Comparison of fatty acid α‐oxidation by rat hepatocytes and by liver microsomes fortified with NADPH, Fe 3+ and phosphate

Rat liver microsomes, when fortified with NADPH, Fe 3+ and phosphate, can catalyze the oxidative decarboxylation (α‐oxidation) of 3‐methyl‐substituted fatty acids (phytanic and 3‐methylheptadecanoic acids) at rates that equal 60–70% of those observed in isolated hepatocytes (Huang, S., Van Veldhoven, P.P., Vanhoutte, F., Parmentier, G., Eyssen, H.J., and Mannaerts, G.P., 1992, Arch. Biochem. Biophys. 296 , 214–223). In the present study we set out to identify and compare the products and possible intermediates of α‐oxidation formed in rat hepatocytes and by rat liver microsomes. In the presence of NADPH, Fe 3+ and phosphate, microsomes decarboxylated not only 3‐methyl fatty acids but also 2‐methyl fatty acids and even straight chain fatty acids. The decarboxylation products of 3‐methylheptadecanoic and palmitic acids were purified by highperformance liquid chromatography and identified by gas chromatography/mass spectrometry as 2‐methyl‐hexadecanoic and pentadecanoic acids, respectively. Inclusion in the incubation mixtures of glutathione plus glutathione peroxidase inhibited decarboxylation by more than 90%, suggesting that a 2‐hydroperoxy fatty acid is formed as a possible intermediate. However, we have not yet been able to unequivocally identify this intermediate. Instead, several possible rearrangement metabolites were identified. In isolated rat hepatocytes incubated with 3‐methylheptadecanoic acid, the formation of the decarboxylation product, 2‐methylhexadecanoic acid, was demonstrated, but no accumulation of putative intermediates or rearrangement products was observed. Our data do not allow us to draw conclusions on whether the reconstituted microsomal system is representative of the cellular α‐oxidation system. However, the results we obtained with [3‐ 3 H]‐labelled fatty acids indicate that during α‐oxidation in intact cells the hydrogen at carbon‐3, which carries the methyl branch, is not attacked.