A rapid, automated micromethod for measuring free fatty acids in plasma/serum

The investigation of mobilization and utilization of fat as fuel in human subjects requires the quantification of free fatty acids (FFA) in the circulating plasma. Methods in current use involve tedious extraction and titration, or enzymatic reaction with colorimetric or fluorometric detections. A rapid and reliable micro-technique is needed. We have adapted the manual enzymatic method of the Wako NEFAC kit to an automated rapid assay performed with a micro-centrifugal analyzer. This method depends upon the specific acylation of CoA by FFA, followed by oxidation and condensation to form a purple adduct measurable at 550 nm. The acylation step requires incubation at 37 degrees C for 10 min, a critical step for serum/plasma analysis. Only 4 microL of plasma is needed, and 20 tests can be performed in 20 min. The precision (CV) of sample analysis is within 2%. The results for the samples analyzed by this technique are within 4% (SEM 1%) of results by the manual method. Thus accurate results are achieved at reduced cost, time, and sample size.