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 dryne...

Mitochondrial very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) was purified from human liver. The molecular masses of the native enzyme and the subunit were estimated to be 154 and 70 kD, respectively. The enzyme was found to catalyze the major part of mitochondrial palmitoylcoenzyme A dehydrogenation in liver, heart, skeletal muscle, and skin fibroblasts (89-97, 86-99, 96-99, and 78-87%, respectively).Skin fibroblasts from 26 patients suspected of having a disorder of mitochondrial a-oxidation were analyzed for VLCAD protein using immunoblotting, and 7 of them contained undetectable or trace levels of the enzyme. The seven deficient fibroblast lines were characterized by measuring acyl-coenzyme A dehydrogenation activities, overall palmitic acid oxidation, and VLCAD protein synthesis using pulse-chase, further confirming the diagnosis of VLCAD deficiency. These results suggested the heterogenous nature of the mutations causing the deficiency in the seven patients.Clinically, all patients with VLCAD deficiency exhibited cardiac disease. At least four of them presented with hypertrophic cardiomyopathy. This frequency (> 57%) was much higher than that observed in patients with other disorders of mitochondrial long-chain fatty acid oxidation that may be accompanied by cardiac disease in infants. (J. Clin. Invest. 1995Invest. . 95:2465Invest. -2473

SIGNR1, a member of a new family of mouse C-type lectins, is expressed at high levels in macrophages (Mphi) within the splenic marginal zone, lymph node medulla, and in some strains, in peritoneal cavity. We previously reported that SIGNR1 captures gram-negative bacteria, such as Escherichia coli and Salmonella typhimurium, as well as Candida albicans. We have now investigated the precise ligands and innate responses that involve SIGNR1. The interaction of SIGNR1 with FITC-dextran and E. coli was completely inhibited by LPS from E. coli and Salmonella minnesota. Using LPS from various types of rough mutants of Salmonella, we found that SIGNR1 primarily recognizes oligosaccharides in the non-reductive end of the LPS core region. In transfectants, expression of SIGNR1 enhanced the oligomerization of Toll-like receptor (TLR) 4 molecules as well as the degradation of IkappaB-alpha after stimulation with E. coli under low-serum conditions. The enhanced TLR4 oligomerization was inhibited by pre-treatment of the cells with anti-SIGNR1 mAb or with mannan. A physical association between SIGNR1 and the TLR4-MD-2 complex was also observed by immunoprecipitation. Finally, we found that transfection of SIGNR1 into the macrophage-like RAW264.7 cells resulted in significant augmentation of cytokine production. These results suggest that SIGNR1 associates with TLR4 to capture gram-negative bacteria and facilitate signal transduction to activate innate M responses.

ABSTRACT. Long-chain acyl-CoA dehydrogenase (LCAD) deficiency is a disorder of fatty acid 8-oxidation. Its diagnosis has been made based on the reduced activity of palmitoyl-CoA dehydrogenation, i.e., in fibroblasts. We previously showed that in immunoblot analysis, an LCAD band of normal size and intensity was detected in fibroblasts from all LCAD-deficient patients tested. In the present study, we amplified via polymerase chain reaction and sequenced LCAD cDNA from three of these LCADdeficient cell lines, and found perfectly normal LCAD sequences in two of them, indicating that at least these patients were not deficient in LCAD. The third patient was homozygous for an A to C substitution at 997, although it is unknown whether or not 997-C is a normal polymorphism. Although the LCAD sequence data were puzzling, a new enzyme, very-long-chain acyl-CoA dehydrogenase (VLCAD), was recently identified. Because VLCAD also has high activity ~vith palmitoyl-CoA as substrate, it was possible that defective VLCAD may cause reduced palmitoyl-CoA dehydrogenating activity. We performed immunoblot analysis of VLCAD in sis "LCAD-deficient" patients; VLCAD was negative in three of them, two of whom had a normal LCAD cDNA sequence. These results indicated that a considerable number of the patients who had previously been diagnosed as having LCAD deficiency in fact have VLCAD deficiency. (Pediatr Res 34: 11 1-1 13, 1993)

Genetic deficiency of short-chain acyl-coenzyme A (CoA) dehydrogenase activity was demonstrated in cultured fibroblasts from a 2-yr-old female whose early postnatal life was complicated by poor feeding, emesis, and failure to thrive. She demonstrated progressive skeletal muscle weakness and developmental delay. Her plasma total carnitine level (35 nmol/ml) was low-normal, but was esterified to an abnormal degree (55% vs. controls of < 10%). Her skeletal muscle total carnitine level was low (7.6 nmol/mg protein vs. controls of 14±2 nmol/mg protein) and was 75% esterified. Mild lipid deposition was noted in type I muscle fibers. Fibroblasts from this patient had 50% of control levels of acyl-CoA dehydrogenase activity towards butyryl-CoA as substrate at a concentration of 50 ,M in a fluorometric assay based on the reduction of electron transfer flavoprotein. All of this residual activity was inhibited by an antibody against medium-chain acyl-CoA dehydrogenase. These data demonstrated that medium-chain acylCoA dehydrogenase accounted for 50% of the activity towards the short-chain substrate, butyryl-CoA, under these conditions, but that antibody against that enzyme could be used to unmask the specific and virtually complete deficiency of shortchain acyl-CoA dehydrogenase in this patient. Fibroblasts from her parents had intermediate levels of activity towards butyryl-CoA, consistent with the autosomal recessive inheritance of this metabolic defect.