We previously reported that in childhood adrenoleukodystrophy (C-ALD) and adrenomyeloneuropathy (AMN), the peroxisomal fl-oxidation system for very long chain (>C22) fatty acids is defective. To further define the defect in these two forms of X chromosome-linked ALD, we examined the oxidation of [1-14C]lignoceric acid (n-tetracosanoic acid, C24:0) and [1-'4C]lignoceroyl-CoA (substrates for the first and second steps of f8-oxidation, respectively). The oxidation rates of lignoceric acid in C-ALD and AMN were 43% and 36% of control values, respectively, whereas the oxidation rate of lignoceroyl-CoA was 109% (C-ALD) and 106% (AMN) of control values, respectively. On the other hand, the oxidation rates of palmitic acid (n-hexadecanoic acid) and palmitoyl-CoA in C-ALD and AMN were similar to the control values. These results suggest that lignoceroyl-CoA ligase activity may be impaired in C-ALD and AMN. To identify the specific enzymatic deficiency and its subcellular localization in C-ALD and AMN, we established a modified procedure for the subcellular fractionation of cultured skin fibroblasts. Determination of acyl-CoA ligase activities provided direct evidence that lignoceroyl-CoA ligase is deficient in peroxisomes while it is normal in mitochondria and microsomes. Moreover, the normal oxidation of lignoceroyl-CoA as compared with the deficient oxidation of lignoceric acid in isolated peroxisomes also supports the conclusion that peroxisomal lignoceroyl-CoA ligase is impaired in both C-ALD and AMN. Palmitoyl-CoA ligase activity was found to be normal in peroxisomes as well as in mitochondria and microsomes. This normal peroxisomal palmitoyl-CoA ligase activity as compared with the deficient activity of lignoceroyl-CoA ligase in C-ALD and AMN suggests the presence of two separate acyl-CoA ligases for palmitic and lignoceric acids in peroxisomes. These data clearly demonstrate that the pathognomonic accumulation of very long chain fatty acids in C-ALD and AMN is due to a deficiency of peroxisomal very long chain (lignoceric acid) acyl-CoA ligase.The peroxisomal disorders represent a newly characterized group of inherited diseases (1, 2). In the adrenoleukodystrophies (ALD), three forms are recognized: childhood ALD (C-ALD; X chromosome-linked), adult ALD [adrenomyeloneuropathy (AMN); X chromosome-linked], and neonatal ALD (autosomal recessive). C-ALD is the most common form (3, 4) and usually appears between the ages of 4 and 8 years. It is characterized by central nervous system demyelination and adrenal cortical insufficiency. Death occurs during the first or second decade. AMN occurs mainly in adults, progresses more slowly, and affects the adrenal cortex, spinal cord, and peripheral nerves (5). The occurrence of both C-ALD and AMN within the same kindred suggests that these forms of ALD are different clinical manifestations of the same mutation (4). The identification of an identical biochemical defect in both would confirm this assumption. The neonatal form of ALD is a severe disorder that is evident in...

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Daidzin is a potent, selective, and reversible inhibitor of human mitochondrial aldehyde dehydrogenase (ALDH) that suppresses free-choice ethanol intake by Syrian golden hamsters. Other ALDH inhibitors, such as disulfiram (Antabuse) and calcium citrate carbimide (Temposil), have also been shown to suppress ethanol intake of laboratory animals and are thought to act by inhibiting the metabolism ofacetaldehyde produced from ingested ethanol. To determine whether or not daidzin inhibits acetaldehyde metabolism in vivo, plasma acetaldehyde in daidzin-treated hamsters was measured after the administration of a test dose of ethanol.Daidzin treatment (150 mg/kg per day i.p. for 6 days) significantly suppresses (>70%) hamster ethanol intake but does not affect overall acetaldehyde metabolism. In contrast, after administration of the same ethanol dose, plasma acetaldehyde concentration in disulfiram-treated hamsters reaches 0.9 mM, 70 times higher than that of the control. In vitro, daidzin suppresses hamster liver mitochondria-catalyzed acetaldehyde oxidation very potently with an IC50 value of 0.4 ,IM, which is substantially lower than the daidzin concentration (70 ,uM) found in the liver mitochondria of daidzin-treated hamsters. These results indicate that (i) the action of daidzin differs from that proposed for the classic, broad-acting ALDH inhibitors (e.g., disulfiram), and (ii) the daidzin-sensitive mitochondrial ALDH is not the one and only enzyme that is essential for acetaldehyde metabolism in golden hamsters.Daidzin, a glucosylated isoflavone isolated from Radix puerariae, suppresses free-choice ethanol intake by Syrian golden hamsters (1). It also potently and selectively inhibits human mitochondrial aldehyde dehydrogenase (ALDH) (2). Almost half of all Asians have inherited an inactive variant form of mitochondrial ALDH, and in this population group alcohol abuse/alcoholism is rare (3-5). Hence, daidzin might be assumed to act by mimicking the consequences of this mutation of the mitochondrial ALDH gene. Individuals carrying the null mitochondrial ALDH gene are not known to suffer from any physiologic problems, except for a significantly compromised capacity to metabolize acetaldehyde. When they consume even small amounts of ethanol (6) their blood acetaldehyde concentration increases markedly.The premise that acetaldehyde accumulation after ethanol consumption may deter further drinking has attracted considerable attention ever since disulfiram was introduced as a therapeutic agent for human alcohol addiction. It was first proposed as an aversive drug based on the observation that workers in the rubber industry who had been exposed to thiuram compounds experienced unpleasant effects after drinking (7). Since then, it has been established that disulfiram-as well as other ALDH inhibitors-suppresses the metabolism of acetaldehyde and causes it to accumulate in peripheral tissues when ethanol is consumed. As a consequence, it induces a broad spectrum of disagreeable effects (8). Association of these no...

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We found that peroxisomal lignoceroyl-CoA ligase, like palmitoyl-CoA ligase, is present in the peroxisomal membrane whereas the peroxisomal beta-oxidation enzyme system is localized in the matrix. To further define the role of peroxisomal acyl-CoA ligases (membrane component) in providing acyl-CoA for peroxisomal beta-oxidation, we examined the transverse topographical localization of enzymatic sites of palmitoyl-CoA and lignoceroyl-CoA ligases in the peroxisomal membranes. The disruption of peroxisomes by various techniques resulted in the release of a "latent" pool of lignoceroyl-CoA ligase activity while palmitoyl-CoA ligase activity remained the same. Proteolytic enzyme treatment inhibited palmitoyl-CoA ligase activity in intact peroxisomes but had no effect on lignoceroyl-CoA ligase activity. Lignoceroyl-CoA ligase activity was inhibited only if peroxisomes were disrupted with detergent before trypsin treatment. Antibodies to palmitoyl-CoA ligase and to peroxisomal membrane proteins (PMP) inhibited palmitoyl-CoA ligase in intact peroxisomes, and no pool of "latent" activity appeared when antibody-treated peroxisomes were disrupted with detergent. On the other hand, disruption of PMP antibody-treated peroxisomes with detergent resulted in the appearance of a "latent" pool of lignoceroyl-CoA ligase activity. These results demonstrate that the enzymatic site of palmitoyl-CoA ligase is on the cytoplasmic surface whereas that for lignoceroyl-CoA ligase is on the luminal surface of peroxisomal membranes. This implies that palmitoyl-CoA is synthesized on the cytoplasmic surface and is then transferred to the matrix through the peroxisomal membrane for beta-oxidation in the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)