Isovaleric acidemia: a new genetic defect of leucine metabolism.

At present, leucine-induced hypoglycemia and maple-syrup urine disease are the only inborn errors of leucine metabolism recognized in man. In the former disorder, recurrent hypoglycemia is the result of leucine-induced hyperinsulinism.1 In maple-syrup urine disease there is a block in the oxidative decarboxylation of leucine, isoleucine, and valine resulting in the accumulation of the amino acids themselves as well as their a-keto derivatives.2 We have recently obtained evidence for a new genetic disorder of leucine metabolism in which there is a defect in the catabolism of leucine resulting in the accumulation of isovaleric acid. This disease has been observed in two siblings, aged 21/2 and 4, who since the early months of life had a persistent odor to their breath and body fluids described as "cheesy" or "like sweaty feet." In addition, these children experienced recurrent episodes of vomiting often progressing to metabolic acidosis and either stupor or coma. These episodes were usually precipitated by protein ingestion or intercurrent infections; the children actually had a pronounced aversion to dietary proteins. During attacks of acidosis the peculiar odor of the children usually intensified. Since the odor resembled that of short-chain fatty acids, the blood and urine were analyzed for these acids by gas-liquid chromatography (GLC) and mass spectrometry. These studies resulted in the demonstration of markedly increased amounts of isovaleric acid in the body fluids of these patients and evidence for a block in the utilization of this branched-chain fatty acid. The complete clinical details of this syndrome will be presented in full detail elsewhere. Materials and Methods.-Patient material: Biochemical studies were performed on blood, urine, and feces of the two patients, B. A. (male, 21/2 yr) and S. A. (female, 4 yr) during attacks of acidosis and after recovery from such attacks. Comparable determinations were performed on specimens from four adults and four children without known metabolic abnormalities. Gas-liquid chromatography (GLC): An improved GLC method for short-chain fatty acids was used (detailed description to be published). Serum samples were acidified with 0.2 vol of 1.0 N H2SO4, then extracted with 20 vol of chloroform-methanol (v/v 2: 1), and subsequently filtered. Mixing with 0.2 vol of 0.1 N NaOH at 0-5O resulted in the formation of a two-phase system. The upper layer was evaporated to dryness and acidified with o-phosphoric acid and steam-distilled.3 The evaporated residue of the lower layer was hydrolyzed with 1.0 N KOH in methanolwater (v/v 4:1) for 4 hr at 650, and similarly steam-distilled. Preliminary experiments indicated that virtually all (>99%) of short-chain fatty acids (straight and branched) (C2-C8) recovered were in the upper layer, and greater than 96% of neutral lipids (tributyrin, tricaproin, trioctanoin) were present in the lower layer. Urine specimens were alkalinized, evaporated to dryness, then acidified and steam-distilled. Alkalinized distillates were evaporated to dryness and acidified with aqueous formic acid. Samples were then injected into a Barber-Colman gas chromatograph (model 10) equipped with a hydrogen flame detector. Columns used were glass U tubes packed with Anakrom (80-90 mesh) coated with 25% neopentylglycol adipate (NPGA)-2% o-phosphoric acid (PA), 20% dioctylphthalate (DOP)-5% o-phosphoric acid, or 20% DOP alone. NPGA-PA and DOP-PA columns were used for free fatty acid assay. DOP column was used only for qualitative analysis of the methyl esters. Methyl esters were produced by reacting the fatty acids with a slight excess of etherial diazomethane. Quantitative analysis was done on NPGA-PA columns,