REDUCTION PATHWAY OF CIS-5 UNSATURATED FATTY-ACIDS IN INTACT RAT-LIVER AND RAT-HEART MITOCHONDRIA - ASSESSMENT WITH STABLE-ISOTOPE-LABELED SUBSTRATES

Besides the conventional isomerase-mediated pathway, unsaturated fatty acids with odd-numbered double bonds are also metabolized by reductio n 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 amou nts of unlabelled cis-5-enoyl-CoA and C-13(4)-labelled acyl-CoA of equ al chain lengths was incubated with mitochondria. The isotope distribu tion of 3-hydroxy fatty acids produced from the first cycle of beta-ox idation 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 cont ent serves to indicate the extent of reduction pathway. Rotenone treat ment was used to switch the pathway completely to reduction. The exten t of m+0 enrichment in untreated mitochondria normalized to the m+0 en richment of rotenone-treated mitochondria was the percentage of reduct ion pathway. With this technique, cis-4-decenoate was found to be meta bolized completely by the reduction pathway in both liver and heart mi tochondria. cis-5-Dodecenoate was metabolized essentially by the reduc tion pathway in liver mitochondria, but only to 75% in heart mitochond ria. When the chain length was extended to cis-5-tetradecenoate, the r eduction pathway in liver mitochondria decreased to 86% and that in he art mitochondria to 65%. The effects of carnitine, clofibrate and othe r conditions on the reduction pathway were also studied. Enrichments o f the label on saturated fatty acids and 3-hydroxy fatty acids indicat ed 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 t hat does not involve forming a reduced saturated fatty acid first.