A quantitative gas chromatography-mass spectrometry (GC/MS) method was developed to measure nanomolar quantities of long-chain saturated beta-hydroxy fatty acids (12, 14, 16, and 18 carbons long) produced by isolated ischemic heart. Only beta-hydroxymyristate (25-40 nmol/g dry) was found in fresh heart. Isolated rabbit heart perfused with fatty acid by the nonrecirculating Langendorff technique produced negligible beta-hydroxy fatty acids. Ischemic perfusion with 0.25-0.75 mM palmitate prompted heart beta-hydroxy fatty acid accumulation, beta-hydroxypalmitate greater than beta-hydroxystearate, up to 100 nmol x g dry-1 x 10 min-1. beta-Hydroxy fatty acid production was proportional to coronary effluent lactate-to pyruvate ratio, did not continue beyond 10 min of ischemia, was dependent on exogenous fatty acid, and was inhibited by coperfusion with 10 mM acetate. Reperfusion for 5-10 min dissipated accumulated beta-hydroxypalmitate. Hypoxic perfusion prompted beta-hydroxy fatty acid production comparable to that with severe ischemia. These data show that during oxygen deficiency heart fatty acid beta-oxidation is not only depressed but is also incomplete; beta-hydroxy fatty acyl intermediates accumulate and contribute to the increased intracellular fatty acid content characteristic of the ischemic myocardium.

Long chain saturated beta-hydroxy fatty acid content and oxidative metabolism were studied in hearts of diabetic mice (C57BL/KsJ db/db) with a progressive cardiomyopathy at intervals of 7, 10, 16, and 26 wk of age. Total beta-hydroxy fatty acid (BHFA) content increases progressively with age in diabetic hearts with a mean value of 143.5 nmol/g dry wt as compared with a mean of 59.6 nmol/g dry wt in control hearts. There was also a redistribution of BHFA in myocardium of diabetic mice when compared with controls, with a relative decrease in beta-hydroxymyristate and an increase of beta-hydroxypalmitate. Oxidative phosphorylation studies using isolated mitochondria from diabetic mice demonstrated depressed state 3 oxidation rates with both palmityl carnitine and pyruvate as substrates. While mitochondrial NADH-oxidase activity was not statistically different from that of controls, there was a significant decrease in mitochondrial total NAD + NADH content in diabetic hearts. In addition, treatment of myocardial tissue with lanthanum demonstrated an abnormal permeability of sarcolemmal, intercalated disc as well as mitochondrial membranes in myocytes of diabetic mice. The data indicate that deficiencies in total NAD + NADH content can account for the depressed state 3 oxidation of palmitylcarnitine and pyruvate in diabetic mice that in turn may explain the abnormal accumulation of BFHA. The latter could play a role in altering the permeability of cardiac cell membranes.

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A restriction endonuclease cleavage map is presented for mouse mitochondrial DNA. This map was constructed by electron microscopic measurements on partial digests containing fixed D-loops, and by electrophoretic analysis of partial and complete single enzyme digests, and of double digests. No map differences were detected between mitochondrial DNA from cultured LA9 cells and an inbred mouse line for the six endonucleases used. Three cleavage sites recognized by HpaI, five sites recognized by HincII, two sites recognized PstI and four sites recognized by BamI were located with respect to the origin of replication and the EcoRI and HinIII sites previously determined by others. No cleavages were produced by KpnI or SalI. The migration of linear DNA with a molecular weight greater than 1 X 10(6) was not a linear function of log molecular weight in 1% agarose gels run at 6.6 volts/cm.