A 7-day oral supplementation with branched-chain amino acids was ineffective to prevent muscle damage during a marathon

The aim of this study was to determine the effectiveness of a 7-day oral supplementation with branched-chain amino acids (BCAA) to prevent muscle damage during a marathon. Forty-six experienced runners were randomly divided into two groups, one with BCAA supplementation (n = 25, supplemented with 5 g day^?1 of powdered 1:0.5:0.5 leucine:isoleucine:valine, during the 7 days prior to the competition) and the other as a control group (n = 21, supplemented with an isocaloric placebo). Before the marathon race and within 3 min of finishing, leg muscle power was measured with a maximal countermovement jump and a urine sample was obtained. During the race, running pace was measured by means of a time-chip. Myoglobin concentration was determined in the urine samples as an indirect marker of muscle damage. A visual analog scale (0–10 points) was used to assess leg muscle pain during the race. In the BCAA group, the mean running pace during the marathon was similar to the control group (3.3 ± 0.4 vs. 3.3 ± 0.5 m s^?1, respectively, 0.98). The pre- to post-race reduction in muscle power was similar in both BCAA and control groups (?23.0 ± 16.1 vs. ?17.3 ± 13.8 %, P = 0.13). Post-race urine myoglobin concentration was similar in both BCAA and control groups (5.4 ± 7.5 vs. 4.5 ± 8.6 μg mL^?1, P = 0.70). Finally, there were no differences between groups in the perceived muscle pain during the race (6 ± 1 vs. 5 ± 1 points, P = 0.80). A 7-day supplementation of BCAA (5 g day^?1) did not increase the running performance during a marathon. Furthermore, BCAA supplementation was ineffective to prevent muscle power loss, muscle damage or perceived muscle pain during a marathon race.     

Amino acid sequence of Salmonella typhimurium branched-chain amino acid aminotransferase

The complete amino acid sequence of the subunit of branched-chain amino acid aminotransferase (transaminase B, EC 2.6.1.42) of Salmonella typhimurium was determined. An Escherichia coli recombinant containing the ilvGEDAY gene cluster of Salmonella was used as the source of the hexameric enzyme. The peptide fragments used for sequencing were generated by treatment with trypsin, Staphylococcus aureus V8 protease, endoproteinase Lys-C, and cyanogen bromide. The enzyme subunit contains 308 residues and has a molecular weight of 33,920. To determine the coenzyme-binding site, the pyridoxal 5-phosphate containing enzyme was treated with tritiated sodium borohydride prior to trypsin digestion. Peptide map comparisons with an apoenzyme tryptic digest and monitoring radioactivity incorporation allowed identification of the pyridoxylated peptide, which was then isolated and sequenced. The coenzyme-binding site is the lysyl residue at position 159. The amino acid sequence of Salmonella transaminase B is 97.4% identical with that of Escherichia coli, differing in only eight amino acid positions. Sequence comparisons of transaminase B to other known aminotransferase sequences revealed limited sequence similarity (24-33%) when conserved amino acid substitutions are allowed and alignments were forced to occur on the coenzyme-binding site.     

Effects of liver failure on the enzymes in the branched-chain amino acid catabolic pathway

Branched-chain alpha-keto acid dehydrogenase (BCKDH) complex catalyzes the committed step of the catabolism of branched-chain amino acids (BCAA). The liver cirrhosis chemically induced in rats raised the activity of hepatic BCKDH complex and decreased plasma BCAA and branched-chain alpha-keto acid concentrations, suggesting that the BCAA requirement is increased in liver cirrhosis. Since the effects of liver cirrhosis on the BCKDH complex in human liver are different from those in rat liver, further studies are needed to clarify the differences between rats and humans. In the valine catabolic pathway, crotonase and beta-hydroxyisobutyryl-CoA hydrolase are very important to regulate the toxic concentration of mitochondrial methacrylyl-CoA, which occurs in the middle part of valine pathway and highly reacts with free thiol compounds. Both enzyme activities in human and rat livers are very high compared to that of BCKDH complex. It has been found that both enzyme activities in human livers were significantly reduced by liver cirrhosis and hepatocellular carcinoma, suggesting a decrease in the capability to dispose methacrylyl-CoA. The findings described here suggest that alterations in hepatic enzyme activities in the BCAA catabolism are associated with liver failure.     

Effects of different branched-chain amino acids supplementation protocols on the inflammatory response of LPS-stimulated RAW 264.7 macrophages

Although branched-chain amino acids (BCAA) are commonly used as a strategy to recover nutritional status of critically ill patients, recent findings on their role as immunonutrients have been associated with unfavorable outcomes, especially in obese patients. The present study aimed to explore the effects of different BCAA supplementation protocols in the inflammatory response of LPS-stimulated RAW 264.7 macrophages. Cell cultures were divided into five groups, with and without BCAA supplementation, (2 mmol/L of each amino acid). Then, cell cultures followed three different treatment protocols, consisting of a pretreatment (PT), an acute treatment (AT), and a chronic treatment (CT) with BCAA and LPS stimulation (1 µg/mL). Cell viability was analyzed by MTT assay, NO production was assessed by the Griess reaction and IL-6, IL-10, TNF-α and PGE2 synthesis, was evaluated by ELISA. BCAA significantly increased cell viability in AT and CT protocols, and NO and IL-10 synthesis in all treatment protocols. IL-6 synthesis was only increased in PT and CT protocols. TNF-α and PGE2 synthesis were not altered in any of the protocols and groups. BCAA supplementation was able to increase both pro and anti-inflammatory mediators synthesis by RAW 264.7 macrophages, which was influenced by the protocol applied. Moreover, these parameters were significantly increased by isoleucine supplementation, highlighting a potential research field for future studies.

     

Clofibric acid stimulates branched-chain amino acid catabolism by three mechanisms

Prolonged activation of the c-Jun N-terminal kinase (JNK) has been suggested as a signal for apoptosis in response to a wide variety of stimuli. Using three cytocidal RNA or protein synthesis inhibitors (actinomycin D, anisomycin, and emetine), the potential role of JNK in activation of the mitochondrial apoptotic cascade was investigated in A549-S cells. Protein synthesis inhibition per se was not the cause of cell death as cycloheximide induced only growth arrest. All the cytocidal inhibitors induced cytochrome c release and caspases 9 activation within hours, but only anisomycin caused persistent JNK activation. Although, the JNK inhibitor, SP600125, inhibited JNK-dependent anisomycin-induced c-Jun phosphorylation, it was ineffective in preventing anisomycin-induced caspase activation and cell death. Thus, all three lethal macromolecule synthesis inhibitors can activate the mitochondrial apoptotic machinery independent of JNK activation, demonstrating that the mitochondrial apoptotic pathway can be activated independently of the JNK pathway in the absence of protein synthesis.     

Effect of chronic supplementation with branched-chain amino acids on the performance and hepatic and muscle glycogen content in trained rats

The objective of this study was to evaluate the effects of a diet supplemented with branched-chain amino acids (BCAA; 3.57% and 4.76%) on the performance and glycogen metabolism of trained rats. Thirty-six adult male Wistar rats received the control diet (AIN-93M) (n=12) and two diets supplemented with BCAA (S1: AIN-93M+3.57% BCAA, n=12, and S2: AIN-93M+4.76% BCAA, n=12) for 6 weeks. The training protocol consisted of bouts of swimming exercise (60 min day(-1)) for 6 weeks at intensities close to the lactate threshold. On the last day of the experiment, all groups were trained for 1 h (1H) or were submitted to the exhaustion test (EX). The time to exhaustion did not differ between groups. The groups submitted to the exhaustion test presented a reduction in plasma glucose and an increase in plasma ammonia and blood lactate concentrations compared to the 1H condition. In the 1H condition, hepatic glycogen concentration was significantly higher in group S2 compared to the control diet and S1 groups (132% and 44%, respectively). Group S2 in the 1H condition presented a higher muscle glycogen concentration (45%) compared to the control diet group. In the EX condition, a significantly higher hepatic glycogen concentration was observed for group S2 compared to the control diet and S1 groups (262% and 222%, respectively). Chronic supplementation with BCAA promoted a higher hepatic and muscle glycogen concentration in trained animals, with this effect being dose dependent.     

Regulation of branched-chain amino acid transport in Escherichia coli.

The repression and derepression of leucine, isoleucine, and valine transport in Escherichia coli K-12 was examined by using strains auxotrophic for leucine, isoleucine, valine, and methionine. In experiments designed to limit each of these amino acids separately, we demonstrate that leucine limitation alone derepressed the leucine-binding protein, the high-affinity branched-chain amino acid transport system (LIV-I), and the membrane-bound, low-affinity system (LIV-II). This regulation did not seem to involve inactivation of transport components, but represented an increase in the differential rate of synthesis of transport components relative to total cellular proteins. The apparent regulation of transport by isoleucine, valine, and methionine reported elsewhere was shown to require an intact leucine, biosynthetic operon and to result from changes in the level of leucine biosynthetic enzymes. A functional leucyl-transfer ribonucleic acid synthetase was also required for repression of transport. Transport regulation was shown to be essentially independent of ilvA or its gene product, threonine deaminase. The central role of leucine or its derivatives in cellular metabolism in general is discussed.     

Isolation of mutants lacking branched-chain amino acid transaminase.

Variants of the Chinese hamster ovary cell have been isolated which can no longer grow when valine, leucine, or isoleucine is replaced in the culture medium by its respective alpha-keto acid: alpha-ketoisovaleric acid, alpha-ketoisocaproic acid, or alpha-keto-beta-methylvaleric acid. These variants lack branched-chain amino acid transaminase activity. Evidence is presented indicating these variants to be single gene mutants. Genetic evidence is also presented confirming previous biochemical evidence that a single enzyme carries out transaminase functions on valine, leucine, and isoleucine. The branched-chain transaminase-deficient (trans-) mutants can be reverted to wild-type behavior by treatment with mutagenic agents. These mutants promise to be useful in exploring regulatory mechanisms in biochemical, genetic, and cancer research.     

Mechanisms responsible for regulation of branched-chain amino acid catabolism

The branched-chain amino acids (BCAAs) are essential amino acids and therefore must be continuously available for protein synthesis. However, BCAAs are toxic at high concentrations as evidenced by maple syrup urine disease (MSUD), which explains why animals have such an efficient oxidative mechanism for their disposal. Nevertheless, it is clear that leucine is special among the BCAAs. Leucine promotes global protein synthesis by signaling an increase in translation, promotes insulin release, and inhibits autophagic protein degradation. However, leucine's effects are self-limiting because leucine promotes its own disposal by an oxidative pathway, thereby terminating its positive effects on body protein accretion. A strong case can therefore be made that the proper leucine concentration in the various compartments of the body is critically important for maintaining body protein levels beyond simply the need of this essential amino acid for protein synthesis. The goal of the work of this laboratory is to establish the importance of regulation of the branched chain alpha-ketoacid dehydrogenase complex (BCKDC) to growth and maintenance of body protein. We hypothesize that proper regulation of the activity state of BCKDC by way of its kinase (BDK) and its phosphatase (BDP) is critically important for body growth, tissue repair, and maintenance of body protein. We believe that growth and protection of body protein during illness and stress will be improved by therapeutic control of BCKDC activity. We also believe that it is possible that the negative effects of some drugs (PPAR alpha ligands) and dietary supplements (medium chain fatty acids) on growth and body protein maintenance can be countered by therapeutic control of BCDKC activity.     

Properties of bacterial and archaeal branched-chain amino acid aminotransferases

Branched-chain amino acid aminotransferases (BCATs) catalyze reversible stereoselective transamination of branched-chain amino acids (BCAAs) L-leucine, L-isoleucine, and L-valine. BCATs are the key enzymes of BCAA metab- olism in all organisms. The catalysis proceeds through the ping-pong mechanism with the assistance of the cofactor pyri- doxal 5′-phosphate (PLP). BCATs differ from other (S)-selective transaminases (TAs) in 3D-structure and organization of the PLP-binding domain. Unlike other (S)-selective TAs, BCATs belong to the PLP fold type IV and are characterized by the proton transfer on the re-face of PLP, in contrast to the si-specificity of proton transfer in fold type I (S)-selective TAs. Moreover, BCATs are the only (S)-selective enzymes within fold type IV TAs. Dual substrate recognition in BCATs is imple- mented via the “lock and key” mechanism without side-chain rearrangements of the active site residues. Another feature of the active site organization in BCATs is the binding of the substrate α-COOH group on the P-side of the active site near the PLP phosphate group. Close localization of two charged groups seems to increase the effectiveness of external aldimine for- mation in BCAT catalysis. In this review, the structure-function features and the substrate specificity of bacterial and archaeal BCATs are analyzed. These BCATs differ from eukaryotic ones in the wide substrate specificity, optimal tempera- ture, and reactivity toward pyruvate as the second substrate. The prospects of biotechnological application of BCATs in stereoselective synthesis are discussed.     

A novel branched-chain amino acid metabolon. Protein-protein interactions in a supramolecular complex

The catabolic pathways of branched-chain amino acids have two common steps. The first step is deamination catalyzed by the vitamin B(6)-dependent branched-chain aminotransferase isozymes (BCATs) to produce branched-chain alpha-keto acids (BCKAs). The second step is oxidative decarboxylation of the BCKAs mediated by the branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKD complex). The BCKD complex is organized around a cubic core consisting of 24 lipoate-bearing dihydrolipoyl transacylase (E2) subunits, associated with the branched-chain alpha-keto acid decarboxylase/dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3), BCKD kinase, and BCKD phosphatase. In this study, we provide evidence that human mitochondrial BCAT (hBCATm) associates with the E1 decarboxylase component of the rat or human BCKD complex with a K(D) of 2.8 microM. NADH dissociates the complex. The E2 and E3 components do not interact with hBCATm. In the presence of hBCATm, k(cat) values for E1-catalyzed decarboxylation of the BCKAs are enhanced 12-fold. Mutations of hBCATm proteins in the catalytically important CXXC center or E1 proteins in the phosphorylation loop residues prevent complex formation, indicating that these regions are important for the interaction between hBCATm and E1. Our results provide evidence for substrate channeling between hBCATm and BCKD complex and formation of a metabolic unit (termed branched-chain amino acid metabolon) that can be influenced by the redox state in mitochondria.     

Single amino acid supplementation in aminoacidopathies: a systematic review

Aminoacidopathies are a group of rare and diverse disorders, caused by the deficiency of an enzyme or transporter involved in amino acid metabolism. For most aminoacidopathies, dietary management is the mainstay of treatment. Such treatment includes severe natural protein restriction, combined with protein substitution with all amino acids except the amino acids prior to the metabolic block and enriched with the amino acid that has become essential by the enzymatic defect. For some aminoacidopathies, supplementation of one or two amino acids, that have not become essential by the enzymatic defect, has been suggested. This so-called single amino acid supplementation can serve different treatment objectives, but evidence is limited. The aim of the present article is to provide a systematic review on the reasons for applications of single amino acid supplementation in aminoacidopathies treated with natural protein restriction and synthetic amino acid mixtures.     

Effects of branched-chain amino acids on plasma amino acids in amyotrophic lateral sclerosis

Summary Although the cause of amyotrophic lateral sclerosis (ALS) remains unknown, biological findings suggest that the excitatory amino acid glutamate contributes to the pathogenesis of ALS. In previous studies of ALS, the therapeutic effect of the branched-chain amino acids (BCAAs) leucine, valine and isoleucine has been evaluated. The present study aimed at investigating the acute effect of BCAAs on plasma glutamate levels in ALS patients. Following two oral doses of BCAAs, significantly increased plasma levels were seen for valine (500%), isoleucine (1,377%) and leucine (927%), however the plasma level of glutamate was not affected. The plasma level of several other amino acids (tryptophan, tyrosine, phenylalanine and methionine) were found decreased after oral BCAAs, which may indicate a diminution in the rate of degradation of muscle protein and/or an increase in tissue disposal of amino acids.     

Regulation of branched-chain amino acid biosynthesis in Streptomyces fradiae,a producer of tylosin

Synthesis of threonine dehydratase in Streptomyces fradiae was positively influenced by valine and negatively by isoleucine. However, these two amino acids had no effect on the activity of this enzyme. Synthesis of threonine dehydratase in -aminobutyrate resistant mutants of S. fradiae was pronouncedly less sensitive to the positive effect of valine and this change in regulation led to valine overproduction. Synthesis of acetohydroxy acid synthase is regulated in a similar manner to that of threonine dehydratase, however a lower level of expression was detected in -aminobutyrate resistant mutants. And again, no effect of branched-chain amino acids on acetohydroxy acid synthase activity was observed. It follows that in S. fradiae synthesis of threonine dehydratase is the main regulatory mechanism governing production and the mutual ratio of synthesized valine and isoleucine.Abbreviations -AB -aminobutyrate - AHAS acetohydroxy acid synthase - -KB -ketobutyrate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - TD threonine dehydratase - Trans. B. transaminase of branched-chain amino acids - VDH valine dehydrogenase