ANALYSIS OF INDIVIDUAL PHOSPHOLIPIDS
Some phospholipids are so important in specialized studies, as in clinical investigations, that rapid and specific procedures were developed to quantify them easily in biological fluids or tissue extracts.
Sphingomyelin is important in cell membranes and in circulating lipoproteins where its determination is required for the study of diseases linked to the well known deficient activity of sphingomyelinase (Niemann-Pick disease).
One of the most efficient method is based on the direct treatment of the lipid extract by a bacterial sphingomyelinase which generates choline, subsequently quantified by the enzymatic generation of a fluorescent product (He X et al., Anal Biochem 2002, 306, 115). This procedure allows quantification of sphingomyelin over a large range (0.02-10 nmol) on a small volume of plasma (10 ml) and may be modified in an automated assay using a plate reader and a fluorescence detector.
Phosphatidic acid determination
The ability of negatively charged phosphatidates to form complexes with Fe3+ ions was used to design a simple spectrophotometric assay for the quantitative determination of phosphatidic acid (Dippe M et al., Anal Biochem 2009, 392, 169). In the reaction with the purple iron(III)–salicylate, phosphatidic acid extracts Fe3+ ions and decreases the absorbance at 490 nm. Neutral phospholipids such as phosphatidylcholine and phosphatidylethanolamine showed no influence on the absorbance of the iron(III) complex. The detection limit of the method on a microplate scale was 10 mM phosphatidic acid. Based on these results, an assay for determining the activity of phospholipase D toward natural phospholipids such as PC, PE, and PG was developed. In contrast to other spectroscopic, this method is able to determine phospholipase D activity toward different lipids or even lipid mixtures.