Reduction pathway of cis-5 unsaturated fatty acids in intact rat-liver and rat-heart mitochondria: assessment with stable-isotype-labelled substrates.
Besides the conventional isomerase-mediated pathway, unsaturated fatty acids with old-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 beta-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.
Tserng
K Y
KY
Medical Research Service, Veterans Affairs Medical Center, Cleveland, Ohio 44106.
Jin
S J
SJ
Chen
L S
LS
D000818:Animals; D002247:Carbon Isotopes; D002331:Carnitine / Q000494:pharmacology; D002994:Clofibrate / Q000494:pharmacology; D003065:Coenzyme A / Q000737:chemistry / Q000378:metabolism; D004041:Dietary Fats / Q000008:administration & dosage; D004140:Dinitrophenols / Q000494:pharmacology; D005215:Fasting; D005231:Fatty Acids, Unsaturated / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D008297:Male; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley
D002247:Carbon Isotopes; D004041:Dietary Fats; D004140:Dinitrophenols; D005231:Fatty Acids, Unsaturated; D002994:Clofibrate; D002331:Carnitine; D003065:Coenzyme A