Enzymatic assay for 3-hydroxyacyl-CoA and 2-trans-enoyl-CoA intermediates of beta-oxidation.

An enzymatic assay is presented for determining 3-hydroxyacyl-CoA and 2-trans-enoyl-CoA esters in tissue samples. The procedure includes extraction of acyl-CoA esters from frozen tissue samples with chloroform/methanol, stochiometric oxidation of the acyl esters to 3-keto-acyl-CoAs with 3-hydroxyacyl-CoA dehydrogenase in the presence or absence of enoyl-CoA hydratase, and an enzymatic cycling amplification of NADH produced for fluorometric determination. The procedure allows measurement of these intermediates of beta-oxidation at the picomole level. The method has been used successfully to measure the concentrations of 3-hydroxyacyl-CoA and 2-trans-enoyl-CoA esters in isolated rat hearts perfused with glucose or oleate or under anoxia.