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4th Amino Acid Assessment Workshop

Brain Amino Acid Requirements and Toxicity: The Example of Leucine^1,2

Children’s Hospital of Philadelphia, Division of Child Development, Rehabilitation and Metabolic Disease, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

³To whom correspondence should be addressed. E-mail: yudkoff{at}email.chop.edu.

	ABSTRACT

TOP ABSTRACT LITERATURE CITED

 
Glutamic acid is an important excitatory neurotransmitter of the brain. Two key goals of brain amino acid handling are to maintain a very low intrasynaptic concentration of glutamic acid and also to provide the system with precursors from which to synthesize glutamate. The intrasynaptic glutamate level must be kept low to maximize the signal-to-noise ratio upon the release of glutamate from nerve terminals and to minimize the risk of excitotoxicity consequent to excessive glutamatergic stimulation of susceptible neurons. The brain must also provide neurons with a constant supply of glutamate, which both neurons and glia robustly oxidize. The branched-chain amino acids (BCAAs), particularly leucine, play an important role in this regard. Leucine enters the brain from the blood more rapidly than any other amino acid. Astrocytes, which are in close approximation to brain capillaries, probably are the initial site of metabolism of leucine. A mitochondrial branched-chain aminotransferase is very active in these cells. Indeed, from 30 to 50% of all -amino groups of brain glutamate and glutamine are derived from leucine alone. Astrocytes release the cognate ketoacid [-ketoisocaproate (KIC)] to neurons, which have a cytosolic branched-chain aminotransferase that reaminates the KIC to leucine, in the process consuming glutamate and providing a mechanism for the "buffering" of glutamate if concentrations become excessive. In maple syrup urine disease, or a congenital deficiency of branched-chain ketoacid dehydrogenase, the brain concentration of KIC and other branched-chain ketoacids can increase 10- to 20-fold. This leads to a depletion of glutamate and a consequent reduction in the concentration of brain glutamine, aspartate, alanine, and other amino acids. The result is a compromise of energy metabolism because of a failure of the malate–aspartate shuttle and a diminished rate of protein synthesis.

KEY WORDS: • brain metabolism • leucine • glutamate • maple syrup urine disease • inborn errors of metabolism • amino acids

Brain glutamate metabolism: the glutamate–glutamine cycle

    The purposes of brain glutamate metabolism. Glutamic acid is a crucial excitatory neurotransmitter in the mammalian brain (1). Accordingly, a large number of biochemical systems have evolved to ensure the integrity of glutamatergic neurotransmission. Examples of these systems include the excitatory amino acid transporters that mediate the uptake and release across various membranes, including the plasma membrane and those of internal organelles like mitochondria and the glutamate-rich vesicles of nerve endings. The family of glutamatergic receptors is yet another key component of the system of glutamatergic neurotransmission. There is a great variety of such receptors, but they can be classified into 2 types: the ionotropic receptors, which induce ionic currents in the postsynaptic neurons, and the metabotropic receptors, which lead to transduction of second messenger systems.

The "goal" of brain glutamate metabolism is to accommodate the 2 primary requirements of glutamatergic neurotransmission:

1. The first need is to ensure that the external glutamate concentration, particularly that in the intrasynaptic space, is kept to a minimum to maximize the signal-to-noise ratio after the Ca²⁺-dependent release of glutamate from presynaptic terminals. In addition, the maintenance of a relatively low intrasynaptic concentration of glutamate minimizes the risk of excessive glutamatergic stimulation of postsynaptic terminals, or excitotoxicity (2–4). A great deal of research now clearly implicates excitotoxicity as a component in mediating brain injury in diverse disorders, including epilepsy; brain hypoxia and trauma; inborn errors of metabolism; and various neurodegenerative diseases, such as Parkinson’s disease and Alzheimer’s disease.

2. The second requirement of efficient glutamatergic neurotransmission is to ensure that neurons have access to precursors from which to produce glutamic acid. Little, if any, glutamate (or glutamine) is transported from blood to brain. It therefore is essential that the brain synthesize glutamate. This requirement presents a complex problem, because glutamate serves not only as a neurotransmitter but as a key metabolic intermediate. Glutamate is not a metabolic substrate for the brain in the manner of glucose or the ketone bodies, but a substantial portion of the carbon of either glucose or the ketone bodies must pass through a large pool of brain glutamate before the final oxidation of this carbon in the tricarboxylic acid cycle (5–7).

The data in Table 1, which shows the glutamate concentration in different brain compartments, underscore the remarkable efficiency with which brain glutamate metabolism accomplishes these 2 goals. A glutamate level of 100 mmol/L exists in the vesicles that fuse with the presynaptic membrane before the Ca²⁺-dependent release of glutamate during depolarization. In contrast, the basal glutamate concentration in the extracellular fluid may be as low as 1 µmol/L. Glutamate levels of the neuronal cytoplasm are 10 mmol/L or 10,000 times greater than the extracellular fluid. The brain therefore effectively maintains an extremely high internal glutamate concentration and a very low external concentration. Indeed, there are few comparable examples of so extreme variation in the internal–external concentration gradient for any other amino acid.

View larger version (17K): [in this window] [in a new window]   FIGURE 1 The glutamate–glutamine cycle. Glutamate is released from nerve endings into the synapse, where it has an excitatory effect. Astrocytes rapidly take up the released glutamate, thereby maintaining a low intrasynaptic level. Within astrocytes, glutamate is converted to glutamine, which astrocytes release via a specialized transport system. Neurons take up glutamine and hydrolyze it to glutamate via phosphate dependent glutaminase, a mitochondrial enzyme. In this manner neurons have a steady supply of precursor for the synthesis of glutamate.

Astrocytes have a steady supply of ammonia for this reaction, because they wrap "foot processes" around brain capillaries, and, within seconds, they convert to glutamine essentially all ammonia that enters the brain (14). Astrocytes then release glutamine so formed via a specific carrier (15). Neurons take up glutamine, again with a specific transport system (16), and hydrolyze this amino acid to glutamate via mitochondrial phosphate-dependent glutaminase (EC 3.5.1.2), thereby restoring to neurons the glutamate that had been released during depolarization.

The glutamate–glutamine cycle accommodates the 2 cardinal purposes of brain glutamatergic neurotransmission: a) extracellular glutamate is kept extremely low, and b) neurons resynthesize glutamate from a readily available precursor (glutamine). In effect, glutamic acid is restored to the neurons in a "camouflaged," non-neuroactive form.

    Limitations of the glutamate–glutamine cycle. The glutamate–glutamine cycle has proved a powerful heuristic device in terms of enabling understanding of brain amino acid metabolism. However, all models necessarily simplify intricate systems, and all models require constant reinterpretation and modification if they are to maintain value.

With respect to the model of the glutamate–glutamine cycle, recent research has highlighted 3 phenomena that the model ignores. a) There is no simple stoichiometric relation between glutamine synthesis and glutamate consumption. b) The brain extensively oxidizes glutamic acid. As a result, the brain needs to replenish glutamate carbon via anaplerotic reactions, the most important of which is pyruvate carboxylase (EC 6.4.1.1). c) The glutamate–glutamine cycle fails to acknowledge the pivotal relation between metabolism in the brain and that in the periphery. In particular, the model does not explain how the brain replaces the nitrogen that is lost in the course of amino acid metabolism.

Glutamate handling involves more than glutamine synthesis and hydrolysis. Considerable evidence now suggests that brain glutamate metabolism involves more than glial glutamine synthesis and neuronal glutamine hydrolysis. In particular, the following reactions are essential to brain glutamate handling. 1) Glutamate dehydrogenase (GDH)⁴ (EC 1.4.1.2): the oxidative deamination of glutamate via GDH is thought to be particularly important to glial disposal of this amino acid (17,18). Indeed, a congenital deficiency or mutation of GDH can result in human neurologic disease (19,20). 2) Malic enzyme (EC 1.1.1.40): when the glutamate "load" that is presented to astrocytes is relatively high, it is likely that the complete oxidation of glutamate involves malic enzyme, which favors formation of pyruvate, thereby replenishing pools of the latter metabolite if glucose is low (17,21). 3) Aminotransferases: abundant evidence underscores the significance of the various aminotransferase reactions in supporting overall brain metabolism. For example, the aspartate aminotransferase (EC 2.6.1.1) reaction is essential to overall brain metabolism, including maintaining the integrity of the malate–aspartate shuttle and neurotransmitter synthesis (6,7,22).

Anaplerosis is essential to brain metabolism. The oxidative capacity of the brain is very great: oxygen consumption is 35 mL · min^–1 · kg^–1, or 20% of whole-body oxygen consumption. As a result, there is a steady formation of CO₂ and a loss of carbon from the system. The brain replenishes this loss, as do most tissues, by using the pyruvate carboxylase reaction to "fix" carbon dioxide as oxaloacetate (9,23–26). It is probable that this role is subserved primarily by astrocytes, which are enriched in pyruvate carboxylase activity (26,27). Glial pyruvate carboxylation is a relatively robust process, with as much as 20 to 30% of glutamate carbon deriving from this pathway (26).

Glutamate–glutamine cycle and external sources of nitrogen. The model of the glutamate–glutamine cycle acknowledges only the internal exchange of glutamate and glutamine between neurons and astrocytes. It makes no statement about the putative relation of the system to the periphery. In particular, the model fails to describe the external nitrogen sources that the brain uses to synthesize glutamate and glutamine. There can be no doubt that such sources must exist. Little glutamate or glutamine is transported from blood to brain. Indeed, there probably is a net efflux of glutamine (28–32), to some extent in exchange for neutral amino acids that enter the brain from the blood (33,34). The release of glutamine, together with the release of ammonia during periods of heightened neuronal depolarization, probably are mechanisms for the maintenance of brain nitrogen balance.

The BCAAs as brain nitrogen donors

Several investigators have entertained the possibility that the branched-chain amino acids (BCAAs), and particularly leucine, might serve in the brain as important donors of amino groups for the purpose of glutamate synthesis (35–37). A reason for entertaining this hypothesis is that these amino acids rapidly cross into the central nervous system, with the entry of leucine exceeding that of any other amino acid (38,39). At usual blood amino acid concentrations, the carrier system for neutral amino acids is 50% saturated with phenylalanine and leucine (39). Furthermore, because leucine is not a neuroactive substance, it can safely be "trafficked" through the brain and be used as a source of –NH₂ groups for the purpose of glutamate synthesis.

The importance of the BCAAs as nitrogen donors in peripheral tissues has long been understood. Skeletal muscle is a major site of transamination of these compounds (40) because of its high content of BCAA transaminase. When catabolism of muscle protein increases, there is an increased supply of BCAAs, thereby favoring the generation by myocytes of glutamate, which becomes available to the alanine aminotransferase reaction (EC 2.6.1.2):

The liver converts alanine so generated to glucose in an important adaptation to the stress of starvation. In vivo studies with stable isotopes indicate that as much as 25 to 30% of alanine nitrogen is formed from the BCAAs (41).

BCAA transaminase activity is abundant in the brain, with both the cytosolic and mitochondrial enzymes being present, albeit with a differential cellular distribution (42). The mitochondrial and cytosolic proteins are distinct from one another. The respective weights are 41 kDa (mitochondrial) (43) and 50 kDa (cytosolic), with the latter probably existing as a homodimer in the native state (44). Most tissues express the mitochondrial form, but, in the brain, testis, and ovary, the cytosolic species is prominent (44). The kinetic constants for both species are similar, with the K_m values for leucine (1–1.3 mmol/L) and -ketoglutarate (0.6 mmol/L) exceeding the endogenous brain concentration of either substrate (0.2 mmol/l) (45,46).

The product of BCAA transamination is glutamate and a branched-chain -ketoacid, in the case of leucine -ketoisocaproate (KIC) (Fig. 2). The brain can oxidize both leucine and KIC to CO₂ (47,48), but available evidence suggests that the rate of this oxidation is far lower than that of transamination (49–51). Instead, the preferred fate of the KIC is reamination back to leucine (51). In contrast, decarboxylation of the branched-chain ketoacid is much more active in the heart and kidney (52).

View larger version (13K): [in this window] [in a new window]   FIGURE 2 Outline of BCAA metabolism. The initial step is transamination by BCAT. Reaction products are glutamate acid and a branched-chain -ketoacid. The latter is decarboxylated to an acyl-CoA ester (R-CoA) via BCAD (EC 1.2.4.4) in the rate-limiting step in the degradation of BCAAs. A specific kinase inactivates BCAD and a specific phosphatase activates this enzyme. Adapted from Shimomura et al. (81).

View larger version (19K): [in this window] [in a new window]   FIGURE 3 Metabolism of [15N]Leucine in cultured astrocytes. Cells were incubated in a buffered medium that contained [15N]leucine (0.1 mmol/L), 15 unlabeled amino acids (0.1 mmol/L each), 0.1 mmol/L NH4Cl, and glucose (10 mmol/L). At the indicated times GC-MS was used to determine isotopic enrichment in [15N]leucine, [2-15N]glutamine, [15N]valine, and [15N]isoleucine. Taken from Yudkoff et al. (55).

We proposed a model (Fig. 4) to account for the fate of the KIC that astrocytes release (35). According to this model, neurons take up the KIC and rapidly convert it back to leucine, in the process consuming glutamic acid. Studies in synaptosomes (61) indicated that flux from KIC to leucine (in medium with -ketoglutarate 0.5 mmol/L and KIC 0.5 mmol/L) was far greater than that from leucine to glutamate: 1.0 nmol · min^–1 · mg protein^–1 vs. 0.3 nmol · min^–1 · mg protein^–1. Flux from KIC to leucine was still high (0.8 nmol · min^–1 · mg protein^–1) when the KIC in the medium was reduced to 0.025 mmol/L (61). With [2-¹⁵N]glutamine instead of [¹⁵N]glutamate as precursor, the flux in this direction was 2–3 times greater, probably reflecting the fact that glutamine enters all terminals, whereas glutamate enters primarily glutamatergic terminals.

View larger version (19K): [in this window] [in a new window]   FIGURE 4 Schematic of brain leucine handling. Leucine in brain capillaries enters astrocytes via the neutral amino acid transporter. In capillaries leucine is transaminated via BCAT, in the process forming glutamate and KIC. Glia release KIC to neurons, which "reverse" transamination and regenerate leucine, which neurons release to astrocytes, thereby completing a "leucine–glutamate cycle."

Transamination reactions generally are construed as equilibrium reactions that are sensitive primarily to the relative concentrations of the reactants. The model of Figure 4, however, posits a "polarization" of brain leucine metabolism, such that leucine transamination in astrocytes proceeds in the "forward" direction and that of neurons with a "reverse" vector. Astrocytes readily dispose of glutamate via glutamine synthetase (12,13), and these cells have a relatively low glutamate concentration, thereby favoring the forward flux of the BCAT reaction. The fact that human brain avidly extracts leucine and releases glutamine (31) supports this rendering. In neurons, we noted a tendency for [¹⁵N]leucine produced from [¹⁵N]glutamate to be released to the medium, thereby restricting the concentration of the former amino acid and enhancing the reverse flux (61). The fact that the brain concentration of KIC is kept quite low (<1 µmol/L) (64,65) implies rapid consumption of this ketoacid.

Brain metabolism in maple syrup urine disease

Maple syrup urine disease (MSUD) is caused by a congenital deficiency of branched-chain ketoacid decarboxylase (EC 1.2.4.4) (66,67), the rate-limiting step in the metabolism of the BCAAs (Fig. 2). Newborns who are screened for this inborn error of metabolism usually receive a special diet that is purposefully low in these amino acids. Most of these youngsters can enjoy normal physical and cognitive development, although dietotherapy must be maintained indefinitely to avoid brain damage (68). A continual risk to well-being is metabolic decompensation in association with acute stress, usually intercurrent infection, when the catabolism of endogenous protein results in a sudden increase in body fluids (both intracellular and extracellular) of both BCAAs and ketoacids. Youngsters then manifest a neurologic syndrome of stupor, anorexia, irritability, ataxia, vomiting, hallucinations, and abnormal movements. A frequent finding is diffuse edema and swelling of the subcortical gray matter. These episodes of decompensation may be fatal if the brain swelling is so severe as to cause transtentorial herniation. If the decompensation is of sufficient frequency or duration, permanent brain damage and mental retardation may ensue (67,69).

The pathophysiology that causes brain damage in MSUD has received considerable attention. The internal [leucine] may become so elevated as to overwhelm the usual brain mechanisms of water homeostasis and lead to cell swelling (67). A high blood leucine would tend to inhibit the entry into the brain of other large neutral amino acids (39), thereby resulting in amino acid deficiency and a compromised rate of protein synthesis (70). Indeed, the fact that leucine is a key modulator of the rates of overall protein anabolism and catabolism (71) makes it likely that a severe disturbance of leucine degradation would have a deleterious effect on body nitrogen metabolism. Metabolites that accumulate in MSUD appear to induce oxidant injury in the cerebral cortex of 30-d-old rats (72). There also appear to be effects on brain energy metabolism (73) and on brain development (74).

The neurologic disarray of MSUD also can be interpreted in the context of the model of brain leucine handling that we have developed (Fig. 4). A recent ¹H magnetic resonance spectroscopy study of a patient during acute decompensation revealed an increase of brain lactate and branched-chain ketoacids and a diminution of brain creatine, suggesting a disturbance of oxidative metabolism (75). The finding of increased lactate in the brain of this child is of interest in light of the report (63) that exposure of cultured astrocytes to KIC (1 or 5 mmol/L) was associated with a profound (50%) reduction of internal aspartate, as well as an increase (2x) of [¹³C]lactate production when the cells were incubated in the presence of [U-¹³C]glutamate.

These findings are consistent with the formulation that the untoward accumulation of KIC is associated with a net flux of the BCAT reaction in the reverse direction and a consequent shift in the equilibrium of the reaction, thereby resulting in a diminution of the size of the pools of both glutamate and aspartate:

According to this rendering, excessive levels of KIC drive the BCAT (Reaction 1) to the right, in the process increasing the -ketoglutarate:glutamate ratio. As a result, more -ketoglutarate becomes available to the aspartate aminotransferase reaction (Reaction 2), thereby consuming aspartate.

A likely effect of a relative depletion of aspartate and glutamate would be a compromise in function of the malate–aspartate shuttle (Fig. 5), which transfers to mitochondria the reducing equivalents formed from glycolysis in the cytosol. There is no mitochondrial uptake system for NADH. Thus, cells must transport reducing equivalents in the form of malate, which in mitochondria is oxidized to oxaloacetate, in the process generating intramitochondrial NADH.

View larger version (21K): [in this window] [in a new window]   FIGURE 5 Interaction in MSUD of the malate–aspartate shuttle with BCAT. The shuttle transports from cytosol to mitochondrion, the reducing equivalents formed during glycolysis. It involves reduction of cytosolic oxaloacetate (OAA) to malate, in the process converting NADH to NAD. OAA is generated via AAT. After transport into the mitochondrion in exchange for -ketoglutarate (-KG), malate is reoxidized to OAA, in the process converting NAD back to NADH, which is oxidized in the electron transport chain. Mitochondrial AAT reconverts OAA to -KG. If KIC accumulates, as it does in MSUD (branched-chain ketoacid decarboxylase deficiency), there is increased consumption of glutamic acid (Glu) and increased production of both leucine (Leu) and -KG via BCAT. (The BCAT system is represented within the 2 squares at the bottom). Intracellular glutamate then is diminished and -KG is increased, leading to enhanced consumption of aspartate via AAT. As a result, overall flux through AAT disrupted and the malate-aspartate shuttle no longer can effectively transfer reducing equivalents from cytosol to mitochondrion.

This rendering of altered brain metabolism is consistent with clinical findings. Thus, the cerebrospinal fluid of affected patients during a period of metabolic decompensation shows a marked increase in leucine and a relative depletion of both glutamate and glutamine (76). A depletion of intracellular glutamate is reflected also in the fact that the leucine:alanine ratio is low, even before the development of overt clinical symptoms (67). The presumed mechanism would be a shift in the equilibrium of the aminotransferase reactions:

Experimental data support the notion that the pathophysiology of MSUD might be related to a relative deficiency of glutamate, aspartate, and other amino acids. As noted above, aspartate is depleted when cultured astrocytes are exposed to KIC (63). We demonstrated that exposure of astrocytes to KIC could reduce the internal glutamate concentration to a level below the K_m for glial glutamine synthetase (56). In addition, clinical studies imply that an untoward concentration of branched-chain ketoacids can deplete the concentration of glutamate and amino acids that derive from it, including alanine. Thus, several investigators have called attention to the relative diminution of plasma alanine, especially during a period of clinical decompensation (67,77). Indeed, the suggestion has been made (67,77) that during a crisis patients ought to receive supplemental alanine, which may have an anabolic effect on protein turnover (78).

Summary

The skein of biochemical reactions and transport systems that constitute brain glutamate handling presupposes the coordinated action of discrete cellular and subcellular compartments to accommodate 2 goals: a) the maintenance of external glutamate at a very low level to minimize the risk of excitotoxicity and to maximize the signal-to-noise ratio upon the release of glutamate from nerve endings, and b) the provision to neurons of a steady supply of precursors from which to resynthesize glutamate, which is steadily lost from neurons via release and via oxidative processes.

The BCAAs, particularly leucine, figure prominently in this arrangement. These compounds provide 30 to 50% of all the –NH₂ groups that the brain draws upon for the purpose of glutamic acid synthesis. Leucine is aptly suited for this role. It crosses rapidly into the brain, first passing into astrocytes, where it is swiftly transaminated, giving rise to glutamate and glutamine, as well as a branched-chain -ketoacid. The ketoacid is not oxidized at the same rate as it is produced. Instead, it is released from astrocytes to neurons, which are capable of reversing the transamination process and reforming leucine, in the process consuming glutamic acid and, conceivably, conferring on the system a "glutamate buffering system" should levels of this excitatory amino acid become excessive. Leucine formed in neurons can be released back to astrocytes, thereby constituting a "leucine–glutamate cycle" that, like the glutamate–glutamine cycle, serves to shuttle nitrogen between astrocytes and neurons.

In MSUD, or a congenital deficiency of branched-chain ketoacid decarboxylase, these relations become exaggerated. The systemic inability to oxidize branched-chain ketoacids results in an accumulation of these compounds that, during periods of metabolic decompensation, may be as much as 10–20 times normal. As the brain transaminates branched-chain ketoacids to the parent amino acids, it consumes glutamate (and aspartate, glutamine, and alanine) at an exaggerated rate, thereby interfering with several critical biochemical processes, e.g., the malate–aspartate shuttle, which serves to transfer reducing equivalents from cytosol to mitochondria. The result is a failure of energy metabolism, as well as a cerebral lactic acidosis. To some extent, this toxicity can be relieved by administration of alanine and other amino acids that help to replenish the relative depletion of the internal glutamate concentration.

The neurotoxicity of MSUD likely is secondary to various factors, not all of which are directly related to brain energy metabolism. It is possible, for example, that extreme elevations of blood leucine may affect transport of amino acids and other nutrients at the blood–brain barrier (62,70,79).

Our experience with MSUD indicates that a near complete block in the oxidation of the BCAAs can have devastating consequences for the central nervous system. However, this experience does not support the conclusion that any increase in blood BCAA concentration is toxic. Elevations of BCAA in plasma occur commonly in diabetes or fasting, when insulin levels are low and the uptake of the BCAA into peripheral tissues, particularly skeletal muscle, is diminished. There is no evidence for neurotoxicity in these states. Indeed, the ketosis of fasting may be beneficial to neural function by diminishing the frequency and the severity of seizures in individuals with epilepsy. Nor is there a cogent reason to be concerned about the neurotoxicity consequent to the administration of these amino acids in reasonable dosage to individuals who have no congenital deficiency of BCAA oxidation. Recent studies in rats demonstrate no obvious toxicity, even with the administration of BCAA in doses that greatly exceed probable human intake (80).

Moderate elevations of plasma BCAA are compatible with normal neurologic development and function. Thus, even in patients with MSUD, who have almost no residual capacity to oxidize BCAAs, an increase of the blood leucine concentration to about twice the normal range (0.1–0.2 mmol/L) is compatible with intact brain function. There is a need for long-term human studies of potential beneficial or detrimental consequences of BCAA supplementation to healthy individuals.

	FOOTNOTES

 
¹ Published in a supplement to The Journal of Nutrition. Presented at the conference "The Fourth Workshop on the Assessment of Adequate Intake of Dietary Amino Acids" held October 28–29, 2004, Kobe, Japan. The conference was sponsored by the International Council on Amino Acid Science. The Workshop Organizing Committee included Dennis M. Bier, Luc Cynober, David H. Baker, Yuzo Hayashi, Motoni Kadowaki, and Andrew G. Renwick. Guest editors for the supplement publication were David H. Baker, Dennis M. Bier, Luc Cynober, John D. Fernstrom, Yuzo Hayashi, Motoni Kadowaki, and Dwight E. Matthews.

² Supported by grants HD26979 and NS37915 from the NIH.

⁴ Abbreviations used: AAT: aspartate aminotransferase; BCAD: branched-chain acyl-CoA dehydrogenase; BCAT, branched-chain aminotransferase; BCAT_c: cytosolic BCAT; -KG: -ketoglutarate; KIC: -ketoisocaproic acid; MSUD, maple syrup urine disease; OAA: oxaloacetate.

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