Peroxisomal fatty acid oxidation disorders and 58 kDa sterol carrier protein X (SCPx): activity measurements in liver and fibroblasts using a newly developed method

Sterol carrier protein X (SCPx) play a crucial role in the peroxisomal oxid ation of branched-chain fatty acids. To investigate whether patients with a n unresolved defect in peroxisomal beta-oxidation are deficient for SCPx, w e developed a novel and specific assay to measure the activity of SCPx in b oth liver and fibroblast homogenates. The substrate used in the assay, 3 al pha,7 alpha, 12 alpha-trihydroxy-24-keto-5 beta-cholestanoyl-CoA (24-keto-T HC-CoA), is produced by preincubating the enoyl-CoA of the bile acid interm ediate THCA with a lysate from the yeast Saccharomyces cerevisiae expressin g human D-bifunctional protein. lifter the preincubation period, liver or f ibroblast homogenate is added plus CoASH, and the production of choloyl-CoA is determined by HPLC. The specificity of the assay was demonstrated by th e finding of a full deficiency in fibroblasts from an SCPx knock-out mouse, In addition to SCPx activity measurements in fibroblasts from patients wit h a defect in peroxisomal beta-oxidation of unresolved etiology, we studied the stability and activity of SCPx in fibroblasts from patients with Zellw eger syndrome, which lack functional peroxisomes. We found that SCPx is not only stable in the cytosol, but displays a higher activity in fibroblasts from patients with Zellweger syndrome than in control fibroblasts. Furtherm ore, in all patients studied with a defect in peroxisomal beta-oxidation of unknown origin, SCPx was found to be normally active, indicating that huma n SCPx deficiency remains to be identified.