Maple syrup urine disease (MSUD) is an inherited disorder of branched-chain amino acid metabolism presenting with life-threatening cerebral oedema and dysmyelination in affected individuals. Treatment requires life-long dietary restriction and monitoring of branched-chain amino acids to avoid brain injury. Despite careful management, children commonly suffer metabolic decompensation in the context of catabolic stress associated with non-specific illness. The mechanisms underlying this decompensation and brain injury are poorly understood. Using recently developed mouse models of classic and intermediate maple syrup urine disease, we assessed biochemical, behavioural and neuropathological changes that occurred during encephalopathy in these mice. Here, we show that rapid brain leucine accumulation displaces other essential amino acids resulting in neurotransmitter depletion and disruption of normal brain growth and development. A novel approach of administering norleucine to heterozygous mothers of classic maple syrup urine disease pups reduced branched-chain amino acid accumulation in milk as well as blood and brain of these pups to enhance survival. Similarly, norleucine substantially delayed encephalopathy in intermediate maple syrup urine disease mice placed on a high protein diet that mimics the catabolic stress shown to cause encephalopathy in human maple syrup urine disease. Current findings suggest two converging mechanisms of brain injury in maple syrup urine disease including: (i) neurotransmitter deficiencies and growth restriction associated with branched-chain amino acid accumulation and (ii) energy deprivation through Krebs cycle disruption associated with branched-chain ketoacid accumulation. Both classic and intermediate models appear to be useful to study the mechanism of brain injury and potential treatment strategies for maple syrup urine disease. Norleucine should be further tested as a potential treatment to prevent encephalopathy in children with maple syrup urine disease during catabolic stress.

Background: Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism caused by a deficiency of branched-chain keto acid dehydrogenase. MSUD has several clinical phenotypes depending on the degree of enzyme deficiency. Current treatments are not satisfactory and require new approaches to combat this disease. A major hurdle in developing new treatments has been the lack of a suitable animal model.

Maple syrup urine disease (MSUD; OMIM 248600) is an inborn error of metabolism of the branched chain alpha-ketoacid dehydrogenase (BCKDH) complex that is treated primarily by dietary manipulation of branched-chain amino acids (BCAA). Dietary restriction is lifelong and compliance is difficult. Liver transplantation significantly improves outcomes; however, alternative therapies are needed. To test novel therapies such as hepatocyte transplantation (HTx), we previously created a murine model of intermediate MSUD (iMSUD), which closely mimics human iMSUD. LacZ-positive murine donor hepatocytes were harvested and directly injected (10(5) cells/50 microl) into liver of iMSUD mice (two injections at 1-10 days of age). Donor hepatocytes engrafted into iMSUD recipient liver, increased liver BCKDH activity, improved blood total BCAA/alanine ratio, increased body weight at weaning, and extended the lifespan of HTx-treated iMSUD mice compared to phosphate-buffered saline (PBS)-treated and untreated iMSUD mice. Based on these data demonstrating partial metabolic correction of iMSUD in a murine model, coupled to the fact that multiple transplants are possible to enhance these results, we suggest that HTx represents a promising therapeutic intervention for MSUD that warrants further investigation.

Skvorak and coworkers [1] demonstrated the therapeutic efficacy of HTx in a murine model of iMSUD, confirming significant metabolic improvement and survival. To determine the effect of HTx on extrahepatic organs, we examined the metabolic effects of HTx in brain from iMSUD animals. Amino acid analysis revealed that HTx corrected increased ornithine, partially corrected depleted glutamine, and revealed a trend toward alloisoleucine correction. For amino acid and monoamine neurotransmitters, decreased GABA was partially corrected with HTx, while the L-histidine dipeptide of GABA, homocarnosine, was decreased in iMSUD mice and hypercorrected following HTx. Elevated branched chain amino acids (BCAA; leucine, isoleucine, valine) in MSUD can deplete brain tyrosine and tryptophan (the precursors of monoamine neurotransmitters dopamine (DA) and serotonin (5-hydroxytryptamine; 5-HT)) through competition via the large neutral amino acid transporter. HTx corrected decreased DA levels and the DA metabolite, 3-methoxytyramine, and partially corrected the DA intermediate 3,4-dihydroxyphenylacetate (DOPAC) and 5-HT levels, despite normal tyrosine and tryptophan levels in iMSUD mouse brain. We further observed enhanced intracellular turnover of both DA and 5-HT in iMSUD mouse brain, both of which partially corrected with HTx. Our results suggest new pathomechanisms of neurotransmitter metabolism in this disorder and support the therapeutic relevance of HTx in iMSUD mice, while providing proof-of-principle that HTx has corrective potential in extrahepatic organs.

A B S T R A C T Treatment of hvperlipidemia withclofibrate may result in development of a muscular svndrome. Our previous investigation (1979. J. ClitG. Ivce.st. 64: 405.) showed that chronic administration of clofibrate to rats causes mvotonia and decreases glucose and fatty acid oxidation and total protein of skeletal muscle. In the present experimiients we have investigated amino acid and protein metabolism in these rats. Clofibrate administratioin decreased the concenitration of all three branched-chain amino acids without affecting those of others in muscle. Studies to examine the mechanism of decreases in muscle concenitrationis of branched-chain amiiino acids showed the followiing: (a)