Rapid, sensitive fluorometric determination of serum triglyceride by measuring lipase-liberated fatty acids

Abstract

A continuous fluorescence displacement assay was developed for the measurement of long-chain fatty acids and utilized in the study of triglyceride lipase-catalyzed reactions (Wilton, DC. Biochem J 1991;276:129-33). We now describe a method for the rapid measurement of triglyceride in serum with the fluorescence displacement assay. The method involves the hydrolysis of a diluted sample equivalent to 0.1 microL of original serum with excess lipase (EC 3.1.1.3) from Rhizopus arrhizus and measuring the fatty acid released after a set time interval, normally 1 min. Under the conditions of the timed assay, about 2 mol of fatty acid are released per mole of triglyceride. The released fatty acid is monitored by fluorescence change and is proportional to the concentration of triglyceride in the original serum sample. The effective range of serum triglyceride concentration that could be measured was 0.5-10 mmol/L (0.44-8.8 g/L), based on triolein standard. The assay is unique in that it measures lipase-liberated fatty acids rather than liberated glycerol and as such is not subject to many of the criticisms of the glycerol-based methods. Comparison of fasted serum samples established a high correlation between the fatty acid and glycerol methods.