Peroxisomal P-oxidation of branched chain fatty acids in human skin fibroblasts

Human skin fibroblasts in suspension are able to degrade [1-'4C]-labeled aand y-methyl branched chain fatty acids such as pristanic and homophytanic acid. Pristanic acid was converted to propionyl-CoA, whereas homophytanic acid was @-oxidized to acetyl-coA. Incubation of skin fibroblasts with [l-i4C]-labeled fatty acids for longer periods produced radiolabeled carbon dioxide, presumably by further degradation of acetyl-coA or propionyl-CoA generated by @-oxidation. Under the same conditions similar products were produced from very long chain fatty acids, such as lignoceric acid. Inclusion of digitonin ( > 10 pg/ml) in the incubations strongly inhibited carbon dioxide production but stimulated acetyl-coA or propionylCoA production from fatty acids. ATP, Mg2+, coenzyme A, NAD' and L-carnitine stimulated acetyl-coA or propionyl-CoA production from [l-'4C]-labeled fatty acids in skin fibroblast suspensions. Branched chain fatty acid @-oxidation was reduced in peroxisome-deficient cells (Zellweger syndrome and infantile Refsum's disease) but they were @-oxidized normally in cells from patients with X-linked adrenoleukodystmphy (ALD). Under the same conditions, lignoceric acid @-oxidation was impaired in the above three peroxisomal disease states. I These results provide evidence that branched chain fatty acid, as well as very long chain fatty acid, @-oxidation occurs only in peroxisomes. As the defect in X-linked ALD is in a peroxisomal fatty acyl-CoA synthetase, which is believed to be specific for very long chain fatty acids, we postulate that different synthetases are involved in the activation of branched chain and very long chain fatty acids in peroxisomes.-Singh, H., M. Brogan, D. Johnson, and A. Poulos. Peroxisomal &oxidation of branched chain methyl group, phytanic acid can only be degraded by aoxidation, i.e., one carbon cleavage. However, the branched chain fatty acids with aor y-methyl groups such as pristanic acid and homophytanic acid (4,8,12,16-tetramethylheptadecanoic acid) can theoretically be degraded by @-oxidation. Our in vitro studies with rat liver indicate that mitochondria lack VLCFA-CoA synthetase activity (2, 5). As VLCFA cannot be converted to VLCFA-CoA at the mitochondrial membranes, their oxidation is directed to peroxisomes. In contrast, we find that branched chain fatty acids can be converted to coenzyme A esters at the mitochondrial membranes (H. Singh, K. Beckman, D. Johnson, and A. Poulos, unpublished results), yet these fatty acids accumulate in some peroxisomal diseases (9). @-Oxidation of VLCFA has been investigated in several laboratories, yet there is limited information on the @oxidation of branched chain fatty acids. We and others reported (10-12) previously that a VLCFA oxidation defect can be demonstrated in X-linked adrenoleukodystrophy (ALD) using cell suspensions. However, the assay conditions were not investigated and the VLCFA oxidation was measured in the presence (10, 12) or absence (11) of digitonin in the incubations. In order to understand the molecular defect in peroxisomal diseases, we optimized fatty acids in human skin fibroblasts. J. Lipid Res. 1992. 33: 1597-1605. supplementary key words adrenoleukodystrophy Zellweger syndrome rhizomelic chondrodysplasia punctata . mitochondria . peroxisomes the assay conditions for the measurements of fatty acid oxidation in skin fibroblast suspensions. This is the first report to date providing detailed studies on fatty acid oxidation, including branched chain fatty acids, in cell suspensions. The data clearly indicate that the oxidation of fatty acids in cell suspensions is several-fold greater than that observed in cell culture (6, 13) and the cells in suspension retain the ability to produce carbon dioxide from fatty acids. In the present paper we describe the condiVery long chain fatty acids (VLCFA), i.e., > 2 2 carbons in length, accumulate in several peroxisomal diseases. Regest that VLCFA are degraded exclusively in peroxisomes cent studies from Our laboratory and Others sugtions for separate measurement of water-soluble products (1-8). In some of the peroxisomal diseases, in addition to VLCFA, branched chain fatty acids with aor @-methyl ____ groups such as pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) and phytanic acid (3,7,11,15-tetramethYlhexadecanoic acid) also accumulate (9). Due to the @Abbreviations: ALD, adrenoleukodystrophy; RCDP, rhizomelic chondrodysplasia punctata; LCFA, long chain fatty acids; VLCFA, very long chain fatty acids; TLC, thin-layer chromatography. 'To whom correspondence should be addressed. Journal of Lipid Research Volume 33, 1992 1597 by gest, on O cber 8, 2017 w w w .j.org D ow nladed fom and carbon dioxide from straight chain and branched chain fatty acids. We report that, in addition to VLCFA, branched chain fatty acid @-oxidation is defective in several peroxisomal diseases.