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 determination

 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.

Phosphatidylcholine determination

Phosphatidylcholine (or lecithin) is the most widespread phospholipid in living materials but is also used as an indicator in clinical diagnoses. Thus, there are several currently available methods for its quantitative estimation. Among them, methods include the molybdate-blue method or enzymatic analysis, thin-layer chromatography, HPLC, nuclear magnetic resonance and ultraviolet spectrometry.
A very sensitive method developed for the determination of trace amounts of lecithin in plasma (Bian W et al., Clin Chim Acta 2006, 368, 144). Norfloxacin can form a binary complex with Tb³⁺, which emits a peak at 545 nm. Lecithin can combine with this complex because of the phosphate radical, and can remarkably reduce its fluorescence intensity at 545 nm. The reduction in the fluorescence intensity of the Tb³⁺ ion is proportional to the concentration of lecithin. blanks. The linear range was 1.08x10^-6–3.02x10^-5 mol/l with a limit of detection of 2.54x10^-7 mol/l. This method has been successfully applied to the determination of serum samples.

Phosphoinositide determination

A novel method was reported for detecting and quantifying individual phosphoinositides via phosphoinositide-binding domains that exhibit high specificity and affinity toward this lipid (Furutani M et al., Anal Biochem 2006, 355, 8). Enzyme-linked immunosorbent assay wells are modified with alkyl chains (C16), which enables more uniform and quantitative immobilization of phosphoinositide-containing liposomes onto the well surfaces. Phosphoinositides, as the substrate or the product, are detected by pleckstrin homology domains that speciWcally bind to each phosphoinositide. By this method, phosphoinositide contents are measured with higher sensitivities than those by conventional methods. More importantly, both phosphoinositide kinase and phosphatase activities can be measured for purified enzymes and crude cellular lysates.