Assay of short-chain acyl coenzyme a intermediates in tissue extracts by high-pressure liquid chromatography

Abstract

A high-pressure liquid chromatographic method for the measurement of short- and medium-chain-length acyl-CoA compounds is described. Compounds are separated on a reverse-phase μBondapak C18 column with the order of elution based on differences in lipophilicity. The mobile phase consisted of variable mixtures of methanol and 50 mm KH2PO4, pH 5.3. Conditions are described that allow isocratic separation of groups of compounds of similar lipophilicity. With increasing methanol concentration, the more lipophilic compounds are eluted earlier. This has the effect of sharpening the peaks and improving quantitation. Detection of acyl-CoA intermediates is achieved using a uv detector and is based on the high absorbance of CoA-containing compounds at 254 nm. Neutralized perchloric acid extracts of tissues can thus be analyzed directly without further purification or derivatization. A mobile phase consisting of a 9:1 phosphate buffer-to-methanol mixture is used to separate CoASH, methylmalonyl-CoA, succinyl-CoA, β-hydroxy-β-methylglutaryl-CoA and acetyl-CoA. Increasing the methanol concentration to a 4:1 mixture allows separation of acetyl-CoA, propionyl-CoA, and isobutyryl-CoA, while with a 7:3 mixture of phosphate buffer to methanol, β-methylcrotonyl-CoA and isovaleryl-CoA are readily separated. Examples of results obtained using extracts from isolated hepatocytes, rat liver mitochondria, and perfused rat hearts incubated with α-ketoisocaproate, α-ketoisovalerate, or propionate are presented. In addition, methods and optimal conditions are presented for the analysis of malonyl-CoA, glutathione-CoA, dephospho-CoA, and oxidized CoA in tissue extracts. © 1981.