Reduction pathway of cis-5 unsaturated fatty acids in intact rat-liver and rat-heart mitochondria: assessment with stable-isotype-labelled substrates Available to Purchase

Besides the conventional isomerase-mediated pathway, unsaturated fatty acids with odd-numbered double bonds are also metabolized by reduction pathways with NADPH as cofactor. The relative contributions of these pathways were measured in intact rat-liver and rat-heart mitochondria with a novel stable isotope tracer technique. A mixture of equal amounts of unlabelled cis-5-enoyl-CoA and ¹³C₄-labelled acyl-CoA of equal chain lengths was incubated with mitochondria. The isotope distribution of 3-hydroxy fatty acids produced from the first cycle of β-oxidation was analysed with selected ion monitoring by gas chromatograph-mass spectrometer. 3-Hydroxy fatty acids produced from the reduction pathway of unsaturated fatty acids were unlabelled (m+0) whereas those produced from saturated fatty acids were labelled (m+4). The m+0 content serves to indicate the extent of reduction pathway. Rotenone treatment was used to switch the pathway completely to reduction. The extent of m+0 enrichment in untreated mitochondria normalized to the m+0 enrichment of rotenone-treated mitochondria was the percentage of reduction pathway. With this technique, cis-4-decenoate was found to be metabolized completely by the reduction pathway in both liver and heart mitochondria. cis-5-Dodecenoate was metabolized essentially by the reduction pathway in liver mitochondria, but only to 75% in heart mitochondria. When the chain length was extended to cis-5-tetradecenoate, the reduction pathway in liver mitochondria decreased to 86% and that in heart mitochondria to 65%. The effects of carnitine, clofibrate and other conditions on the reduction pathway were also studied. Enrichments of the label on saturated fatty acids and 3-hydroxy fatty acids indicated that the major pathway of reduction was not by the direct reduction of the cis-5 double bond. Instead, it is most probably by a pathway that does not involve forming a reduced saturated fatty acid first.