monia responses to prolonged exercise in

red dietary intakes prior to

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D. A. MA CLEAN,^ L. L. SPWET,AND T. EE.GRAHAM School of H u m n Biology, University of Guelph, GuelpgPh, &b. , Canada IVH G 2 W l Received Aprd 1 1, 1991 MACLEAN,D. A., S P ~ E TL., L., and GRAHAM, T. E. 1992. P l a s m amino acid and ammonia responses to altered dietary intakes prior to prolonged exercise in humans. Can. S. Physiol. P h a m c o l . 70: 420-4241. This study examined the e f k t s of altered dietary intakes on amino acid and ammonia (NH,) responses prior to and during prolonged exercise in humans. Six male recreational cyclists rode to exhaustion at 75% of Vo,max following 3 days on a low carbohydrate (LC), mixed (M), or high carbohydrate (HC) diet in a latin square design. There were differences ( p < 0.65) in exercise times among all treatments (58.8 +_ 3.7, B 12. H 1 '9.3, and 152.9 + 10.3 rnin for the LC, ha, and HC treatments, respectively). The rate of increase in plasma NH, during exercise was greater ( p < 0.05) during the LC trial. The LC trial was also characterized by higher (p < 6.05) resting plasma concentrations of branched chain amino acids (BCAA) and a greater decrease in these amino acids during exercise ( p < 0.05), as compared with the other two treatments. Both plasma BCAA and NH, were susceptible to dietary manipulations. These findings suggest that limited carbohydrate availability in association with increased BCAA availability results in enhanced BCAA metabolism during exercise. This is reflected in a greater rate of increase in plasma NH, and is consistent with the hypothesis that a significant fraction of the NH, released during a prolonged, submaximal exercise bout is from amino acid catabolism. Key words: AMP deaminase, branched chain amino acids, branched chain keto acid dehydragenase, glycogen, purine nucleotide cycle. MACLEAN,D. A., SPMET?E. L., et GRAHAM, TsE. 1992. P l a s m amino acid and ammonia responses to altered dietary intakes prior to pro10ng& exercise in humans. Can. J. Physiol. Pkarmcol. 70 : 428-42'7. Cette Cmde examine les effets d'une alttration de I'apport alimentaire sur les rkponses de l'arnmoniac (Nhf,) et des acides arninks, av!nt et durant un exercice prolong6 chez les humains. Six cyclistes amateurs, rnlles, ont @dalC jusqu'i Cpuisement h 75% du Vo,max, apr&s3 jours d'une di&tehypoglucidique (HYPO), mixte (M) ou hyperglucidique (HYPER), conformCrnent a un plan expkrimental en carrC latin. On a observC des diffkrences ( p < 0,05) entre les temps d'exercice des difftrents traikmesats (58,$ f 3,7, 1H2,f 7'3 et 152,9 +_ f0,3 min pour les traitements HYPO, M et HYPER, respectivement). LIZtaux d'augmentation de NH, plasrnatique durant l'exercice a kt6 plus Clevd ( p < 6,65) durant I'kpreuve HYPO. Celle-ci a aussi dt6 caract6isCe par de plus fortes concentrations plasmatiques ( p < 0,051 au repos d'acides amints B chafne ramifike (AACR) et par une plus forte diminution de ces acides aminCs durant l'exercice (g < 0,845), comparativement aux deux autres traitements. Le NH, et les AACR plasmatiques on%Ctt sensibles aux fluctuations alimentaires. Ces rksultats suggkrent que la disponibilitd limit& en glucides, associ6e B la dispnibilid accrue en AACR, augmente le mdtabolisme en AACR durant l'exercice. Ceca est refl6tC par une plus forte augmentation du taux de NH, plasrnatique et est en accord avec l'hypothkse qu'une fraction significative du NH, libkre durant une Nriode d'exercice submaximal, prolongke, rtsulte du catabolisme des acides aminks. h a cl&s : AMP dQarninase, acides amin6 ii chaine ramiGCe, cCtoacide dCshydrogtnase h chafne ramifiCe, glycog&ne, cycle des nuclCotides puriques. [Traduit par la rCdaction]

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Introduction Ammonia (NH3)2 is released from skeletal muscle during both intense and prolonged, submaximal exercise (Broberg and Sahlin 1989; Graham et al. 1990; Graham et al. 1998; Wagenmakers et d. 1998). The rate of NH3 release from skeletal muscle continually increases during prolonged, submaximal exercise (Broberg and Sahlin 1989) and is reflected by a continually increasing venous plasma NH3 concentration (MacLean et al. 1998). During intense exercise the dominant source of NH3 in human skeletal muscle is the deamination of AMP to IMP and NH3 by AMP deaminase as one s f the reactions of the purine nucleotide cycle (PNC) (Graham et al. 1990). It has been suggested that the same mechanism is also responsible for the NH3 produced by human skeletal muscle during prolonged, submaximal exercise (Broberg and Sahlin 1989). However, another possible source of NH3 formation in skeletal muscle is from the deamination of the branched 'Author for correspondence. ,In physiological solutions both ammonia and the ion, ammonium exist. In this paper NH, will represent the sum of both forms. Printed in Canada I ImprimC au Canada

chain amino acids (BCAA), isoleucine, leucine, and valine. This invo%vesthe coupling of a BCAA transferase with glutamate dehydrogenase (GDH) forming a transdeamination reaction and subsequently producing NH3. The oxidation of amino acids has been suggested to be an additional source of energy for skeletal muscle during fasting (Goldberg and Odessey 1972), low energy diets (Munro 195I), and prolonged exercise (Brooks 1987). Furthermore, studies have shown that amino acid uptake and oxidation by skeletal muscle increases in response to exercise (Ahlborg et al. 1974; Brooks 1987; Dohm 1886; Hagg et ah. 1982; Wolfe et al. 1982). Thus, it is reasonable to suggest that a portion of the NH3 produced during prolonged, submaximal exercise may originate from BCAA catabolism. Altering the availability of muscle glycogen or blood glucose as a substrate also influences the rate of protein catabolism during exercise (Lemon and Mullin 1980; Wagenmakers et al. 1991). It has been proposed that NW3 production, via the PNC, is greater when initial muscle glycogen content is low (Broberg and Sahlin 1989). However, the declining concentration of muscle glycogen may progressively enhance BCAA catabolism and NH3 production, because skeletal muscle NH3

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MACLEAN ET AL.

42 1

production continudly increases during submaximal exercise (Graham e t idnE. 1991) and the highest plasma NH3 concentrations occur at exhaustion when muscle glycogen i s depleted. The present study was undedaken to examine the relationship between plasma m i n o acid and NH3 responses to altered dietary intakes prior to prolonged, s u b r n a x h d exercise. We hypothesize that a major source of NW3 formation during prolonged, submaximd exercise may b e from the deanination s f the BCAA. Furthermore, by limiting dietary carbohydrate intake there will be a greater need to metabolize the BCAA, resulting in an even greater plasma NH3 level than normally observed during exercise.

4 mL was transferred to a wontreated Vacutainer tube for serum. The plasma was analyzed fluorometrically in triplicate for NH, (Kun a d Kearney 19741, glucose, and lactate (Bergrneyer 1974) using enzymatic techniques. The amino acids were derivatized with pheraylisohimyanate and analyzed in duplicate by Waters HHPEC according to the method sf Heinrikson and Meredith (1984). The serum was analyzed enzymatically with a fluorometer for free fatty acids (FFA) (Miles et al. 1983) and glycerol (Boobis and Maughaan 1983). Serum urea nitrogen was determined with a S i g m Urea Nitrogen Kit (No. 640) using a B e c h a n Du-78 spectrophotorneter. Hematocrit was determined by high speed centrifuge to document changes in p l a s m volume. However, with the constant infusion of saline there were no significant differences between treatments in the shifts in hematocrit during exercise.

Material and methods

CaHculatihans The BCAA concentration was calculated by summing the concentrations of valine, isoleucine, and leucine. The total essential amino acid (EAA) concentration was calculated by summing the concentrations of threonine, methionine, BCAA, phenylalanine, tryptophan, and lysine.

Subjects The experimental protocol was approved by the School of Human Biology Ethics Committee and six healthy, male volunteers were i n f o r d of the purposes and risks of the study. All subjects were active, recreational cyclists between the ages of 19 and 32 years (X = 25.5 f 2.3 years; X f SEM), weight 63.6-87.0 kg (X = 75.0 f 4.61 kg), and height 170.5-183.0 crn (X = 197.3 f 2.0 cm). Pre-experirnen~a!procedure Prior to participating in the actual experiment, the subjects underwent two incremental maximum oxygen consumption tests (F"o,max) on a Momrk cycle ergometer. The subjects' F"02max ranged from = 52.1 f 3.4) mL - kg-' 44.3 to 62.9 mL - kg-I min-"3 min-I). The second ~ 0 2 m a test x was followed by 95 min of exercise consisting of 75 rnin at 60%, IS min at 3076,and 2 min at 120%of Vo,max, designed to deplete muscle glycogen. The subjects were then randomly assigned a low carbohydrate (LC), mixed (M,norm&), or high carbohydrate (HC) diet. The M diet was not controlled, while the assigned LC diet was calculated to contain 10 8 18 W/day with 2.9% carbohydrates, 50.7% fat, and 46.4% protein. The assigned HC diet was calculated to contain 10 193 W/day with 85.6 % carbohydrates, 3.6% fat, and 10.8 % pmtein. The intake of water and noncaloric material was not controlled in any phase of the study. The subjects remained on the assigned diet for 3 days, while maintaining a f d diary, and refrained from any strenuous activity or ingestion of caffeine.

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Experimental prot~k:o& The subjects reported to the laboratory after a 12-h fast; a catheter was placed percutaneously into a medial antecubid vein and a saline drip was started. After resting approximately 20 rnin a venous blood sample was obtained. The subjects h e n cycled at 75 % Vo2max until exhaustion (i.e., unable to maintain the pedal cadence) and blood samples, expired gas, and heart rates were collected every 15 min and at exhaustion. Following this trial the subjects were assigned one of the two remaining diets in a specific order to complete a latin square (i.e., LC, HC, M) design and returned to the laboratory every 3 days until all three trials were completed. During the exercise trials the subjects were unaware of the exercise duration, and performance outcomes were not revealed until the study was completed. Analyses The food diary analysis was assessed by processing 18 diary records with a popular 886 f w d item system developed by the Department of National Hedth and Welfare Canada (Dibblee and Graham 1983). The fractions of expired 8, and CO, were Qetermined with an Applied Electrochemical S-3A O2 analyzer and a Sensor Medics LB-2 C02 detector, respectively, and expired volume was determined with a Parkinson-Cowan volumeter. The analyzers were calibrated with known gas concentrations, previously determined by Scholander9s microtechnique and the volumeter was calibrated with a Tissot spirometer. Each blood sample was separated into two aliquots: 5 mL was transferred into a sodium heparinizd Vacutainer tube for plasma and

Statistics All data are means f SEM, unless otherwise noted. The data were dependent over time and because each subject exercised for a different duration, there were unequal sample sizes within and between treatments. As a result, the presented means for the LC treatment are rz = 6 until 60 min and for the last time point, n = 2. The means presented for the M treatment are pm = 6 until 90 min and for the remaining three time points, PZ = 4, 3, and 1, respectively. The means presented for the HC treatment are n = 6 subjects until 128 min and for the remaining four time points, n = 5, 4, 2, and 2, respectively. These differences in the number of data points within and between trials cause some of the presented graphs to be skewed toward the end of the trials. The corresponding SEM associated with these last time points may also be large. To overcome this problem a summary statistic of the data over time was obtained using multiple regression analysis. This was accomplished using the persona1 computer version of the SAS software package. The data were fitted with either a cubic, quadratic, or linear tern depending on if that t e r n was present or significantly different between treatments. When the line fitting was complete (either linear, quadratic, or cubic) the corresponding coefficients were tested for treatment effects using a randomized complete block design analysis of variance. If significant treatment effects were indicated, the least squares means were compared by using protected least significant differences to determine where the significant differences a m n g the slopes and intercepts occurred (i.e., among the LC, M, and HC treatments). The strengths of this statistical approach are that it overcomes the difficulty of unequal sample sizes within and between treatments. It brings all the desired parameters together in one test and allows the analysis of these parameters over time. 'This procedure is a powerfaal test for significant trends and allows the discussion of treatment effects over time (Hslbert et a%.1990). Significance was accepted at p < 0.05.

Results Diet T h e total estimated energy content of the actual LC diet was significantly lower than both the M and H@ diets, but there were n o significant differences in the total energy content between the M and HG diets (Table 1). The estimated protein, fat, and carbohydrate content s f the three diets were dl significantly different. T h e BCAA content of the three diets were dl significantly different from each other (Table 2).

~er$ormnceand cardiorsspirarkory data There were significant differences in exercise times among all three treatments with means sf58.8 f 3.7, 112.1 d 7.3,

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TABLE1 . Estimated total energy content and the percentage of energy from the different constituents for the LC, M, and WC diets Diet

LC M

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HC

Total energy (U/day)

Protein (%,

9113f1B43a B6476&2072b 13394f712"

Fat (%)

32f2" Hi-2" 18f2-6f2' 12f l c 22f3c

Carbohydrate

120 AMMONIA

100 88

41 1" 4 9 f 3' 6 6 f 3"

NOTE: All values are means f SEM, n = 6. LC, low carbohydrate; M, mixed; HC, high carbohydrate diet. Within a column, significant ( p < 0.05) differencesare denoted by a different letter.

60

4Q 20

8 0

15

30

48

80

90 $85 120 135 150 165 180

75

TIME (MlN)

TABLE2. The BCAA content of the LC, M, and HC diets Diet

Valine (g/day)

Issleaacine (dday)

Leucine @/day)

Total BCAA @/day)

UREA NITROGEN

(mM)

NOTE:Ail values are means & SEM, PL = 6 . Abbreviations as in Table I . Within a column, significant ( p < 0.05) differences are denoted by a different letter.

and 152.9 f 10.3 min for the LC, M, and HC treatments, respectively. There were no significant pifferences in heart rate between treatments. The percent Vo2max for the LC treatment (78 & 3%) was significantly greater than those of the M (73 + 1%) and HC (70 i 1%) treatments, but there were no other significant differences. The respiratory exchange ratio (RER) for the LC treatment (0.85 f 0.01) was significantly lower than those of the M (0.96 f 0.01) and HC (0.97 f 0,02) treatments throughout the experiment. Blood metabolites There were no significant differences in the venous plasma NH3 at rest across all treatments (29 4, 28 f 3, and 28 f 3 pM for the LC, M. and HC treatments, respectively). The plasma concentration of NH3 rose significantly faster from rest for the LC treatment and remained significantly higher throughout exercise as compared with the M and HC treatments (Fig. 1). The resting semm urea nitrogen concentrations were all significantly different from each other (9.1 8.9, 6.5 f 0.5, and 5.5 0.7 mM for the LC, M, and HC treatments, respectively) and remained significantly different throughout exercise (Fig. 1). The subjects demonstrated a significantly lower resting plasma glucose concentration and a significantly greater decrease in glucose during exercise for the LC as compared with the M and HC treatments (Fig. 2). Similarly, the subjects had a significantly lower resting plasma lactate concentration for the LC treatment as compared with the M and HC treatments. Plasma lactate concentration increased in all conditions during exercise but remained significantly lower during the LC trial (Fig. 2). The resting semm FFA and glycerol concentrations were significantly higher for the LC as compared with the M and HG treatments (Fig. 2). The LC treatment produced a significantly greater increase in FFA and glycerol during exercise as compared with the other treatments. There were no significant differences between the M and HC treatments at rest or during exercise. P l a s m amins acids There were no significant differences in the total plasma amino acid concentration at rest or during exercise across all

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FIG. 1 . All values are means f SEM. Plasma ammonia (NH,) and serum urea nitrogen responses during exercise for the low carbohydrate (LC), mixed (M), and high carbohydrate (He)diets. (A) Plasma NH, continually increased throughout exercise and was significantly ( p < 0.05) greater for the LC as compared with the M and HC treatments. (B) Resting serum urea nitrogen concentrations were all significantly different from each other and remained significantly different throughout exercise.

treatments (Fig. 3A). The LC treatment manifested a higher resting EAA (1295 k 88 pM) as compared with both the M (884 f 72 pM) and HC (794 36 $eM) treatments (Fig. 3B). There was also a significantly greater decrease in the EAA concentration during exercise for the LC as compared with the M and HC treatments. However, there were no significant differences in the EAA minus the BCAA (EAA -BCAA) concentrations at rest or during exercise across all treatments (Pig. 3C). The LC treatment produced significantly higher resting plasma valine and isoleucine concentrations as compared with the M and HC treatments (Fig. 4). The resting valine csncentrations were 498 39,265 i 22, and 209 f 10 pM and the resting isoleucine concentrations were 157 f 11, 1838 f 19, and 81 8 pM for the LC, M, and HC treatments, respectively. The LC treatment also produced a significant decrease in the plasma valine and isoleucine concentrations during exercise as compared with no change for the M and HC treatments. There were no significant differences in the plasma valine and isoleucine concentrations at rest or during exercise between the M and HC treatments. Leucine was the only BCAA to be significantly different between d l treatments at rest: 252 + 17, 147 i-14, and 123 12 pM for the LC, M, and HC treatments, respectively (Fig. 4). The LC treatment produced a significant decrease in the plasma leucine concentration during

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MACLEAN ET AL. LACTATE (mM)

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FIG. 2. All values are means f SEM. Plasma glucose and lactate and serum FFA and glycerol responses during exercise are shown for the low carbohydrate (LC), mixed (M), and high carbohydrate (HC) diets. (A)The LC treatment had a significantly ( p < 0.05)Bower resting glucose concentration and a significantly greater decrease in glucose during exercise as compared with the M and HC treatments. (B) The LC treatment had a significantly lower resting lactate concentration and it remained significantly lower throughout exercise as compared with the other two treatments. (C and D) The resting serum FFA and glycere~lconcentrations were significantly higher for the LC treatment and rose significantly faster during exercise as compared with the M and HC treatments.

exercise as compared with no change for the M and He treatments. The M and HC treatment remained significantly different throughout the experiment. The resting plasma alanine concentration was significantly higher for the HC treatment $490 26 pM) as compared with the M (406 f 42 pM) and LC (352 37 pM) treatments. The response of danine was similar in nature for all three diets suggesting that the balance between the rate of accumulation and removal of danine, as reflected by its plasma concentration, was similar for all three treatments. There were no significant differences across all treatments in the plasma glutamate or glutamine concentrations at rest or during exercise. The following m i n o acids did not exhibit any significant differences at rest or during exercise across all treatments: aspartate, glycine, histidine, arginine, tyrosine, ornithine, and hydroxyproline (data not shown).

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There are very little data available on plasma or muscle amino acids during prolonged, moderate exercise in humans. This study is one of the first to examine amino acid and NH3 responses every 15 min (up to 3 h) in humans and the possible impact altered dietary intakes may have on these metabolites during prolonged, submaximal exercise. Similarly, this is one sf the first studies to suggest that BCAA may be a source of NH3 in muscle during this type of exercise, and that their

contributisn to NH3 formation may be enhanced when carbohydrates are limited. It was found that the resting plasma BCAA concentration was significantly higher for the LC treatment and was significantly decreased during exercise. Meanwhile, there was a significantly greater rise in the plasma NH3 concentration during exercise for the LC treatment as compared with when exercise was performed after ingestion of a M or HC diet for 3 days. Although we are limited to only plasma concentrations, these findings strongly suggest that BCAA contribute to NH3 formation. The diet manipulation in this study followed the traditional method used to alter initial muscle glycogen (Hultman sand Spriet 1988). Each subject underwent a muscle glycogen depletion exercise regime followed by ingestion of a LC, M, or HC diet for several days. Because muscle biopsies were not taken it is impossible to conclude that initial muscle glycogen was significantly altered. However, the performance, blood, and respiratory data all support the premise that muscle glycogen was altered in the desired direction. For example, manipulation sf initial muscle glycogen prior to submaximal exercise has been shown to affect endurance performance (Bergstrsm et al. 1967). In the present study the exercise times and RER were increased for the HC treatment and decreased for the LC treatment. The LC treatment produced lower resting plasma glucose and lactate concentrations while manifesting higher resting FFA and glycerol concentrations. The converse was found for the HC treatment

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TOTAL AMINO ACIDS (pM) 5000

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Time (min)

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FIG. 3. All values are means f SEM. Plasma total. total essential (EAA), and total essential minus branched chain amino acid (EAA-BCM) responses during exercise are shown for the low carbohydrate (LC), mixed (M), and high carbohydrate (HC) diets. (A) There were no significant differences in the plasma total amino acids between treatments; the %EMwere small and omitted for clarity. (B)The LC treatment had a significantly ( p < 0.05) higher resting EAA concentration, which was significantly decreased during exercise as compared with that of the M and HC treatments. ( C ) There were no significant differences in the EAA -BCAA concentrations between treatments; the SEM were small and omitted for clarity.

FIG.4. All values are means f SEM. Plasma valine, isoleucine, and lelrcine responses during exercise are shown for the low carbohydrate (LC), mixed (M), and high carbohydrate (HC) diets. (A and B) The resting plasma valine and isoleucine concentrations were significantly ( p e: 0.05) higher for the LC treatment atad were significantly decreased during exercise as compared with the M and PIC treatments. (6)The resting plasma leucine concentrations were all significantly different at rest. The M and HC treatments remained unchanged and significantly different from each other during exercise, while the plasma leucine concentration was significantly decreased during exercise for the LC treatment as compared with the other two treatments.

and the results are in strong agreement with earlier findings (Bergstrorn et al. 1967). These data support the assumption that resting muscle glycogen was decreased for the LC trial

manipdate total energy, carbohydrate availability, and (or) muscle glycogen differ not only in the amount of carbohydrate they contain but also in the amount of fat and protein. It is therefore impossible to attribute a11 the observed differences ts only cal-bohydrates. In this study the amaunts s f carbohydrate,

and increased for the HC trial. The diets traditionally used in this and sther studies to

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fat, and protein were d l significantly different between diets as was the BCAA profile sf the ingested protein. It is well established that ingestion of different protein contents will alter plasma BCAA levels (Harper et al, 1984). As a result, there were significantly more BCAA available in the circulating blood prior to exercise for the LC treatment as compared with the M and HC treatments. The present study d s s showed that the increased BCAAs were removed from the plasma during exercise in conjunction with a greater rate of NH3 increase for the LC trid as compared with the M and HC trials. However, it must be noted that this trid was d s o associated with a decreased carbohydrak and increased FFA availability. Another effect of the altered dietary intakes was a significantly higher resting FFA concentration and a significantly greater increase in FFAs during exercise for the LC treatment as c o m p a ~ dwith the other two treatments. It has been demonstrated that increased octanoate availability increases vdine oxidation (Spydevold 1979). Therefore, the increased FFA concentrations observed during the LC trid may have increased the capacity ofthe muscle to metabolize BCAA. Similarly, the tot& energy content of the diets consumed has been shown to influence m i n o acid oxidation, such that when dietary energy is low, amino acid metabolism is increased (Munro 1951). The LC diet had a significantly lower energy content t h the M and HC diets. This may have influenced the rate of BCAA utilization in muscle causing increased BCAA utilization. The resting and exercise plasma NH3 levels in all trials are consistent with results reported by other researchers (Broberg and SAlin 1989; Machan et al. 1991; Wagenmakers et al. 1991). Broberg and Sahlin (1989) demonstrated that muscle continually releases NH3 during exercise and that the rate of release increases as exercise progresses. The progressively increasing plasma NH3 concentrations in this study are consistent with this finding m d the greater NH3 levels during the LC trial strongly suggest an even greater rate of NH3 release than in the other trials. The only investigators to examine the effects of alkred muscle glycogen and NH3 responses are Broberg and Sahlin (1989). They demonstrated h a t significantly more NM3 was formed during exercise when initial muscle glycogen was limited and concluded this was exclusively due to the reactions s f the PNC. It should be noted that the authors ordered their trials such that the second, or glycogen-depleted bout, followed a previous bout of exercise to e f i a u s ~ o n .Recently Graham et d. (1991) repn%ed NH3 fluxes during one leg extensor exercise at 80% Vo2max that are similar to those of Broberg and SaHin (1989), despite abundant muscle glycogen. Therefore, it is quite possible that the PNC may be o d y a partial contributor to NH3 production during prolonged submaximal exercise. The PNC plays an important role in maintaining the energy state of the cell when the rate of ATP synthesis cannot match the rate of ATP utilization. This occurs predominantly during intense, exhaustive exercise. The AMP deminase enzyme is activated by imacreased levels of free ADP and AMP and thus IMP and NH3 are formed. By activating AMP deaminase the adenylate base reaction QADP ++ ATP + AMP) can continue to produce ATB and help maintain a high ATPIABP ratio weeded predominantly during intense exercise. On the other hand, AMP deaminase is effectively inhibited by normal concentrations of ATP and GTB, but activated by increased concentrations of free ADP and AMP (Goodman and Lowenstein 1977;Wheeler and Lowenstein 1979). Although

we are limited to total rather than free concentrations, previqus human studies during exercise at approximately 70% Vo2max have demonstrated no significant changes in the muscle content of ATP, ADP, and IMP after 45 min or in ATP, ADP, AMP, and ATPIADP ratio at 60 min, even in glycogendepleted fibers (MacEean et al. 1991; Norman et al. 198'7, 1988). Therefore, it is unlikely that these modulators would play an important role in activating AMP deaminase until the muscle approaches exhaustion. When AMP deaminase has been shown to be active in human skeletd muscle, such as at exhaustion, its activity was modest and twofold higher in fast twitch (HT) as compared with slow twitch (ST) muscle fibers (Norman et d. 1988; Sahlin et. d. 1989). Because a submaximal exercise bout of 75% Vo2max recruits primarily ST muscle fibers (Gsllnick et al. 1974) (at least during the majority of the bsut) and the modulators of AMP deaminase are not significantly altered, the activity of AMP deaminase should be minimal. Therefore, caution should be taken when implying that the PNC is the only contributor to NH3 formation during prolonged, submaximd exercise such as that of the present study. Another factor that must be addressed is that the % h 2 m a x was significantly greater, despite the same power output, for the LC trial as compared with the M and HC trials. In the glycogen-depleted state this may have resulted in a greater proportion sf FT fibers being recruited and an increased amount of NH3 produced from these fibers. However, the data clearly show that there is a substantial difference in NM3 concentrations among treatments by 15 min. It is unlikely that the ptential difference in the fiber recruitment pattern for the LC treatment played a significant role early in the exercise bsut. It is possible that this difference may be more important as exhaustion approaches, but by then significant differences in NH3 levels have dready been established. It has been demonstrated that amino acid oxidation increases in response to prolonged exercise and including gluconeogenesis may contribute up to 18% of the energy required for sustained exercise (Brooks 1987; Dohm 1986; Hagg et al. 1982; Rennie et al. 1881; Wolfe et d. 1982). As a result, amino acid metabolism may contribute more to NH3 formation as exercise continues, explaining the progressively increasing NH3 concentration observed in this and other studies (Broberg and Sahlin 1989; Graham et al. 1981; M a c k m et d. 1991; Wagenmakers et al. 1991) and this contribution may change depending on the availability of other carbon substrates (Lemon and Mullin 1980). During prolonged exercise there is a net brmkdown of whole body protein accomplished by a decrease in the rate of protein synthesis a d an increase in the rate of protein degradation in the liver (Dohm 1986) and a net increase in the rate of noncontractile protein degradation in active muscle (Kasperek and Snider 1989). The produced amino acids can then be metabolized. In the present study, the plasma EAA csncentration was significantly greater for the LC treatment than the other two treatments. When the BCAA concentations were sa~btractedfrom the EAA (EAA-BCAA) there were no significant differences between treatments. This indicates that the diet effect was very specific, because changes in the EBB pool were a function of changes in the BCAA. This m u r r e d because the LC diet had a significantly higher BCAA content than the M and HC diets and the BCAA are h s w n to selectively escape uptake by the liver (Whren et al. 1976). Furthermore, neither the totd nor the EAA-BCAA concen-

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trations were significantly dtered during exercise for d l treatments. This suggests that the degree s f endogenous protein breakdown was not significantly different among the three treatments. The unchanging serum urea concentrations during exercise for the three trials is also consistent with this. The BCAA are the m i n o acids of choice for oxidation in skeletal muscle (Dohm 1986) and originate from both diet and endogenous protein breakdown. During prolonged exercise there is an increase in the uptake and oxidation sf B C M by exercising leg tissues and a significant output of BCAA from the liver (Ahllborg et d. 1974). Furthermore, Gelfand et d. (11986) reported that after infusion of an amino acid load in humans, resting skeletal muscle was responsible for the removal of 65 -70 % of the BCAA load. The present study and previous work have demonstrated that the BCAA do not accumulate in the plasma or muscle (Bergstrom et d. 1985; MacLean et d.1991) during exercise, despite protein catabolism. In the present study the LC trid was characterized by a significantly higher resting plasm BCAA concentration and a significantly greater decrease in these m i n o acids during exercise. It is reasonable to assume that they were taken up and metabolized by active muscle. The first step in the degradation of BCAA in muscle involves the liberation of NW?by trmsdeamination catalyzed by aminotransferase and GDH: ~t has been suggested that- skeletal muscle GDH activity is insufficient to produce free NH3 (Broberg and Sahlin 1989). Recently, Wibom and Hultman (1990) reported the first human GDH activities for sedentary and trained individuals. Their data indicate that even sedentary individuds had a GDH activity severdfold greater than that required to produce all the NH; during a prolonged, submaxim d exercise bout. Furthermore, trained subjects had almost twice the activity of the sedentary subjects and thus, skeletal muscle has the ability to produce free NH3. The rate-limiting step in BCAA oxidation in muscle is the dmarbsxylatisn of the bmched chain keto acids by branched chain keto acid dehydrogesaase (BCUDH). The BCWDH complex is dmost totally in the inactive form in resting skeletal muscle, while dmost totally in the active form in liver (Kasprek et d. 1985; Wagenmakers et d. 8984). It has been shown that exercise greatly increases the activity of the B C M D H complex in skeletal muscle [Kasprek et d - 1985). Furthermore, Wagenmakers et al. (11991) reported h a t the B C U D H complex was activated to a significantly greater degree during exercise when muscle glycogen was depleted. It is evident that muscle can change its capacity to metabolize BCAA during exercise and this capacity can be influenced by the availability of other energy substrates. In the present study the LC trial was characterized by a significantly greater rate sf increase in plasm NH3. Wagenmakers et d. (1990) demonstrated that administration of BCAA to a McArdle9spatient prior to exercise resulted in significantly greater NH3 concentrations. Similarly, Lemon and Mullin (1980) showed that significantly more urea was formed during exercise when cahohydrate depleted &an when carbohydrate loaded and conclude that protein is utilized during exercise to a greater extent than is generally assumed. Furthermore, Goldberg and Odessey (1972) demonstrated that BCAA oxidation was greater in diaphragms from fasted than fed rats. Therefore, the greater plasma NH3 concentrations observed when carbohydrate availability was limited and B C M concentrations were high may reflect augmented B C M deamination and (or) metabolism. As noted earlier the dietary treatments

may have d t e r d the NH3 responses by changing carbohydrate and (or) BCAA availability- 'This study can not resolve which factor was most important, but does clearly demonstrate that NH3 metabolism is sensitive to diet and is associated with increased BCAA availability. In summary, this study clearly demonstrates that altering dietary intake affects the BCAA profile of the plasma prior to exercise and d s o amino a ~ i dand NH3 responses during prolonged exercise at 75% Vo2rnax. When carbohydrate availability was limited, and BCAA were abundant, a significantly greater increase in plasma NH3 levels was observed during exercise. PNC activity should be minimal or constant and produce little NH3 during submaximal exercise. Therefore, it was postulated that a significant component of the NH3 produced during prolonged exercise may come from BCAA. When the BCAA were available and carbohydrate availability was limited, their uptake, deamination, and subsequent metabolism may be enhanced, resulting in significantly greater plasma NH3 concentrations. Lastly, additional research is needed to determine whether decreased carbohydrate availability, increased BCAA availability, or both are important in stimulating increased NH3 formation.

Acknowledgements The authors thank Prernila Sathasivam for her excellent technical assistmce and the Department of Nutrition, University of Gael@ for access to their HPLC. This work was supported by operating grants (A6466 and A2320) from the Natural Science and Engineering Research Council of Cmada. Ahlborg, G., Hellg, P., Hagenfeldt, L., HendIer, W., and Wahren, J. 1974. Substrate turnover during prolonged exercise In man. J. Clin. Invest. 53: 1088-1090. Bergmeyer, H. U. 1974. Methods of enzymatic analysis. 2nd d. Academic Press, New York. Bergstrom, J., Hermansen, L., Hultman, E., and Saltin, B. 1967. Diet, muscle glycogen and physical perfomasace. Acta Phy siol. Scand. 71: 148- 150. Bergstrom, b., Furst, P., and Hulman, E. 1985. Free amino acids in muscle tissue and plasma during exercise in man. Clin. Physiol. 5: 155-160. Boobis, E. H.,and Maughan, W. J. 1983. A simple one-step enzymatic flaorometriic method for the determination sf glycerol in 20 pL of plasma. Clin. Chem. Acta, 132: 173- 179. Wroberg, S., and Shlin, K. 1989. Adenine nucleotide degradation in human skeletal muscle during prolonged exercise. J. Appl. Physiol. 67: 116 - 122. Brooks, G . A. 1987. Amino acid and protein metabolism during exercise and recovery. Med. Sci. Sports Exercise, 19: S150 S156. Bibblee, L., a d Graham, %. E. 1983. A longitudinal study s f changes in aerobic fitness, body composition and energy intake in primigravid patients. Am. J. Bbstet. Gynecol. 144: 908 -914. Dshm, G . E. 1986. Protein as a fuel for endurance exercise. Exercise Sport Sci. Rev. 14: 143- 173. Gelfand, R. A., Glicbaan, M. G., Jacob, R., Sherwin, R. S., and DeFronzo, R. A, 1986. Removal of infused amino acids by splanchnic and leg tissues in humans. Am. I. Physiol. 250: WQ7-M13. Goldberg, A. L., and Odessey, R. 1972. Oxidation of amins acids by diaphragms from fed and fasted rats. Am. J. Physisl. 223: 1384-1391. Gollnick, P., PieHal, K., and Saltin, B. 1974. Selective glycogen depletion pattern in human muscle fibers after exercise of varying intensity and at varying pedal rates. J. Physiol. (London), 241: 45-58.

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