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Phytanic acid is broken down by alpha-oxidation in three steps finally yielding pristanic acid. The first step occurs in peroxisomes and is catalysed by phytanoyl-CoA hydroxylase. We have studied the second step in the alpha-oxidation pathway, which involves conversion of 2-hydroxyphytanoyl-CoA to pristanal catalysed by 2-hydroxyphytanoyl-CoA lyase. To this end, we have developed a stable isotope dilution gas chromatography-mass spectrometry assay allowing activity measurements in rat liver homogenates. Cell fractionation studies demonstrate that in rat liver 2-hydroxyphytanoyl-CoA lyase is localised in peroxisomes. This finding may have important implications for inherited diseases in man characterised by impaired phytanic acid alpha-oxidation.

To illustrate the clinical and biochemical heterogeneity of peroxisomal disorders, we report our experience with 27 patients seen personally between 1982 and 1997. Twenty patients presented with a phenotype corresponding either to Zellweger syndrome, neonatal adrenoleukodystrophy, or infantile Refsum disease, 3 of whom had a peroxisomal disorder due to a single enzyme defect. One patient had a mild form of rhizomelic chondrodysplasia punctata, 1 had classic Refsum disease. Finally, 5 patients presented with clinical manifestations that were either unusually mild or completely atypical, and initially did not arouse suspicion of a peroxisomal disorder. They showed multiple defects of peroxisomal functions with one or several functions remaining intact, suggesting a peroxisome biogenesis disorder. The defect in peroxisome biogenesis was further characterized by variable expression in different tissues and/or individual cells in 5 patients. Studies restricted to fibroblasts failed to identify abnormalities in this group. We demonstrate that clinical manifestations of peroxisomal disorders may be very mild or completely atypical, and therefore, peroxisomal disorders should be considered in a variety of clinical settings. Furthermore, we suggest performing extensive peroxisomal investigations in every patient suspected of suffering from a peroxisomal disorder, even when the clinical presentation is typical.

The degradation of the first intermediate in the alpha-oxidation of phytanic acid, 2-hydroxyphytanoyl-CoA, was investigated. Human liver homogenates were incubated with 2-hydroxyphytanoyl-CoA or 2-hydroxyphytanic acid, after which formation of 2-ketophytanic acid and pristanic acid were studied. 2-Hydroxyphytanic acid was converted into 2-ketophytanic acid and pristanic acid. When ATP, Mg2+, and coenzyme A were added to the incubation medium, higher amounts of pristanic acid were formed, whereas the formation of 2-ketophytanic acid strongly decreased. When 2-hydroxyphytanoyl-CoA was used as substrate, there was virtually no 2-ketophytanic acid formation. However, pristanic acid was formed in higher amounts than with 2-hydroxyphytanic acid as substrate. This reaction was stimulated by NAD+ and NADP+. Pristanic acid, and not pristanoyl-CoA was found to be the product of the reaction. These results suggest the existence of two pathways for decarboxylation of 2-hydroxyphytanic acid. The first one, starting from 2-hydroxyphytanic acid, involves the formation of 2-ketophytanic acid with only a small amount of pristanic acid being formed. The second pathway, which starts from 2-hydroxyphytanoyl-CoA, does not involve 2-ketophytanic acid and generates higher amounts of pristanic acid. The first pathway, which is peroxisomally localized, was found to be deficient in Zellweger syndrome, whereas the second pathway, localized in microsomes, was normally active. We conclude that the second pathway is predominant under in vivo conditions.

The structure and enzymology of the phytanic acid alpha-oxidation pathway have long remained an enigma. Recent studies have shown that phytanic acid first undergoes activation to its coenzyme A ester, followed by hydroxylation to 2-hydroxyphytanoyl-CoA. In this paper we have studied the mechanism of decarboxylation of 2-hydroxyphytanoyl-CoA in human liver. To this end, human liver homogenates were incubated with 2-hydroxyphytanoyl-CoA in the presence or absence of NAD+. Hereafter, the medium was analyzed for the presence of pristanal and pristanic acid by gas chromatography mass spectrometry. Our results show that pristanal is formed from 2-hydroxyphytanoyl-CoA. Pristanal is subsequently oxidized to pristanic acid in a NAD+ dependent reaction. These results finally resolve the mechanism of the phytanic acid alpha-oxidation process in human liver.