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Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada A1B3X9
2To whom correspondence should be addressed. E-mail: lbrosnan{at}mun.ca.
The session was chaired by Professor J. T. Brosnan and Professor K. Kishi. The discussion focused on the broad range of issues raised by the presentations on branched-chain amino acid (BCAA) metabolism, as well as on the effects of supplementary intake of these compounds. It also sought to identify areas where our knowledge base is lacking, as well as to consider studies that would directly address the issue of excess intake.
J. T. Brosnan: Introductory comments
Professor Brosnan indicated a number of areas where the general discussion might be most profitably focused. These included
He asked the participants to make their remarks as relevant as possible to the human situation. The discussion ranged broadly and engaged most of the participants. The comments of the various discussants have been paraphrased and grouped under unifying headings.
General discussion
Brain metabolism. Amino acid handling at the blood–brain barrier. It is now well established that the large neutral amino acids (which include the BCAAs) are transported into the brain via a common mechanism, the large neutral amino acid transporter. Furthermore, the affinities of this transporter for the different amino acids are in the range of their physiological plasma concentrations. As demonstrated by Fernstrom, changes in the plasma levels of the BCAAs in rats can affect the brain uptake of the neurotransmitter precursors, tyrosine and tryptophan, and this can affect rates of neurotransmitter synthesis. That the same phenomenon can occur in humans is suggested by the studies of Scarna et al. (1), which showed that BCAAs can reduce mania symptomatology, consistent with an action to lower brain tyrosine and thus catecholamine synthesis. In addition, Delgado et al. (2) have reported that ingestion of a mixture of amino acids containing BCAAs but lacking tryptophan can precipitate depressive symptomatology in humans; this is consistent with an effect on brain tryptophan uptake and the synthesis of serotonin. These effects are consistent with the data, particularly those from Partridge’s laboratory, that suggest that the kinetic properties of this brush-border transporter are quite similar in humans and in rats. Nevertheless, one of the remarkable aspects of Fernstrom’s presentation was the number of studies in which substantial quantities of BCAAs have been administered, both acutely and chronically, and have produced high plasma levels of BCAAs without adverse effects.
The discussion also focused on potential tests that might detect any neurological consequences of BCAA supplementation. A wide variety of views were expressed. One view was that it would be important to monitor long-term neurological outcomes; this would require longitudinal psychological studies. On the other hand, there is now a battery of sophisticated techniques in the neurological armamentarium that may be applied to the delineation of regional brain function. These techniques include magnetic resonance spectroscopy, functional MRI, and positron emission tomography. Electroretinography would permit the exploration of one discrete nervous tissue. These techniques could be used in experiments where alterations in plasma BCAA levels could be carefully varied, either by ingestion or by infusion. Such studies could be modeled after a substantial body of work that has explored the effects of small decreases in blood glucose on performance in specialized cognitive tests and other tests. The work with glucose provides proof-of-principle that such an approach is feasible. The choice of which brain region to examine could also be guided by a knowledge of where the enzymes of BCAA metabolism are expressed. The point was also made that it will be very difficult to find psychological or cognitive tests that would pinpoint a particular kind of neurotransmitter or neuron. For example, glutamate controls 60% of all synapses in the brain. It is conceivable that excess BCAAs could cause a subtle change in the handling or the compartmentation of glutamate in the brain. However, it will be exceedingly difficult to find a psychological or cognitive test that will be sufficiently precise and sensitive to serve as a reliable indicator of glutamergic function in a specific brain region.
MSUD.
This inborn error of metabolism is caused by a specific impairment of BCAA catabolism at the level of the branched-chain 
-ketoacid dehydrogenase complex. It results in high circulating levels of both the BCAAs and -ketoacids and is associated with mental retardation. A key question is whether MSUD can provide us with lessons that may be useful in determining potential consequences of excessive levels of the BCAAs or -ketoacids that might be brought about by supplementation. The disease does provide us with considerable data, because it has been examined in detail. The occurrence of an MSUD crisis, when there is an acute onset of symptoms paralleled by marked increases in plasma and cerebrospinal fluid levels of the BCAAs, might provide guidance as to unacceptable levels of these amino acids. However, such information should be used with caution. It was generally felt that the 2 situations are quite different. MSUD is brought about by a block in metabolism; this does not apply to the supplementation paradigm. Children with MSUD have been exposed to high levels of BCAAs from early stages in their development. The disease is associated with greatly decreased rates of BCAA catabolism, whereas supplementation with these amino acids will result in increased flux through the catabolic pathway. Downstream metabolites of the branched-chain -ketoacids will be depleted in MSUD but are likely to be increased in concentration upon supplementation.
Clearly, excessive levels of BCAAs and -ketoacids are associated with profound symptomatology in MSUD patients, but the disease probably provides little guidance as to safe levels of these metabolites in unaffected individuals with normal genotypes.
It was suggested that there are now opportunities for novel work on MSUD models. Besides overt acute toxicity, children with this disease often display subacute consequences. Many MSUD children exhibit subtle neurologic dysfunction, such as attention-deficit disorder or learning disabilities. This is true even of children who have been identified prospectively and who appear to be well controlled. It is difficult to explain these observations. One of the sources of variation may be the degree of metabolic block. Endo’s presentation showed that different patients have different residual activities of the branched-chain -ketoacid dehydrogenase complex. It should now be possible to apply transgenic technology to produce mice with different degrees of dehydrogenase loss; it should also be possible to do this in a tissue-specific manner. Such models may provide novel information as to the origin of the variability of symptoms in children suffering with MSUD.
Factors that affect BCAA metabolism.
BCAA metabolism throughout the life cycle.
There are important differences in the handling of BCAAs throughout the life cycle. Leucine is an insulin secretagogue but the magnitude of the ß-cell response to leucine is highly age dependent. Leucine can cause hypoglycemia in children. However, the ß-cell response to leucine is greatly attenuated in adults and virtually nonexistent in the elderly. It is well known that the responsiveness of many enzyme systems to physiological demands declines with age. Work from Nair’s laboratory has shown a progressive decline in human mitochondrial function. Mitochondrial DNA copy numbers decline with age, as do rates of ATP synthesis (3). This is of particular importance to BCAA me-tabolism, be-cause the rate-limiting step of their catabolism, the branched-chain -ketoacid dehydrogenase, is exclusively mitochondrial. An additional feature of aging is a decline in muscle mass. This represents a decline in the total-body capacity to oxidize BCAAs. Muscle mass seems to have been relatively ignored as an important factor in determining total branched-chain oxidative capacity. Yet, a physically active individual has a higher capacity to oxidize these amino acids. There will also be increased rates of BCAA catabolism during exercise. As much as 5 g BCAA/h can be oxidized during moderately strenuous exercise. There was a consensus among the discussants that not enough is known about the effects of BCAAs or of the rates of BCAA catabolism throughout the life cycle. Future work should address this issue.
Effects of gender on BCAA metabolism.
Most of the animal work, to date, has been carried out on male animals. It is known that, during exercise, women partition their macronutrients differently than men. This provoked a discussion on the effects of gender on BCAA metabolism. Women tend to have lower muscle mass than men, which may decrease their total oxidative capacity for these amino acids. Recent work from Shimomura and Harris’ laboratories has shown that female rats display a marked diurnal variation in the activity state of the branched-chain -ketoacid dehydrogenase; this results from estrogenic control of the dehydrogenase kinase (4). No such diurnal rhythm is evident in male rats. A gender difference was also reported by Tsubuku et al. (5). These workers established the no-adverse-effect levels for the 3 BCAAs in male and female rats. No gender difference was observed for leucine or isoleucine. However, a growth depression was evident in female rats at the 5% dietary level of valine that was not evident in males. The discussants felt that gender differences in human BCAA metabolism have not been sufficiently explored.
Nutritional status and BCAA metabolism.
BCAA metabolism is sensitive to nutritional status. In common with other aminotransferases, the enzyme that initiates the catabolism of the BCAAs contains pyridoxal phosphate as its prosthetic group. In common with other ketoacid dehydrogenases, the branched-chain -ketoacid dehydrogenase contains tightly bound thiamine pyrophosphate, lipoic acid, and flavin adenine dinucleotide as prosthetic groups. Thus, adequate tissue levels of thiamin, riboflavin, vitamin B-6, and lipoic acid are necessary for normal BCAA metabolism. This may be particularly important in the elderly, who have a higher risk of vitamin insufficiency.
The effect of habitual protein intake on the metabolic disposal of BCAAs was also discussed. In experiments with rats, the potential for adverse effects of excess BCAAs is much greater in animals fed marginal protein intakes than in those fed generous protein intakes. Excess leucine, acting through -ketoisocaproate, will activate the branched-chain -ketoacid dehydrogenase complex, and this tends to decrease plasma levels of valine and isoleucine. The decrease in these amino acids may be sufficiently severe to compromise protein synthesis. This effect is most marked in growing animals who have higher protein requirements. However, this effect is not found in animals fed generous protein intakes, presumably because of the high intakes of valine and isoleucine. The traditional protein of choice for rat nutrition is casein. Because casein is highly soluble and quickly digested its use makes it rather easy to show amino acid imbalances, leading to subse-quent growth depression. These imbalances are not as readily apparent when protein sources, such as egg albumin or lactalbumin, are used. Thus, habitual protein intake and the nature of the protein source are important features to be taken into account in investigations of potential adverse effects of supplementary BCAA intake.
BCAAs, insulin action, and glucose metabolism. It was appreciated by the discussants that potential interactions between supplementary BCAA intake and insulin action deserve exploration. On the one hand, Nair’s presentation suggested that leucine infusion could decrease both endogenous glucose production as well as the metabolic clearance of glucose. On the other hand, Garlick characterized the stimulatory effect of leucine on muscle protein synthesis as an increase in insulin sensitivity. These issues have been largely unexplored in individuals ingesting supplementary BCAAs. The necessity for such exploration was brought into focus by the near-epidemic increase in obesity and of type 2 diabetes in the developed world. Such an exploration should be relatively straightforward in that we have at our disposal sophisticated and powerful methodology for studying human glucose metabolism. Some of the simplest studies could investigate the effects of supplementary BCAA intake on oral glucose tolerance tests and on intravenous glucose tolerance tests. The combination of glucose and insulin measurements would provide useful information on the responsiveness of muscle to insulin-stimulated glucose removal. Analysis of C-peptide would permit a measure of insulin secretion. However, a more precise measurement of potential insulin resistance would be provided by the euglycemic, hyperinsulinemic clamp. It would also be important to determine any possible effects of BCAAs on glucose production. The application of stable-isotope methodology would permit measurement of endogenous glucose production. The discussants felt that it would be important to determine both acute and chronic effects of BCAAs. In addition, it was considered important to determine whether there would be differences in the response of young adults and of the elderly.
BCAA excess: studies and biomarkers. One of the objectives of the general discussion section was to consider possible studies and biomarkers that could be used to examine BCAA excess. The discussants were immediately faced with 2 challenges.
First, they recognized that generic approaches that could be applied to virtually any amino acid would have the greatest general utility; yet, they were specifically focused on the BCAAs. Second, they recognized that regulatory bodies would probably require studies of specific commercial products or formulations, whereas studies driven by scientific curiosity might use different experimental designs. As a specific example of the latter point, it was suggested that scientific curiosity might prompt a study of the effect of leucine alone. However, most commercial products contain all 3 BCAAs. The study by Tsubuku et al. (5) was considered to be an excellent example of a generic study that could be generally applicable to other amino acids. The broad scope of the study and the inclusion of groups of both male and female rats were strong points. Obviously, not all features of this study could be carried out in humans, but basic features of it could. Thus, monitoring routine clinical chemistry and markers of organ function (e.g., serum alanine aminotransferase for liver function and serum creatinine for renal function) would be very straightforward. The discussants were also very interested in Kimura’s application of microarray analysis of hepatic gene expression to the armamentarium available for investigating the consequences of amino acid excess. One particular strength of the microrray approach is its global reach, and therefore its potential for uncovering alterations in the expression of genes whose relation to amino acid metabolism is unanticipated. The application of the microarray approach to human studies will be limited by the tissues that may be sampled; for practical purposes, its utility may be limited to monitoring changes in mRNA abundance in white blood cells. Nevertheless, such studies can reveal novel biomarkers that may be monitored in future studies.
It was suggested that we need to know more about the basic pharmacokinetics of BCAA metabolism. However, the paper by Marchesini et al. (6) provides very useful information on plasma clearances, volumes of distribution, half-lives etc., of the individual BCAAs. The idea of a BCAA tolerance test was advanced. Such a test would reflect the balance between intake or absorption and oxidative capacity and would integrate the effects of such influences as metabolic regulation, gene expression, muscle mass, etc. The application of such a test to different demographic groups, genders, etc., should reveal gross differences in their ability to metabolize supplementary amino acids.
BCAAs, unlike most pharmaceutical agents, are a normal dietary constituent and are effectively metabolized by the body. A commonsense approach would therefore compare the supplementary intake with the habitual dietary intake. Because BCAAs are major components of dietary proteins, we can estimate a dietary intake by most adults of some 15–25 g/d. A supplementary intake of 5–10 g/d should be readily accommodated by normal human metabolism. This is not to suggest that such an intake would elicit no metabolic signature. The BCAAs essentially escape splanchnic first-pass metabolism so that increased intakes would be expected to result in an elevation of the peripheral concentrations of these amino acids. The spectrum of supplementary BCAA use covers a wide range of intakes. On the one hand, a commercially available product that contains the 3 BCAAs (as well as some other amino acids) has been reported to be useful in reducing muscle pain experienced by athletes after vigorous exercise. The recommended intake of BCAAs via this product only amounts to about 5 g. In addition, the target group tends to have a relatively high habitual protein intake and, also, a relatively large muscle mass, which would favor rapid metabolic clearance of these amino acids. On the other hand, Fernstrom’s presentation (7) reviewed studies in which large quantities of BCAAs (from 14–60 g/d) were given without reports of adverse effects.
There seems to be little information available on the provision of high BCAA doses to elderly subjects. The elderly tend to have a reduced muscle mass and are at a higher risk of vitamin insufficiency. A deficiency of one of the vitamins required for BCAA metabolism would result in decreased metabolic clearance of the supplementary BCAA.
The ideal marker for identifying excess should have very specific dose-response characteristics. In particular, its variation with intake should display an inflection point that would identify the onset of the excess situation. However, the discussion revealed no such biomarker for humans. However, biomarkers of exposure are also required. In this connection it was suggested that urinary BCAAs (and, also, of branched-chain -ketoacids) may serve as an accurate indicator of exposure. In this case, the lack of an inflection point is an advantage. The best index of integrated exposure may be provided by the 24-h urinary excretion of these compounds. One caveat is that urinary excretion will also depend on renal function. Therefore, the decreased glomerular filtration rates that are found in elderly populations may mean that excretion data obtained in young adults may not be directly applicable to the elderly. It was also pointed out that urinary excretion could also be used as an index of intake (i.e., of compliance) in studies of BCAA ingestion.
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FOOTNOTES |
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LITERATURE CITED |
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 TOP LITERATURE CITED |
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1. Scarna, A., Gijsman, H. J., McTavish, S. F., Harmer, C. J., Cowen, P. J. & Goodwin, C. M. (2003) Effects of a branched-chain amino acid drink in mania. Br. J. Psychiatry 182:210-213.
2. Delgado, P. L., Charney, D. S., Price, L. H., Aghajanian, G. K., Landis, H. & Heninger, G. R. (1990) Serotonin function and the mechanism of antidepressant action. Reversal of antidepressant-induced remission by rapid depletion of plasma tryptophan. Arch. Gen. Psychiatry 47:411-418.
3. Rooyackers, O. E., Adey, D. B., Ades, P. A. & Nair, K. S. (1996) Effect of age on in vivo rates of mitochondrial protein synthesis in human skeletal muscle. Proc. Nat. Acad. Sci. U.S.A. 93:15364-15369.
4. Obayashi, M., Shimomura, Y., Nakai, N., Jeoung, N. H., Nagasaki, M., Murakami, T., Sato, Y. & Harris, R. A. (2004) Estrogen controls branched-chain amino acid catabolism in female rats. J. Nutr. 134:2628-2633.
5. Tsubuku, S., Hatayama, K., Katsumata, T., Nishimura, N., Mawatari, K., Smriga, M. & Kimura, T. (2004) Thirteen-week oral toxicity study of branched-chain amino acids in rats. Int. J. Toxicol. 23:119-126.
6. Marchesini, G., Bianchi, G. P., Vilstrup, H., Checchia, G. A., Patrono, D. & Zoli, M. (1987) Plasma clearances of branched-chain amino acids in control subjects and in patients with cirrhosis. J. Hepatol. 4:108-117.[Medline]
7. Fernstrom, J. D. (2005) Branched-chain amino acids and brain function. J. Nutr. 135:1539S-1546S.
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