Alk-1-enylacyl, alkylacyl, and diacyl subclasses of native ethanolamine and choline glycerophospholipids can be quantified directly by phosphorus-31 NMR in solution.

We show that phosphorus-31 nuclear magnetic resonance spectroscopy can be used to distinguish and to quantify the alk-1-enylacyl, alkylacyl, and diacyl glycerophosphoethanolamine (GPE) subclasses, and the respective glycerophosphocholine (GPC) subclasses, in their native form without prior degradation or derivatization, provided the phospholipids are observed in the nonaggregated state. Monomeric phospholipid distribution is ascertained by recording the spectra, after removal of metal ions, on CDCl3/CD3OD/D2O (50:50:15, by vol) solutions. The utility of this approach is exemplified for the ethanolamine glycerophospholipids (EPL) from bovine brain and the choline glycerophospholipids (CPL) from bovine heart. Sharp and well-resolved resonances are obtained for alkylacylGPE (+0.395 ppm; re 1% H3PO4), alkenylacylGPE (+0.353 ppm), and diacylGPE (+0.315 ppm), and for alkylacylGPC (-0.383 ppm), alkenyl-acylGPC (-0.436 ppm) and diacylGPC (-0.451 ppm). Integrated peak areas are shown to closely correlate with dose. Accurate quantitation of EPL and CPL subclasses at submicromolar levels can further be facilitated by use of synthetic dialkylGPE (+0.602 ppm) and dialkylGPC (-0.196 ppm) as internal standards. The method is simple, rapid, sensitive and reproducible, and permits the complete resolution and direct quantitation of all ethanolamine and choline glycerophospholipid subclasses quite independent of fatty chain length and degree of unsaturation.

Lanthanide-induced phosphorus-31 NMR downfield chemical shifts of lysophosphatidylcholines are sensitive to lysophospholipid critical micelle concentration.

Lysophosphatidylcholine (lysoPC) monomers or micelles in water give rise to a narrow, isotropic phosphorus-31 NMR signal (40.6 ppm; v1/2 1.7 Hz; 32.2 MHz). Upon addition of praseodymium ions, the phosphorus signals are shifted downfield. However, the downfield shifts for the longer-chain lysophosphatidylcholines, which exist in the aggregated state, are far greater than those for the shorter-chain homologues, which exist as monomers. At a Pr3+/lysoPC molar ratio of 0.5, the signals of C12lysoPC through C18lysoPC were shifted by 12.1 ppm, whereas the signals of C6lysoPC and C8lysoPC were shifted by only 2.26 ppm. This very pronounced difference in lanthanide-induced downfield shifts between micelles and monomers can be utilized to determine with accuracy lysoPC critical micelle concentrations (CMC) from downfield shift-vs.-concentration plots. The CMC values we determined were 57 mM for C8lysoPC, 5.7 mM for C10lysoPC, and 0.6 mM for C12lysoPC. The shift reagent phosphorus-31 nuclear magnetic resonance technique particularly lends itself to the measurement of CMC values in the millimolar and high micromolar range. The method can equally be used for measuring critical micelle concentrations of short-chain phosphatidylcholines.

Modulation of diacylglycerol acyltransferase by lysophosphatidylcholine and related monochain phospholipids.

Acyl-CoA : 1,2-diacylglycerol O-acyltransferase (EC 2.3.1.20) activity of rat liver microsomes was found to be stimulated by 1-acyl-sn-glycero-3-phosphocholine at low concentrations, but was inhibited above 0.2 mM. Diacylglycerol acylation was optimal at 75 microM lysophosphatidylcholine, resulting in a more than 2-fold activation of the enzyme. Acyltransferase activity disappeared above 0.5 mM lysophosphatidylcholine levels. 0.05% sodium taurocholate supplementation reduced diacylglycerol acyltransferase activity by approx. 2/3 over the entire range of lysophosphatidylcholine concentrations. 1-O-Hexadecylpropanediol-3-phosphocholine was shown to mimic lysophosphatidylcholine at stimulatory and at inhibitory concentrations in the absence and in the presence of sodium taurocholate, thus ruling out acyl-CoA depletion due to lysophosphatidylcholine acylation as a cause of depressed triacylglycerol synthesis at higher lysophosphatidylcholine levels. 1-Acyl-sn-glycero-3-phosphoethanolamine stimulated diacylglycerol acyltransferase to a lesser extent, without showing inhibition at higher concentrations. The data point towards a direct effect of the lysophospholipids on the acyltransferase system.