Sequential microenzymatic assay of cholesterol, triglycerides, and phospholipids in a single aliquot

The assay of multiple analytes in a single aliquot can be advantageous from both measurement and economic standpoints. The objective of this study was to develop a simple and sensitive microenzymatic method for the determination of three biologically important lipids. Triglycerides (as glycerol), phospholipids (as choline), and total cholesterol (as unesterified cholesterol) were assayed, in that order, by sequential addition of sample, reagents, and microbial enzymes directly into a single microtiter plate well, accompanied by continuous monitoring of a common reporter reaction in which hydrogen peroxide is quantified either by colorimetry with 4-aminoantipyrine and 3-hydroxy-2,4,6-triiodobenzoic acid or by ultraviolet fluorometry with p-hydroxyphenylacetic acid. The detection limit of the method is in the subnanomole mass range for all three lipids. Results obtained with either fluorescence or colored endpoints were in excellent agreement with alternative individual chemical and enzymatic procedures.