-acyl-ethanolamine phospholipids in rat brains during post-decapitative ischemia: a 31 P NMR study

Phosphorus-31 nuclear magnetic resonance ( 31 P NMR) spectroscopy has been used to study accumulation of N -acyl-ethanolamine phospholipids in rat brains during post-decapitative ischemia. Lipids were extracted from rat brain homogenates and the extracts were thoroughly washed with aq. potassium ethylenediaminetetraacetic acid (EDTA). The lower organic phases were isolated and evaporated to dryness under a stream of nitrogen and the lipids were redissolved in CDCl 3 –CH 3 OH–H 2 O 100.0:29.9:5.2 (v/v/v) for NMR analysis. Increasing the period of post-decapitative ischemia resulted in an accumulation of two signals in the NMR spectra at 0.18 and 0.22 ppm (relative to the chemical shift of 1,2-diacylsn -glycero-3-phosphocholine (PC DIACYL ) at 2 0.84 ppm). These signals were identified as originating from 1,2-diacylsn -glycero-3-phospho-( N -acyl)-ethanolamine (NAPE DIACYL ) and 1-(1 9 -alkenyl)-2-acylsn -glycero-3-phospho( N -acyl)-ethanolamine (NAPE PLAS ), respectively, by spiking with authentic materials. Additionally, the identification was verified by thin-layer chromatography, which also showed the accumulation of N -acyl-ethanolamine phospholipids. The use of K-EDTA instead of the commonly used Cs-EDTA in the preparation of the NMR samples allowed the separation of the chemical shifts of N -acyl-ethanolamine phospholipids from those of the ethanolamine phospholipids. Moreover, the chemical shift of cardiolipin was moved from 0.15 ppm observed with Cs-EDTA to about 0.31 ppm with KEDTA. The present study demonstrates that it is possible to detect and quantify post-decapitative accumulation of NAPE subclasses (NAPE DIACYL and NAPE PLAS ) in rat brains by the use of 31 P NMR spectroscopy.— Moesgaard, B., J. W. Jaroszewski, and H. S. Hansen. Accumulation of N -acyl-ethanolamine phospholipids in rat brains during post-decapitative ischemia: a 31 P NMR study. J. Lipid Res. 1999. 40: 515– 521. Supplementary key words N -acyl-phosphatidylethanolamine • 1,2diacylsn -glycero-3-phospho-( N -acyl)-ethanolamine • 1-(1 9 -alkenyl)-2-acylsn -glycero-3-phospho-( N -acyl)-ethanolamine • cardiolipin • rat brain • ischemia • phosphorus nuclear magnetic resonance • plasmalogen The accumulation of N -acyl-ethanolamine phospholipids (NAPE) under the condition of post-decapitative ischemia has earlier been reported in rat brains using a chromatographic method (1). This rare lipid is of biological interest because it is the precursor for N -acyl-ethanolamines, some of which are ligands for the cannabinoid receptors (2), as well as because this class of phospholipids by itself may have membrane stabilizing properties and their accumulation may indicate neuronal injury (2, 3). Phosphorus-31 nuclear magnetic resonance ( 31 P NMR) spectroscopy is a convenient method for the determination of phospholipids (4), and the chemical shifts of many phospholipids extracted from biological tissues, including cardiolipin (CL), 1,2-diacylsn -glycero-3-phosphoethanolamine (PE DIACYL ), 1-(1 9 -alkenyl)-2-acylsn -glycero-3-phosphoethanolamine (PE PLAS ), ethanolamine lysophospholipids (LPE), 1-2-diacylsn -glycero-3-phosphocholine (PC DIACYL ), 1-(1 9 -alkenyl)-2-acylsn -glycero-3-phosphocholine (PC PLAS ), 1-alkyl-2-acylsn -glycero-3-phosphocholine (PC AA ), choline lysophospholipids (LPC), sphingomyelin (SPH), serine phospholipids (PS), inositol phospholipids (PI), and phosphatidic acid (PA) have been characterized (4–13). To our knowledge, NAPE has not previously been detected in biological samples by use of 31 P NMR. It was our objective to identify and quantify NAPE in extracts of animal tissue by the use of 31 P NMR. Abbreviations: CL, cardiolipin; EDTA, ethylenediaminetetraacetic acid; FID, free induction decay; LPC, choline lysophospholipids; LPE, ethanolamine lysophospholipids; NAPE, N -acyl-ethanolamine phospholipids; NAPE AA , 1-alkyl-2-acylsn -glycero-3-phospho-( N -acyl)-ethanolamine; NAPE DIACYL , 1,2-diacylsn -glycero-3-phospho-( N -acyl)-ethanolamine; NAPE PLAS , 1-(1 9 -alkenyl)-2-acylsn -glycero-3-phospho-( N -acyl)-ethanolamine; NAPE TRIOLEOYL , 1,2-dioleoylsn -glycero-3-phospho-( N -oleoyl)ethanolamine; NMR, nuclear magnetic resonance; PA, phosphatidic acid; PC, choline phospholipids; PC AA , 1-alkyl-2-acylsn -glycero-3-phosphocholine; PC DIACYL , 1,2-diacylsn -glycero-3-phosphocholine; PC PLAS , 1-(1 9 -alkenyl)-2-acylsn -glycero-3-phosphocholine; PE, ethanolamine phospholipids; PE AA , 1-alkyl-2-acylsn -glycero-3-phosphoethanolamine; PE DIACYL , 1, 2-diacylsn -glycero-3-phosphoethanolamine; PE PLAS , 1-(1 9 -alkenyl)-2-acylsn -glycero-3-phosphoethanolamine; PI, inositol phospholipids; PS, serine phospholipids; SPH, sphingomyelin; T 1 , longitudinal NMR relaxation time; T 2 *, apparent transverse NMR relaxation time; TLC, thin-layer chromatography. 1 To whom correspondence should be addressed. by gest, on S etem er 7, 2017 w w w .j.org D ow nladed fom