A spectrophotometric method for the determination of free fatty acid in serum using acyl-coenzyme A synthetase and acyl-coenzyme A oxidase.

A mixture of Ti(IV) and 4-(2-pyridylazo)resorcinol was found to be useful in the spectrophotometric determination of trace amounts of hydrogen peroxide. The absorbance at 508 nm was proportional to the concentration of hydrogen peroxide added. The reagent was successfully applied to the assay of free fatty acid in serum through the combined use of acyl-CoA synthetase and acyl-CoA oxidase. The latter enzyme produces H2O2. As a result, hydrogen peroxide was produced through the enzymatic oxidation of free fatty acid. It was possible to determine free fatty acid in 50 microliters of serum at concentrations ranging from 0.02 to 1.5 mM. The coefficient of variation was less than 3% at concentrations ranging from 0.1 to 1.5 mM. In the present method, there is the advantage of minimal influence from reducible substances as well as greater simplicity and accuracy.