Novel molecular analogues of phosphatidylchoilines in a lipid extract from bovine brain: I-long-chaiin

A vasodepressor phospholipid fraction named Depressor-IA from a bovine brain lipid extract was analyzed by capillary gas-liquid chromatography-mass spectrometry as tert-butyldimethylsilyl derivatives after hydrolysis with phospholipase C. Results show that Depressor-IA is a mixture of 3 platelet-activating factors and 17 1-acyl analogues. Three platelet-activating factors having sn-1-0-hexadecyl, octadecyl, and octadecenyl groups were suggested to account for the hypotensive activity of Depressor-IA, although the total amount of 1-acyl analogues of platelet-activating factor was much more than that of platelet-activating factor in the purified Depressor-IA. 1-Long-chain acyl-2-short-chain acyl-glycero-3-phosphocholines identified in Depressor-IA included novel molecular analogues having sn-2-propionyl, acryloyl, butyryl, valeryl, caproyl, and hepatonoyl groups. -Tokumura, A., K. Takauchi, T. Asai, K. Kamiyasu, T. Ogawa, and H. Tsukatani. Novel molecular analogues of phosphatidylcholines in a lipid extract from bovine brain: 1-long-chain acyl-2-short-chain acyl-snglycero-3-phosphocholines. J Lipid Res. 1989. 30: 219-224. Supplementary key words molecular composition tot-butyldimethylsilyl derivatives positional isomer of glycerides GLC-MS PAF acyl PAF short-chain acyl group Platelet-activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a phospholipid mediator that is synthesized by a variety of animal tissues (1-3). Because of the very low cellular content of PAF, bioassays based on its in vitro platelet-activating effect or its in vivo vasodepressive effect have been used in many studies to demonstrate its existence in natural sources (1-3). Thus PAF has been characterized chemically in only limited kinds of cells (4-8) or tissue (9-11). Molecular heterogeneity of the sn-lalkyl moiety of PAF (in chain length and degree of unsaturation) has been demonstrated in stimulated and unstimulated neutrophils (5-8), lesional scales of psoriatic patients (9), and isolated rat uterus (10). Furthermore, l-acyl-2acetyl-sn-glycero-3-phosphocholine, an acyl analogue of PAF (acyl PAF) has been reported to coexist with PAF in rabbit (12) and human (13) neutrophils activated with Ca2'-ionophore A23187. Using conventional GLC-MS, we have recently identified more than 10 acyl PAFs including 1-long-chain acyl-2-propionyl-GPC and 1-long-chain acyl 2-butyryl-GPC together with PAF as the major components of a vasodepressor substance, which we tentatively named Depressor-IA in a lipid extract from bovine brain (14). In the present study, we have successfully applied high-resolution capillary GLC-MS for the analysis of Depressor-IA, and found an additional seven compounds of acyl PAF, which have not previously been detected in natural samples. O u r results demonstrate the coexistence with PAF of a family of biologically active choline phospholipids with different combinations of sn-1-long-chain acyl groups and sn-2-short-chain acyl groups. MATERIALS AND METHODS l-O-Hexadecyl-2-acetyl-GPC, l-O-octadecyl-2-acetyl-GPC, and their lyso compounds were obtained from Bachem Feinchemikalien AG, Bubendorf, Switzerland. 1-0-Octadecenyl-2-acetyl-GPC, l-myristoyl-2-lyso-GPC, l-palmitoyl2-lyso-GPC, l-stearoyl-2-lyso-GPC, l-oleoyE-2-lyso-GPC, Abbreviations: PAF, platelet-activating factor; acyl PAF, acyl analogue of platelet-activating factor; IysoPC, lysophosphatidylaholine; GPC, SRglycero-3-phosphocholine; GLC-MS, gas-liquid chromatography-mass spectrometry; tBDMS, Int-butyldimethylsilyl; TLC, thin-layer chromatog raphy; EI-MS, electron impact-mass spectrometry. The nomenclature used to designate various analogues of acyl PAF indicates the total number of carbons in the acyl chain at either SR-1 or sn-2 and the degrees of unsaturation, e.g., 16:0/3:1 represents 1-palmitoyl-2-acryloyl GPC. 'To whom correspondence should be addressed. Journal of Lipid Research Volume 30, 1989 219 by gest, on O cber 8, 2017 w w w .j.org D ow nladed fom 1,2-dipalmitoyl-GPC, 1,2-distearoyl-GPC, and 1,2-dioleoylGPC were purchased from Sigma Chemical Co., St. Louis, MO. Phospholipase C from Bacillus cereus, phospholipase A, from bee venom and lipase from Rhizopus arrhizus were also from Sigma. Acryloyl chloride and n-heptanoic anhydride were from Aldrich Chemicals, Milwaukee, WI. Acetic anhydride, n-propionic anhydride, n-butyric anhydride, nvaleric anhydride, and n-caproic anhydride were from Wako Pure Chemicals, Osaka, Japan. Perdeuterated acetic anhydride was from E. Merck, Darmstadt, West Germany. Other chemicals and solvents used were of analytical grade. Preparation of standard PAF and acyl PAF Various PAFs and acyl PAFs were prepared by treatment of the corresponding lyso compound with appropriate acid anhydride or acid chloride, essentially as described by Kumar et al. (15). LysoPCs with sn-2-palmitoy1, stearoyl, and oleoyl groups were prepared by treatments of 1,2-dipalmitoyl-GPC, 1,2-distearoyl-GPC, and 1,2-dioleoyl-GPC, respectively, with lipase from Rhizopus arrhizw, as described by Slotboom et al. (16) except for an addition of 4 ml diethylether to the reaction mixture (1 ml) to minimize acyl migration of 2-acyl lysoPC. The following acyl PAFs were prepared by the method described above: l-myristoyl-2-acety1(14:0/2:O)-GPC, 1-palmitoyl-2-acetyl(l6:0/2:O)-GPC, l-acetyl-2-palmitoyl(2:0/ 16:O)-GPC, l-stearoyl-2-acetyl(18:0/2:O)-GPC, l-acetyl-2stearoyl(2:0/18:0)-GPC, l-oleoyl-2-acetyl(l8:1/2:O)-GPC, 1acetyl-2-oleoyl(2:0/18:1)-GPC, l-palmitoyl-2-propionyl(l6:0/ 3:O)-GPC, l-palmitoyl-2-acryloyl(l6:0/3:1)-GPC, l-palmitoyl2-butyryl(16:0/4:0)-GPC, l-stearoyl-2-propionyl(l8:0/3:0)GPC, l-stearoyl-2-acryloyl(l8:0/3:1)-GPC, l-stearoyl-2-b~tyryl(l8:0/4:O)-GPC, l-oleoyl-2-butyryl(l8:1/4:0)-GPC, 1palmitoyl-2-valeryl(16:0/5:O)-GPC, l-palmitoyl-2-caproyl(l6:0/ 6:O)-GPC, l-palmitoyl-2-heptanoyl(l6:0/7:0)-GPC, l-palmitoyl-2-perdeuterated acetyl-GPC, and 1-0-hexadecyl2-perdeuterated acetyl-GPC. These phospholipids were converted to the tBDMS derivatives after their hydrolysis with phospholipase C (6). Analysis of PAF and acyl PAF in a lipid extract from bovine brain Lipids were extracted from bovine cerebrum, and Depressor-IA in the extract was purified as described previously (14). The partially purified Depressor-IA was then hydrolyzed with phospholipase C, and the resultant glycer-