Summary: The Wako enzymatic method relies upon the acylation of coenzyme A (CoA) by the fatty acids in the presence of added acyl-CoA synthetase (ACS). The acyl-CoA thus produced is oxidized by added acyl- CoA oxidase (ACOD) with generation of hydrogen peroxide, in the presence of peroxidase (POD) permits the oxidative condensation of 3-methy-N-ethyl-N(β-hydroxyethyl)-aniline (MEFA) with 4-aminoantipyrine to form a purple colored adduct which can be measured colorimetrically at 550 nm.