Enzymic determination of free fatty acids in serum

We describe the enzymic determination of free fatty acids in serum with use of acyl-CoA synthetase (EC l.2.1.3), acyl-CoA oxidase (no EC no. assigned) and peroxidase (EC 1.11.1.7). The free fatty acids are activated by acyl-CoA synthetase in the presence of ATP and coenzyme A. The acyl-CoA formed is oxidized by acyl-CoA oxidase to enoyl-CoA with simultaneous production of hydrogen peroxide, which is oxidatively coupled with 4-amino-antipyrine and 2,4-dibromphenol in the presence of peroxidase to yield a product that absorbs maximally at 505 nm. Standard curves prepared with various kinds of fatty acids are linear to at least 2.0 mmol/L and are practically congruent. Ascorbic acid or bilirubin interferes slightly. Results by the present method correlate well with those by the colorimetric method involving 2-(2-thiozolylazo)-p-cresol (r = 0.98). Replicate analyses of standard solution and of two kinds of control sera demonstrated the following between-assay precision: mean absorbance 0.224 (SD 0.002), CV 0.85%; mean concentration, 326 (SD 7.3) and 1076 (SD 22.7) mumol/L, CV 2.24 and 2.11%, respectively.