Patients with generalized
peroxisomal disorders,
rhizomelic chondrodysplasia punctata, and
Refsum disease are all unable to alpha-oxidize 3,7,11,15-tetramethylhexadecanoic (
phytanic) acid. The exact cause of the oxidation defect in these patients is not well characterized, in part because there is only limited knowledge of the biochemical pathway. In 1969, the alpha-oxidation of
phytanic acid was reported [Tsai, S.-C.,
Avigan, J. & Steinberg, D. (1969) Studies on the alpha-oxidation of
phytanic acid by rat liver mitochondria, J. Biol. Chem. 244, 2682-2692] to involve the formation of an
alpha-hydroxyphytanic acid intermediate prior to removal of the alpha
carbon. Subsequently, most researchers have had difficulty detecting this intermediate. In the present study, cofactors known to form hydroxy intermediates by both
monooxygenase and
dioxygenase reaction mechanisms were incubated with purified rat liver peroxisomes and either [2,3-3H]
phytanic acid or [1-14C]
phytanic acid. Reaction products were separated by reverse-phase HPLC. A single reaction product, identified as
alpha-hydroxyphytanoyl-CoA rather than the
free fatty acid, was detected when
2-oxoglutarate/Fe+2/ascorbate, cofactors associated with a
dioxygenase reaction mechanism, were present. Concomitant with
alpha-hydroxyphytanoyl-CoA production, there was an increased accumulation of
formate and CO2. This increase in alpha-oxidation products is evidence that
alpha-hydroxyphytanoyl-CoA is a true pathway intermediate and that the entire pathway functions in peroxisomes. In contrast,
alpha-hydroxyphytanoyl-CoA was not formed in any quantity in mitochondria. These studies suggest that the alpha-hydroxylation step of
phytanic acid oxidation, which has been shown to be defective in
Refsum disease, is located in peroxisomes.