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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The increase in free fatty acids in the ischemic tissue is a consistent observation and these free fatty acids are considered to play a role in the cellular toxicity. To elucidate the cause of higher levels of free fatty acids in ischemic tissue, we examined the catabolism of fatty acids. The beta-oxidation of lignoceric (24:0), palmitic (16:0) and octanoic (8:0) acids and the peroxidation of fatty acids were measured at different times of renal ischemia in whole kidney homogenate. The enzymatic activities for the oxidation of fatty acids decreased with the increase in ischemia time. However, the lipid peroxide levels increased 2.5-fold of control with ischemic injury. Sixty min of ischemia reduced the rate of oxidation of octanoic, palmitic and lignoceric acids by 57, 59 and 69%, respectively. Almost similar loss of fatty acid oxidation activity was observed in the peroxisomes and mitochondria. These data suggest that loss of mitochondrial and peroxisomal fatty acid beta-oxidation enzyme activities from ischemic injury may be one of the factors responsible for the higher levels of free fatty acids.

Rat kidney microsomal fraction is able to catalyze the enzymatic desaturation of eicosatrienoic acid (20:3n-6) to arachidonic acid (20:4n-6) by the delta 5 desaturase pathway, in the presence of reduced nicotinamide adenine dinucleotide (NADH), adenosinetriphosphate (ATP) and coenzyme A (CoA). The substrate of the reaction [1-14C]eicosa-8,11,14-trienoic acid (20:3n-6), was separated from the product [1-14C]eicosa-5,8,11,14-tetraenoic acid (20:4n-6) by reverse phase high-pressure liquid chromatography (RP-HPLC). These fatty acids were individually collected by monitoring the eluent at 205 nm and their radioactivity was measured by liquid scintillation counting. The delta 5 desaturase activity in kidney microsomes increased linearly with the substrate concentration up to 20 microM. Enzymatic activity was sensitive to pH with the maximum at 7.0 and was proportional with incubation time up to 10 min. The apparent Km and Vmax of delta 5 desaturase were 56 microM and 60 pmoles.min-1.mg-1 microsomal protein, respectively. Neither the cytosolic renal fraction nor the cytosolic liver fraction enhanced the delta 5 desaturase activity. Contrary to a report but in accordance to others, the present results suggest that rat kidneys can synthesize arachidonic acid at least to satisfy partially their needs for eicosanoid production.

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