Enzymatic fluorometric procedure for phospholipid quantification with an automated microtiter plate fluorometer

Abstract

We describe a new enzymatic assay for phospholipids that is rapid, sensitive, convenient, and inexpensive. The method is based on the fluorometric detection of H2O2, generated by the choline oxidase-catalyzed oxidation of choline, after liberation of choline from phospholipids by phospholipase D. Significant advantages over existing methods are that the entire reaction sequence can take place in a single vessel, a 12 × 8 well microtiter plate, and that the fluorescence intensity can be measured automatically with a Fluoroskan II (Labsystems Oy) detector. Extracting samples with organic solvents is unnecessary, although the method can be applied to extracts in isopropanol. The assay is approximately 60-fold more sensitive and has a limit of detection eightfold lower than currently available enzymatic colorimetric methods. Including solvent blanks, eight standards, and three quality-control pools, 34 samples can be pipetted and assayed in duplicate in 60 min. Results obtained by this procedure for total phospholipid choline in lipoproteins in primate plasma agreed well with those obtained for inorganic phosphorus by the Bartlett acid-digestion procedure.