Resolution of phospholipid molecular species by ³¹P NMR

Abstract

Phosphorus-31 NMR chemical shifts of phospholipids (PLs) solubilized in bile salts were studied with respect to variations in the structure of the acyl substituents. The presence of double bonds in the acyl chains of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidic acid (PA) induced small but consistent upfield shifts relative to the corresponding disaturated acyl PL signals. The magnitudes of the unsaturation-related upfield shifts were approximately twice as large in sodium cholate as in sodium deoxycholate. Chemical shift separations of PC, PE, and PG dipalmitoyl-dioleoyl species pairs increased slightly at lower temperatures. Resolution of the PC species pair was maximized and nearly independent of cholate-to-PL ratio at values greater than about 100 in 2% cholate. Only the PA species resolution varied significantly over the pH range 6.5 to 9.5, in the vicinity of its pK2. Shift differences for a homologous series of disaturated acyl PCs showed a logarithmic dependence on chain length. Spectra for a variety of PC standards were used to interpret the composite PC signals from egg yolk extract, soybean extract and whole human amniotic fluid. The 31P NMR analyses were consistent with published chromatographic studies. Interpretation of composite PL signals in the cholate system is simplified for mixtures having restricted acyl chain length and degree of unsaturation.

Analysis of phospholipid molecular species in brain by31P NMR spectroscopy

“…For example, 16:0/15Ј-HPETE-PC refers to the derivative of 16:0/20: 4-PC with a hydroperoxy group at C-15 of the sn-2 acyl substituent (20:4 ϭ ETE ϭ eicosatetraenoic acid) and a double bond at the C-13,14 position due to migration of the double bond originally at C-14,15 into conjugation. Quasispecies format: A partial sorting of species by 31 P NMR chemical shift (␦) occurs in the PL ad system (see below) (12), providing structural information intermediate between the species and headgroup class levels. Groupings are represented by two-letter prefixes.…”

“…The signal from 16:0/16:0-PC furnished a convenient internal chemical shift reference due to its ubiquity, ease of resolution, and previous correlation with the standard 31 P NMR zero shift reference, 85% H 3 PO 4 (10,12,18). A PC shift near Ϫ0.5 ppm, although not a consensus value in the 31 P NMR literature, has been obtained using a variety of approaches (18,19).…”

“…High resolution 31 P NMR has the advantages of simultaneous detection and quantitation of numerous PLs, including unsuspected and unidentified PLs, and its nondestructive nature. By solubilization in an aqueous solution of sodium cholate under very specific conditions (12), we have resolved the 31 P NMR signals of phosphatidylcholine (PC) molecular species of differing unsaturation or chain length. We have applied this technique to measurement of saturated PC in human amniotic fluid (13), for which sample preparation is relatively straightforward.…”

Changes in Cellular and Plasma Membrane Phospholipid Composition after Lipopolysaccharide Stimulation of Human Neutrophils, Studied by31P NMR

“…Ach2GroPCho) and that of the monounsaturated species (e.g. Ole,GroPCho) being located at peak 9 (Pearce and Komoroski, 1993). PtdCho species containing polyunsaturated acyl chains, such as SteA,AchGroPCho gave peak 10.…”

Current and future applications ofin vitromagnetic resonance spectroscopy in hepatobiliary disease

“…It has also been utilized for studying the effects of cholesterol on the fluidity of human gallbladder bile as well as for quantifying micellar PTC concentrations [61,62] . 31 P NMR spectroscopy has been used to quantify phospholipids in red cell membranes and in model bile salts [63] . Such 31 P MRS studies have shown higher concentrations of PTC and Pi in gallbladder bile compared to canalicular bile [64] .…”