Sterol carrier protein X (
SCPx) plays a crucial role in the peroxisomal oxidation of branched-chain
fatty acids. To investigate whether patients with an unresolved defect in peroxisomal beta-oxidation are deficient for
SCPx, we developed a novel and specific assay to measure the activity of
SCPx in both liver and fibroblast homogenates. The substrate used in the assay, 3alpha, 7alpha,12alpha-trihydroxy-24-keto-5beta-cholestanoy l-
CoA (24-keto-THC-CoA), is produced by preincubating the enoyl-
CoA of the
bile acid intermediate THCA with a lysate from the yeast Saccharomyces cerevisiae expressing human D-bifunctional
protein. After the preincubation period, liver or fibroblast homogenate is added plus
CoASH, and the production of choloyl-
CoA is determined by HPLC. The specificity of the assay was demonstrated by the finding of a full deficiency in fibroblasts from an
SCPx knock-out mouse. In addition to
SCPx activity measurements in fibroblasts from patients with a defect in peroxisomal beta-oxidation of unresolved etiology, we studied the stability and activity of
SCPx in fibroblasts from patients with
Zellweger 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. Furthermore, in all patients studied with a defect in peroxisomal beta-oxidation of unknown origin,
SCPx was found to be normally active, indicating that human
SCPx deficiency remains to be identified.