Phospholipids Analysis by 31P NMR

Abstract

Cell membrane phospholipids can be identified and quantitated using ³¹P NMR spectroscopy in conjunction with an analytical reagent composed of chloroform-benzene(d6)/methanol-CsEDTA 2:l ml/ml. 3 ml of this reagent dissolves between 0.01–100 mg crude tissue lipids obtained by the Folch procedure. When the source phospholipids are strongly contaminated with cations, it is necessary to modify the extraction method, backwashing with K-EDTA, 0.6 M, pH 6, instead of KC1. Also if source tissues must be stored for long periods of time, acetone desication is recommended. Using a 500 MHz ³¹PNMR spectrophotometer (magnetic field ?11.75 T), the extracted phospholipids yield narrow Lorenzian signals (1.8–3.2 Hz at half-height), with these widths at half-height corresponding to their 1/πT₂ values. Chemical shifts (δ) at 24 °C, following the IUPAC shift convention and relative to 85% phosphoric acid, were determined as follows:CAEP,21.09;LPG,l,O9;LPA,0.83;LPE plas,0.53;PG,0.50;LPE,0.43; PA,0.25;CL,0.18;LPI,0.10;PE plas,0.07;PE,0.03;PS,?0.O5;SPH,?0.O9; DiMePE,?b.18;LPC plas,?0.20;LPC,?0.28;PI,?0.37; PAF, ?0.70;PC plas,?0.77; PC, ?0.84. This reagent permits assays of high precision and accuracy that use little spectrometer time and that are suitable for automated procedures.