Biochemical Analysis in Peroxisomal Disorders

Abstract

It has become clear in recent years that several genetic diseases in man are caused by mutations leading to impaired peroxisomal functions. The Zellweger syndrome can be considered to be the prototype of this newly recognized group of disorders. The following approach is used to study peroxisomal functions in patients suspected of suffering from a peroxisomal disorder. First, plasma levels of di- and trihydroxycoprostanoic acid, very long chain fatty acids, pipecolic acid and phytanic acid are measured. Secondly, in order to establish whether peroxisomes are present the amount of particle- bound catalase in fibroblasts is determined by digitonin titration. Third, peroxisomal functions (activity of dihydroxyacetone phosphate acyltransferase and alkyl dihydroxyacetone phosphate synthase, de novo plasmalogen biosynthesis, and peroxisomal fatty acid β-oxidation) are measured in cultured skin fibroblasts. Fourth, the biosynthesis of peroxisomal β-oxidation enzyme proteins is studied by means of pulse/chase and/or continuous pulse experiments in fibroblasts. Finally, immunoblotting experiments are carried out in liver to determine whether peroxisomal β-oxidation proteins are deficient or not. The results allow one to distinguish between three groups of peroxisomal disorders. In the first group there is a generalizedloss of peroxisomal functions (Zellweger syndrome; neonatal adrenoleukodystrophy; infantile Refsum disease). In the second group some but not all peroxisomal functions are impaired (the rhizomelic type of chondrodysplasia punctata). In the third group there is a loss of a single peroxisomal function (X-linked adrenoleukodystrophy, adult Refsum disease, acatalasaemia and pseudo Zellweger syndrome).Using immunoblotting analysis and in vitro peroxisomal β-oxidation activity measurements we show that in pseudo—Zellweger syndrome there is a deficiency of peroxisomal 2-oxoacyl-CoA thiolase. Finally, attention will be paid to the defect in X-linked adrenoleukodystrophy. In X-linked adrenoleukodystrophy there is an impairment in the peroxisomal oxidation of lignocerate but not of palmitate. Evidence will be presented that the two fatty acids are activated by different acyl-CoA ligases. Total lignoceroyl-CoA synthetase activity was, however, not deficient in X-linked adrenoleukodystrophy fibroblasts.KeywordsPhytanic AcidPipecolic AcidZellweger SyndromePeroxisomal DisorderRefsum DiseaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.