alpha-Oxidation of 3-methyl-substituted fatty acids in rat liver. Production of formic acid instead of CO2, cofactor requirements, subcellular localization and formation of a 2-hydroxy-3-methylacyl-CoA intermediate.

alpha-Oxidation of 3-methyl-substituted fatty acids in rat liver was studied in intact and permeabilized rat hepatocytes, and in homogenates and subcellular fractions. The experiments revealed that the primary end product of alpha-oxidation is formic acid, which is then converted to CO2. Rates of alpha-oxidation identical to those observed in intact hepatocytes were obtained in the permeabilized hepatocytes and liver homogenates when ATP, Mg2+ and CoA, and Fe2+, 2-oxoglutarate and ascorbate were added, suggesting that alpha-oxidation involves a fatty acid activation reaction and a dioxygenase reaction. Subcellular fractionation by differential and density gradient centrifugation demonstrated that alpha-oxidation is confined to peroxisomes, which produce formic acid that is converted to CO2, mainly in the cytosol. alpha-Oxidation in broken cell systems went hand in hand with the formation of a 2-hydroxy-3-methylacyl-CoA ester. Formation of the metabolite was strictly dependent on the presence of the above-mentioned cofactors, was confined to peroxisomes and was inhibited by fenoprofen and propyl gallate, inhibitors of alpha-oxidation in intact cells, indicating that the 2-hydroxyacyl-CoA ester is a bona fide intermediate of alpha-oxidation. Selective omission of cofactors from the reaction mixture and analysis of the incubation mixtures for 3-methyl fatty acids, 3-methyl fatty acyl-CoAs and their respective 2-hydroxy derivatives revealed that the activation reaction precedes the dioxygenase (hydroxylase) reaction. Our experiments demonstrate that alpha-oxidation is a peroxisomal process that consists of at least three reactions: fatty acid activation, hydroxylation and the reaction(s) involved in the release of formic acid.