Errors in measuring plasma free fatty acid concentrations with a popular enzymatic colorimetric kit

Our goal was to test whether an enzymatic, colorimetric assay, the WAKO NEFA kit, provides information equivalent to liquid chromatography (LC) LC-based measures of free fatty acid (FFA).

We reanalyzed nadir FFA samples from 109 volunteers from a previous study where we demonstrated that maximal suppression of FFA concentrations predicts metabolic abnormalities in humans; the results from the WAKO NEFA kit, which has been widely used for over three decades, could not replicate our findings. We conducted additional studies to directly compare results from this kit to our LC-mass spectrometry (LC/MS) method that was validated by our LC-UV detection method.

Plasma samples with FFA concentrations ranging from 0.015 to 1.813 mmol/L were measured both by LC-mass spectrometry (LC/MS) and by the WAKO NEFA kit. Despite good overall agreement (R² = 0.86), the slope was significantly different from 1.0 and the intercept was significantly different from zero. The results from the kit were especially discrepant with FFA concentrations <0.200 and >1.000 mmol/L. Some of the discrepancy was related to the use of oleate as the standard solution for the kit and the substrate specificity of the kit enzymes for different fatty acids. Despite attempts to improve the kit by modifying the reaction time, sample volume and the types of standard solutions, we could not obtain a satisfactory agreement between the WAKO NEFA results and LC/MS.

The WAKO NEFA kit should not be used when high precision and accuracy of FFA concentrations over a wide range is required.