (17). Plasma glycerol was measured spectrophotometrically by a modified enzymatic method (33) for triglycerides by omitting the saponification step. A kit from Sigma Chemical Co. was used (procedure no. 320-UV). Plasma FFA were measured in triplicate by a colorimetric method (10). Plasma PLP was measured by a modified method of Chabner and Livingston (4).

Plasma catecholamines were measured by HPLC using a modification of the method of Goldstein et al. (14) and were done only in study 2. Catecholamine standards were 1.0, 2.0, 5.0, 10.0, and 20.0 nmol·L−1 for both epinephrine and norepinephrine with an additional standard concentration of 30 nmol·L−1 for norepinephrine. Dihydroxybenzylamine (DHBA) was used as an internal standard and was kept constant at 25 nmol·L−1 in each standard. Samples were eluted on an UltraCarb (Phenomenex Columns, Torrance, CA) 5 ODS (20) reverse-phase C18 column (250 × 4.6 mm) using a Shimadzu HPLC system (Tokyo, Japan) consisting of: an LC-10AD solvent delivery module, an SIL-10A autoinjector, an L-ECD 6A electrochemical detector with a glassy carbon working electrode (potential of +0.72 V), an SCL-10 system controller, and a CR5-1 chromatopac integrator.

Amino acids were determined by HPLC using a modification of the method of Rosenlund (36) and were done only in study 2. A standard amino acid mixture (Pierce Chemical Co., Indianapolis, IN) contained the following L-amino acids at a concentration of 2.5 mmol·L−1: alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and cysteine (1.25 mmol·L−1). Samples were eluted on a Spherex (Phenomenex Columns) 5-μ C8 column (250 × 4.6 mm) using a Shimadzu HPLC system consisting of: an LC-10AD solvent delivery module, an SIL-10A autoinjector, an SPD-10A uv-vis detector (254 nm), an SCL-10 system controller, and a CR5-1 chromatopac integrator. Norleucine was used as an internal standard. Only one specific blood collection sample per person was done in duplicate as a measure of reproducibility. Tryptophan was measured fluorometrically by a modification of the method of Bloxam and Wharen (3), and all samples were done in triplicate.

Hematocrit (Hct) and hemoglobin (Hb) were measured by the micro Hct and cyanomet Hb methods, respectively. Hb was done in triplicate and Hct was done in duplicate. Plasma volume changes were measured by the method of Dill and Costill (8).

Statistics. To increase statistical power the appropriate data (N = 6 study 1; N = 5 study 2) were combined after checking for normality using the univariate procedure. The data were analyzed by the standard statistical methods (Statgraphics Software; Manugistics, Inc., Rockville, MD) of ANOVA and Student's t-test (34). A Student's t-test for paired values was used to determine whether a difference existed between T1C and T2B6 at each specific blood collection time point for all fuel substrates, catecholamines, amino acids, and all other variables studied. ANOVA was used to determine whether changes occurred over time within T1C or T2B6 for each variable studied. Fisher's least significant difference was used for post-hoc analysis in testing multiple comparisons. Null hypotheses were rejected at the 0.05 level of significance. All values reported within the results section are mean ± the standard deviation of the mean.

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RESULTS

Diet. The mean energy consumed during study 1 was 3407 ± 98 kcal·d−1 and in study 2 3845 ± 216 kcal·d−1. The mean carbohydrate consumption in study 1 was 595 ± 35 g·d−1 and in study 2 632 ± 33 g·d−1. We attribute this difference to the higher body weights of the subjects in study 2 compared with study 1 (see Table 1 for subject characteristics). Foods such as cola beverages and candy were provided ad libitum to subjects to maintain body weight. Protein was constant in both studies at 155 g·d−1. There was no difference in the energy or macronutrient content consumed between each of the testing periods within each study.

TABLE 1

TABLE 1

Plasma volume changes. Blood was collected before exercise (pre), 60 min into exercise (during), immediately after exercise (post), and 60 min after exercise (post-60) and analyzed for plasma volume changes. The mean percent plasma volume changes in T1C for the during, post, and post-60 time points are −10.8 ± 2.7, −11.4 ± 5.3, and −4.9 ± 5.5, respectively. The mean percent plasma volume changes in T2B6 for the during, post, and post-60 time points are −13.6 ± 1.9, −13.7 ± 4.8, and −3.7 ± 3.6, respectively. The during-exercise time point resulted in a statistically significant increase in plasma volume change in T2B6 relative to T1C (P = 0.01). The data were not corrected for plasma volume changes.

PLP. The fasting prestudy mean plasma PLP concentrations were 37.6 ± 6.1 nmol·L−1. Preexercise mean plasma PLP concentrations were 31.8 ± 11 nmol·L−1 (31.4 ± 12.5 study 1 and 32.4 ± 10.1 study 2) in T1C and 187 ± 40 nmol·L−1 in T2B6 (199 ± 47.2 study 1 and 172 ± 24.2 study 2), representing a statistically significant difference (P < 0.01). The relatively low prestudy and preexercise plasma PLP concentrations were due to the high protein intakes of the subjects from both their free-living and controlled diets.

Glucose. There was no vitamin B-6 effect on glucose concentrations nor was there a change in mean plasma glucose levels over time in either test (Fig. 2).

Figure 2

Figure 2

Lactate. Vitamin B-6 supplementation had no effect on plasma lactate concentrations. As expected, there was a significant change in mean plasma lactate concentrations over time (P < 0.0001; Fig. 3). For both tests the during-exercise and postexercise mean plasma lactate concentrations were statistically higher than either of their respective pre and post-60 samples.

Figure 3

Figure 3

Glycerol. No vitamin B-6 effect was observed on plasma glycerol concentrations. However, mean plasma glycerol concentrations did change significantly over time (P < 0.0001) (Fig. 4). In both tests, the during and post-60 sample means were statistically higher than their respective preexercise means and in both tests the post means were statistically higher than all other respective means.

Figure 4

Figure 4

Free fatty acids. There were statistically significant differences in the plasma FFA means between T1C and T2B6 at pre (P = 0.03), during (P = 0.05), and post-60 min (P = 0.04) of exercise. In addition, there was a significant change in mean plasma FFA over time in both tests (P < 0.0001; Fig. 5). Plasma FFA concentrations were between 8% and 25% lower at all blood sampling time points in T2B6 relative to T1C. In both T1C and T2B6, the post and post-60 means were higher than each of their respective pre and during-exercise means.

Figure 5

Figure 5

Catecholamines. Vitamin B-6 had no effect on the plasma concentrations of both epinephrine and norepinephrine before, during, and after exercise. There was a significant change in mean plasma epinephrine concentrations in T1C as the during and postexercise means were statistically higher than the preexercise mean (P = 0.02; Fig. 6). In both tests the during and postexercise sample means for norepinephrine were significantly higher than their respective pre and post-60 means (P < 0.0001; Fig. 7).

Figure 6

Figure 6

Figure 7

Figure 7

Amino acids. There were statistically significant differences between the mean plasma levels of tyrosine (P = 0.007 at post-60 min of exercise) and methionine (P = 0.03 at postexercise) between T1C and T2B6, and there were significant changes over time for the mean plasma alanine (T1C, P = 0.01; T2B6, P = 0.01) and plasma histidine concentrations (Table 2). The plasma concentration of no other amino acid differed between the two tests nor did any change statistically significantly over time within a test.

TABLE 2

TABLE 2

Although not statistically significant, at all specific blood collection time points with the exception of post-60 min of exercise, mean plasma tryptophan concentrations were lower in the T2B6 compared with T1C. On an individual basis, in four of five subjects it was observed that plasma tryptophan concentrations were lower in T2B6 at both preexercise time points and at the postexercise time point.

R-values and oxygen consumption. There were no statistically significant differences in mean R-values or mean oxygen consumption between T1C and T2B6 at any of the specific collection time points (recall that gases were collected for three min intervals every 10 min). In addition, there was no significant change in mean R-values or oxygen consumption over time during either of the tests (Table 3). Although it was not statistically significant, it did appear that within the first 90 min of exercise, mean R-values tended to be higher by 0.01-0.02 units in T2B6 relative to T1C when comparing each specific collection time point.

TABLE 3

TABLE 3

Exercise times to exhaustion. There were no statistically significant differences in mean exercise times to exhaustion between T1C (108.9 ± 32.6 min) and T2B6 (109.9 ± 51.2 min). Four subjects showed a decrease in time to exhaustion ranging from 12 to 37 min, whereas four subjects showed an increase in time to exhaustion ranging from 12 to 44 min in T2B6 compared with T1C. The other three subjects showed less than a 5-min difference between T1C and T2B6. Two subjects did not make it to the 60-min time point in T2B6.

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DISCUSSION

Previous studies in the area of vitamin B-6 and exercise have observed either an increase (22), a decrease (28) or no change at all (27) in mean plasma lactate concentrations during exercise following a period of vitamin B-6 supplementation compared with a control situation. The experimental design was different in each of these studies. Vitamin B-6 is thought to possibly enhance carbohydrate metabolism by enhancing a more rapid rate of glycogen metabolism (27). The lactate results of this study do not support this hypothesis. The changes in lactate concentrations in this study are consistent with those observed by others from pre to during exercise (6) and from during exercise to post-60 min of exercise (18).

If carbohydrate metabolism increases, then lipid metabolism should decrease, because the two combined fuels contribute almost exclusively to energy production during exercise (25). Although mean plasma glycerol concentrations were not statistically different between the two tests, at three of the four blood collection time points, the mean plasma FFA levels were significantly (P < 0.05) lower under conditions of vitamin B-6 supplementation compared with the control condition despite a greater plasma volume change in T2B6 relative to T1C. This is consistent with the hypothesis that vitamin B-6 stimulates glycogenolysis, thereby decreasing FFA mobilization due to decreased utilization. Manore and Leklem (27) similarly found there to be statistically significantly lower mean fasting preexercise plasma FFA concentrations following vitamin B-6 supplementation relative to a control situation. These present results could either be reflective of a decrease in total fat oxidation or a result of increased intramuscular triglyceride utilization, thus reducing the reliance on blood-borne fatty acids. Training (19) and diet (30) are both variably believed to enhance one's capacity to utilize intramuscular lipid stores. However, these variables were closely regulated and monitored during both T1C and T2B6. The changes in glycerol (19) and FFA (12) concentrations over time are consistent with those observed in the literature.

Mean plasma glucose concentrations changed very little with vitamin B-6 supplementation and as a result of endurance exercise to exhaustion. Hofmann et al (18). observed a similar finding during a control 2-h cycle ergometer test at 63% of O2max in trained male subjects. This present study was the first that we are aware of to examine the plasma catecholamine response to endurance exercise during a period vitamin B-6 supplementation. No vitamin B-6 effect observed between T1C and T2B6 for plasma catecholamine levels.

Prior work (9,16,20,32) has shown that alterations in dietary vitamin B-6 intake result in plasma changes of several amino acids in humans, possibly due to vitamin B-6's role in the gluconeogenic process. The significance of lower mean plasma tyrosine concentrations in T2B6 relative to T1C is difficult to explain at best and would appear to have no relevance to fuel utilization. However, this finding is in part explained by the results of Donald et al. (9), who found that when young adult women were fed a diet low in vitamin B-6, it resulted in significant increases in the plasma concentration of tyrosine as well as other amino acids (i.e., serine, threonine, glutamic acid, and methionine). The diet low in vitamin B-6 was suggested to result in a temporary shifting of free amino acids into the plasma followed by removal of some amino acids either by redistribution in tissues or by excretion. Possibly with supplemental vitamin B-6, as utilized in this study, plasma tyrosine levels would shift in the opposite direction. Speitling et al. (1988, as cited by Kang-Yoon and Kirksey, 1992) similarly observed that plasma concentrations of serine, tyrosine, and histidine decreased by the end of a 30-d supplementation study of 40 mg·d−1 PN-HCl in men between the ages of 20 and 30 yr. One of the metabolic fates of tyrosine is its conversion to dopamine and further to the catecholamines, norepinephrine, and epinephrine. One of the enzymes involved in this conversion is DOPA decarboxylase. Supplemental vitamin B-6 has been shown to result in elevated DOPA decarboxylase (PLP-dependent) activity in animals (35), which might therefore enhance the flux of tryosine through the pathway, possibly resulting in lower levels of plasma tyrosine. In this study, following an overnight fast and 60 min of recovery from exhaustive endurance exercise, there would obviously be an increased need for elevated plasma catecholamines as lipolysis and gluconeogenesis would be the primary pathways for energy in the body. However, our epinephrine and norepinephrine results do not support this possible conversion of tyrosine to the catecholamines.

By the end of exercise in T2B6, mean plasma methionine concentrations were statistically (P = 0.03) lower in comparison with T1C means despite a 4-5% greater plasma volume change in T2B6 relative to T1C. The significance of this finding, as mentioned in the tyrosine discussion, has limited application to fuel utilization and furthermore to exercise performance. It is difficult to explain why it required a mean exercise time of approximately 93 min for mean plasma methionine levels to show a statistically significant change in T2B6 relative to T1C. Under conditions of vitamin B-6 supplementation, we expected to observe an increase in plasma alanine in the blood during exercise as a result of supplemental vitamin B-6 enhancing the muscle's transaminase capabilities (i.e., pyruvate to alanine). At all blood collection time points, with the exception of post-60 min of exercise, there was a trend for higher mean plasma alanine levels in T2B6 compared with T1C. Recently, Kang-Yoon and Kirksey (20) have observed that after 14 d of vitamin B-6 supplementation of 27 mg·d−1 in young women, plasma alanine concentrations significantly increased by approximately 16%. They suggest this response to supplementation to be a result of accelerated protein and/or amino acid metabolism.

Similar to alanine, histidine plasma concentrations increased significantly over time in both exercise tests. However, there were no statistically significant differences between the plasma concentrations despite the fact that mean plasma histidine concentrations were between 1% and 10% higher at all blood sampling points in T2B6 compared with T1C. Others have observed either no change (20) or a decrease (Speitling et al., 1988, as cited by Kang-Yoon and Kirksey, 1992) in mean plasma histidine concentrations following 2-4 wk of PN supplementation of dosages greater than the 20 mg·d−1 administered in this study. Vitamin B-6 (as PLP) is involved as a coenzyme in the metabolism of histidine in two distinct metabolic pathways. These include the primary pathway of the deamination of histidine with the ultimate formation of glutamate and the decarboxylation of histidine to histamine. During exercise, the deamination pathway would be expected to increase as the body works to maintain a steady output of hepatic glucose.

Originally, fatigue in the exercising muscle was linked to either a depletion of available fuel in the form of carbohydrate or to metabolic acidosis as a result of lactate accumulation within the muscle, thus suggesting peripheral mechanisms of action. However, recently, the concept of central/mental fatigue as dictated by plasma amino acid levels has received interest in the area of exercise and sport physiology as a potential factor in an individual's capacity to perform physical activity for extended periods of time (31). Of particular importance is the ratio of tryptophan relative to the large neutral amino acids (LNAA, i.e., branched-chain amino acids, tyrosine, and phenylalanine), with higher ratios resulting in the formation of the sleep-inducing neurotransmitter serotonin. In T1C and T2B6, the ratios were similar and equalled 0.095 and 0.097, respectively. Although PLP is directly involved as a coenzyme in the metabolism of tryptophan to serotonin and as well is involved in the metabolism of each of the LNAA, there was no significant difference in the tryptophan:LNAA ratio between T2B6 and T1C, thus suggesting no vitamin B-6 effect on central fatigue (as related to amino acid levels).

One of the inconsistencies found in the literature in this area of study is whether plasma volume changes have been measured and whether plasma substrate and hormone concentrations have been corrected based on the observed plasma volume changes. Often, authors claim that they have corrected their data with respect to plasma volume changes, although many times an equation or method for their data manipulation is not provided. In this study plasma volume changes were measured, however, the plasma substrate concentrations were not normalized for the observed plasma volume changes. Plasma volume changes were primarily used to provide a possible explanation for observed changes or lack of these between T1C and T2B6 for a given plasma substrate.

During the first 90 min of exercise, the mean R-values in this study tended to be 0.01-0.02 units higher in T2B6 relative to T1C. If all subjects were exercising below their respective anaerobic/lactate thresholds (AT/LT), then this translates out to as much as a 6-7% increase in carbohydrate utilization with a similar decrease in fat utilization in T2B6 compared with T1C. Previously in our lab (unpublished observations), we found there to be a 9% increase in carbohydrate utilization during exercise in trained male subjects exercising at 72% of O2max following a period of vitamin B-6 supplementation (20 mg PN·d−1), as reflected by higher R-values. Because the AT/LT was not actually measured in the present study and because there was the likelihood that some subjects might have been exercising at an intensity above their AT/LT, an accurate percentage breakdown of carbohydrate and fat cannot be provided. Exercising above one's AT/LT would tend to mistakenly add to the proportion of carbohydrate utilized for energy and falsely decrease the proportion of fat oxidized. In endurance-trained individuals, the AT/LT usually occurs at 70-80% of a persons O2max (11). Although we did not observe a significant difference in R-values between the tests, the trends indicate an enhanced rate of carbohydrate metabolism. A larger N possibly would have resulted in a statistically significant finding.

Vitamin B-6 had no effect upon exercise times to exhaustion as mean times differed by only 1 min between the tests. Thus, based on these studies, endurance athletes can supplement their diets with relatively high intakes (20 mg·d−1 or less) of vitamin B-6 for 3 wk or less without the possibility of a detrimental effect on performance. Although there is some evidence to suggest that vitamin B-6 may be affecting fuel utilization during exercise, the effect does not appear to be of such a magnitude that one's endurance capacity is compromised as measured by times to exhaustion.

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Keywords:

PYRIDOXAL 5′-PHOSPHATE; GLUCOSE; LACTATE; GLYCEROL; FATTY ACIDS; HORMONES; R-VALUES; DIET STUDY; CYCLING; SUBMAXIMAL EXERCISE; ENERGY METABOLISM

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