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...

We have previously reported that the peroxisomal β‐oxidation system for very long chain fatty acids is defective in X‐linked childhood adrenoleukodystrophy [(1984) Proc. Natl. Acad. Sci. USA 81, 4203‐4207]. In order to elucidate the specific enzyme defect, we examined the oxidation of [1‐14C]lignoceric acid, [1‐14C]lignoceroyl‐CoA and (1‐14C)‐labelled α,β‐unsaturated lignoceroyl‐CoA (substrates for the 1st, 2nd, and 3rd steps of the β‐oxidation cycle, respectively). These studies suggest that the pathognomonic accumulation of very long chain fatty acids in X‐linked childhood ALD may be due to the defective activity of peroxisomal very long chain (lignoceroyl‐CoA) acyl‐CoA ligase.

The hepatocellular glutathione content is partitioned into a cytosolic pool, which accounts for about 85% of the cellular glutathione content, and a mitochondrial pool, which accounts for about 15% of the cellular glutathione content. Previous studies indicated that the mitochondrial glutathione pool may play a critical role in cytoprotection against xenobiotic-induced cell damage. Tests of the role of mitochondrial glutathione in cytoprotection have been hampered by the lack of agents that selectively deplete the mitochondrial glutathione pool. To test the hypothesis that mitochondrial glutathione plays a critical role in protecting against cytotoxic agents, we developed a method to deplete selectively mitochondrial glutathione concentrations. (R,S)-3-Hydroxy-4-pentenoate, an analog of (R)-3-hydroxybutanoate, caused a rapid and selective depletion of mitochondrial glutathione concentrations. Incubation of (R,S)-3-hydroxy-4-pentenoate with rat liver mitochondria or with 3-hydroxybutyrate dehydrogenase in the presence of glutathione afforded a glutathione conjugate whose chromatographic properties were identical with synthetic S-(3-oxo-4-carboxybutyl)glutathione, indicating that (R,S)-3-hydroxy-4-pentenoate was oxidized to the Michael acceptor 3-oxo-4-pentenoate, which reacts with glutathione. Exposure of rat hepatocytes to (R,S)-3-hydroxy-4-pentenoate, which was not cytotoxic and did not induce mitochondrial dysfunction, potentiated the cytotoxicity of tert-butyl hydroperoxide. These results establish the critical role of mitochondrial glutathione in cytoprotection and demonstrate and (R,S)-3-hydroxy-4-pentenoate may find utility in exploring mitochondrial glutathione homeostasis.

In Table 1, the room-temperature lifetimes for [ C u ( d ~p ) ~] + and [Cu(tpp)2]+ are incorrect and should be 240 and 190 ns, respectively. The quantum yields are also incorrect and should be as follows: [ C ~( d m p ) ~] + 2.1 x [ C ~( d p p ) ~] + 8.0 x [Cu(bcp)J+ 2.5 X and [ C ~( t p p ) ~] + 10.0 x

S-(3-Oxopropyl)glutathione, the glutathione conjugate of acrolein, has been reported to be nephrotoxic. The objective of the present studies was to investigate the bioactivation mechanism of the analogues S-(3-oxopropyl)-N-acetyl-L-cysteine (1) and S-(3-oxopropyl)-N-acetyl-L-cysteine S-oxide (2) and to test the hypothesis that the cytotoxicity of 1 is associated with its latent potential to release acrolein in kidney cells. Mechanistic considerations indicated that sulfoxidation of sulfide 1 to form S-oxide 2 and a subsequent general-base-catalyzed beta-elimination reaction would release the cytotoxin acrolein. Hence the release of acrolein from 1 and 2 was studied in chemical systems, and their cytotoxicity was investigated in cultured LLC-PK1 cells and in isolated rat renal proximal tubular cells. Acrolein formation from S-oxide 2, but not from sulfide 1, was observed under basic conditions and with phosphate as the base. Kinetic analysis indicated that a general-base-catalyzed reaction was involved. Both S-conjugates 1 and 2 were cytotoxic in LLC-PK1 cells and in isolated rat renal proximal tubular cells, and the cytotoxicity of sulfide 1, but not of S-oxide 2, in isolated renal proximal tubular cells was reduced in presence of methimazole, an inhibitor of the flavin-containing monooxygenase. These findings indicate that the cytotoxicity of S-conjugate 1 is associated with a novel bioactivation mechanism that involves sulfoxidation followed by a general-base-catalyzed elimination of acrolein from S-oxide 2.