A spectrophotometric procedure for rapid and sensitive measurements of beta-oxidation. Demonstration of factors that can be rate-limiting for beta-oxidation.
1. A spectrophotometric direct-reading assay for measurements of beta-oxidation by intact mitochondria is described. The procedure relies on the ability of ferricyanide to trap reducing equivalents generated by the acyl-CoA dehydrogenases (EC 1.3.99.3). The reduction of ferricyanide was recorded by using a dual-wavelength spectrophotometer. 2. Oxaloacetate or acetoacetate was used to stimulate the rate of beta-oxidation by rotenone-blocked mitochondria. Although both were effective with rat liver mitochondria, oxaloacetate gave about 75% more stimulation. With heart or kidney mitochondria, only oxaloacetate gave marked stimulation. Acetoacetate had no stimulatory effect with heart mitochondria, but a small stimulatory effect on the rate of beta-oxidation by kidney mitochondria. 3. The stoicheiometry of beta-oxidation-dependent reduction of ferricyanide was examined, and good correlations were found between experimental and theoretical amounts of ferricyanide reduced. 4. Ferricyanide appears as efficient a final electron acceptor as O2. With ferricyanide the rate of beta-oxidation by heart mitochondria can be measured without interference from the oxidation of tricarboxylic acid-cycle intermediates.
D000090:Acetoacetates / Q000494:pharmacology; D000818:Animals; D002331:Carnitine / Q000031:analogs & derivatives / Q000494:pharmacology; D005292:Ferricyanides; D007700:Kinetics; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D010071:Oxaloacetates / Q000494:pharmacology; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D011506:Proteins / Q000378:metabolism; D051381:Rats; D012402:Rotenone / Q000494:pharmacology; D013053:Spectrophotometry / Q000379:methods*
D000090:Acetoacetates; D005292:Ferricyanides; D010071:Oxaloacetates; D011506:Proteins; D012402:Rotenone; D010088:Oxidoreductases; D002331:Carnitine