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Purified carnitine acetyltransferase was employed to catalyze the reaction:Carnitine+acetylCoA⇌acetylcarnitine+CoA. In the presence of a known excess of acetyl CoA, free CoA released was determined simultaneously in a coupled spectrophotometric assay. CoA formed was shown to be directly related to carnitine initially present. Rat tissue extracts were analyzed using this method, and μmoles of (–)-carnitine per gram dry weight were found to be 4.87 for heart; 3.14 for brown adipose tissue; 2.68 for skeletal muscle; 1.74 for testes; 1.26 for kidney; 0.55 for brain; and 0.051 for serum. The specificity and accuracy of the determinations are described.

Carnitine-H3 or palmitate-C14 incubated with heart muscle preparations was incorporated into a compound that had chromatographic behavior in several systems identical to that of palmitylcarnitine chemically synthesized from palmityl chloride and carnitine. Palmitylcarnitine biosynthesis from palmitic acid and carnitine was dependent upon ATP and CoA in addition to substrates and an enzyme preparation. In contrast, palmityl carnitine formation from palmityl CoA and carnitine did not require ATP or CoA. The following reaction, catalyzed by palmitylcarnitine transferase, was shown to be reversible:Palmitylcarnitine+CoA⇌palmitylCoA+carnitine.General chemical and metabolic properties of palmitylcarnitine were defined. Addition of palmitylcarnitine to heart muscle mitochondria increased respiration more than did addition of palmityl CoA, suggesting that palmitylcarnitine can more readily contribute its acyl group to the fatty acid oxidase system than can exogenous palmityl CoA. Carnitine increased degradation of pa1mityl-l-C14 CoA and steary1-l-C14 CoA to CO2 and increased total oxygen uptake in the absence of ATP if acyl CoA were present. Carnitine did not appreciably enhance respiration or increase conversion of pa1mitate-l-C14 to CO2 in the absence of ATP but did augment palmitate oxidation, as previously reported, when ATP and CoA were added to the system. Results are consonant with the hypothesis that the catalytic stimulation by carnitine of long-chain fatty acid oxidation is mediated via acylcarnitine formation, with subsequent transfer of the acyl group to CoA at the site of the fatty acid oxidase system.