Absolute quantitation of tissue phospholipids using 31P NMR spectroscopy

Abstract

31P NMR spectroscopy has been used to quantitate phospholipids in tissue extracts without requiring their physical separation. Concentrated lipids were dissolved in chloroform-methanol-water 100:36:9 (v/v/v) containing a cesium salt of (ethylenedinitrilo)tetraacetic acid. Tri-n-butyl phosphate was added at the beginning of lipid extraction as an NMR internal standard, permitting the absolute quantitation of phospholipids in mumoles per gram of tissue. For efficient data collection, 10 mM chromium(III) acetylacetonate was included to promote relaxation. It was found that spectral peak separations could be optimized by manipulating the sample temperature. Phospholipid levels determined by NMR agreed with colorimetric measurements and literature values for rat liver and brain. Using a 0.5-1 g tissue sample and 800 averages (2 h acquisition), the coefficient of variation for total phospholipids was 2-3%.