A semiautomated enzymatic method for determination of nonesterified fatty acid concentration in milk and plasma

Abstract  An enzymatic assay for the determination of non-esterified fatty acid concentrations in milk and plasma is described. The procedure is semiautomated for use with a plate luminometer or plate spectrophotometer and enables routine batch processing of large numbers of small samples (≤5 μL). Following the activation of nonesterified fatty acids (NEFA) by acyl-CoA synthetase, the current assay utilizes UDP-glucose pyrophosphorylase to link inorganic pyrophosphate to the production of NADH through the reactions catalyzed by phosphoglucomutase and glucose-6-phosphate 1-dehydrogenase. With this assay sequence the formation of NADH from NEFA is complete within 50 min at 37°C. Enzymatic spectrophotometric techniques were unsuitable for NEFA determination in human milk due to the opacity of the sample. The use of the NADH-luciferase system has overcome this problem, allowing the enzymatic determination of NEFA in human milk. Sample collection and treatment procedures for milk and plasma have been developed to prevent enzymatic lipolysis and to limit interference from enzymes present in milk. The recovery of palmitic acid added to milk and plasma samples was 94.9±2.9 and 100±4.5%, respectively. There was no difference (P=0.13) in plasma NEFA concentrations determined by the current method and a commercially available enzymatic spectrophotometric technique (Wako NEFA-C kit). Plasma NEFA concentrations determined by gas chromatography were 28% higher compared to both the Wako NEFA-C kit and the current method.