Oxidation of cis-5-unsaturated fatty acids in intact rat liver mitochondria: the operation of reduction pathways

Abstract

The metabolism of cis-5 unsaturated fatty acids was studied in intact rat liver mitochondria to assess the operation of a reduction pathway. By using direct quantification of metabolites with a capillary-column gas chromatography, 3-hydroxydodecanoate was identified among other metabolites when cis-5-dodecenoate was metabolized in intact rat liver mitochondria. The formation of 3-hydroxydodecanoate supports the existence of a reduction pathway in the metabolism of cis-5-unsaturated fatty acids. This metabolite cannot be produced from the conventional isomerase-mediated pathway. However, the data also indicated the possible operation of the conventional isomerase-mediated pathway in intact rat liver mitochondria. The reduction pathway appears to account for at least 61% of the pathway for cis-5-dodecenoate. This reduction pathway was likely to proceed from the dehydrogenation to trans-2,cis-5-dodecadienoyl-CoA, which was isomerized to delta 3, delta 5-dodecadienoyl-CoA, then to trans-2,trans-4-dodecadienoate. The reduction was mediated by 2,4-dienoyl-CoA reductase by the conversion of trans-2,trans-4-dodecadienoyl-CoA into trans-3-dodecenoyl-CoA. However, direct reduction of the cis-5 double bond was also shown to be operating, although to a lesser extent.

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

“…The ions m/z 233 and 236 were analysed for studies with cis-5-dodecenoyl-CoA and dodecanoyl-CoA as substrates. Oxidation of an equal mixture of cis-4-decenoyl-CoA and [3,4,5,[6][7][8][9][10][11][12][13] C 4 ]decanoyl-CoA in rat-liver and rat-heart mitochondria 3OHMC10 was the major metabolite detected in the incubation of an equal mixture of cis-4-decenoyl-CoA and [3,4,5,6-"$C % ]decanoyl-CoA in rat-liver and rat-heart mitochondria. As shown in Table 2, the concentration of 3OHMC10 was significantly increased after rotenone treatment in both liver and heart mitochondria.…”

“…On the other hand, saturated 3-hydroxyacyl-CoA is the metabolic intermediate from the reduction pathway. The ratio of 3-hydroxy and 3-hydroxy-cis-5 fatty acids produced in the incubation mixture might be a rough estimate of the percentage of reduction pathway even though its magnitude is expected to be underestimated [8]. Nevertheless, a higher percentage of reduction pathway would result in a higher 3OH-\3OHc5-metabolite ratio.…”

“…However, this estimation was based on assumptions that might not be met under the experimental conditions. An underestimation of the reduction pathway by this approach was suggested [8]. Here we report a novel method of determining the degree of pathways in intact mitochondria by using stable-isotope-labelled substrates.…”

“…As well as the conventional isomerase-mediated pathway [1], the oxidation of cis-5 unsaturated fatty acids also uses reduction pathways that are dependent on NADPH [2]. The major reduction pathway is postulated to mediate through the dehydrogenation of cis-5-enoyl-CoA to trans-2,cis-5-dienoyl-CoA, which is then isomerized to cis-3,cis-5-and trans-3,cis-5-dienoyl-CoAs as shown in Scheme 1 [3][4][5][6][7][8]. This isomerization is catalysed by ∆$,∆#-enoyl-CoA isomerase or peroxisomal tri-functional enzyme [3,5].…”

“…With excess NADPH, the metabolic pathway of cis-5-enoyl-CoA switches to the reduction pathway [7]. In a previous paper [8] we have demonstrated the existence of the reduction pathway in intact rat-liver mitochondria. The magnitude of this pathway was estimated to be at least 61 % on the basis of the ratio of released metabolic intermediates.…”