Release of 1-0-alkylglyceryl 3-phosphorylcholine, O-deacetyl platelet-activating factor, from leukocytes: Chemical ionization

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1 Proc. Natil. Acad. Sci. USA Vol. 77, No. 12, pp , December 1980 Biochemistry Release of 1-0-alkylglyceryl 3-phosphorylcholine, O-deacetyl platelet-activating factor, from leukocytes: Chemical ionization mass spectrometry of phospholipids (platelet aggregation/anaphylaxis mediator/phospholipase A2) JUDTH POLONSKY*, MARTNE TENCE*t, PERRE VARENNE*, BHUPESH C. DAS*, JEAN LUNELt, AND JACQUES BENVENSTEt *nstitut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France; tnstitut National de la Sante et de la Recherche MWdicale, U. 131, 32 rue des Carnets, Clamart, France; and *Rh6ne-Poulenc Recherche et D6veloppement, Centre Nicolas Grillet Vitry, France Communicated by D. H. R. Barton, August 12, 1980 ABSTRACT Evidence is presented for the simultaneous release of platelet-activating factor (PAF-acether) and of its deacetylated derivative (lyso-paf-acether) from hog leukocytes. On the basis of spectroscopy and chemical reactions, the structure of 0-deacetyl-PAF is shown to be 1-aalkylglyceryl 3-phosphorylcholine, an alkyl ether analog of lyso-phosphatidylcholine. Acetylation of lyso-paf yields a compound with biological activity and chromatographical behavior indistinguishable from those of native PAF. Lyso-PAF may be considered to be either the precursor or the enzymatic degradation product of PAF. The usefulness of chemical ionization mass for structural determination of phospholipids is Platelet-activating factor (PAF) is. a mediator of anaphylaxis and inflammation discovered in the early 1970s (1, 2). t aggregates rabbit, rat, guinea pig, and human platelets and liberates their vasoactive amines. PAF is released by blood leukocytes from various mammalian species and by macrophages, under immunological and nonimmunological stimuli (1-7). PAF was also shown to originate from platelets themselves during aggregation provoked by the ionophore A (8). Structural analysis of purified PAF preparations led, in 1977, to postulation of a phospholipidic structure for PAF, unique among the mediators of anaphylaxis (9). The low availability of PAF precluded its structural study by the currently used methods. Some insight into the structure of PAF nevertheless was gained by using different lipases and specific chemical treatments. The results obtained indicated that PAF was a 2- O-acylglyceryl phosphorylcholine not having an ester group at position 1 (9, 10). The absence of a hydroxyl group at position 1 was evidenced by lack of effect of attempted acetylation of highly purified PAF preparations on either the activity or the chromatographical behavior of PAF (10). During these experiments it was discovered that acetylation of partially purified PAF gave rise to a significant increase in PAF activity and that the increased activity was maintained after thorough purification. This observation suggested that a "precursor" of PAF was present in this preparation and that the ester function present in PAF might be an acetate group. This led to the partial synthesis of a highly active platelet-activating component possessing the physicochemical and biological properties of PAF. Because this was obtained by successive methylation, hydrogenation, and acetylation of the commercially available lyso-ethanolamine plasmalogen, the structure 1-O-alkyl-2-O-acetyl-sn-glyceryl 3-phosphorylcholine and the name PAF-acether were proposed (11). The same result has been reached by Hanahan's group (12) for PAF and also by Snyder's group (13) for antihypertensive polar renomedillary lipid (APRL) using choline plasmalogen as starting material. Recently, 1-O-octadecyl-2-O-acetyl-sn glyceryl 3-phosphorylcholine was synthesized and it showed biological properties identical with those of PAF-acether (unpublished data). We herein report the isolation and structural elucidation of the PAF-acether precursor, 1-O-alkylglyceryl 3-phosphorylcholine, which is released along with PAF by hog leukocytes. Acetylation of this glyceryl ether phosphorylcholine, named lyso-paf-acether, produced a compound with biological activity and chromatographical behavior indistinguishable from those of the native PAF-acether. We also demonstrate here the potential of chemical ionization (C) mass spectrometry for structural determination of phospholipids. MATERALS AND METHODS Chemicals. 1-O-Hexadecyl-rac-glycerol 3-phosphorylcholine (; n = 14) was purchased from dmark (Grfinwald, Munich). RED-Al [NaAlH2(OCH2-CH2-O-CH3)2, 70% in benzene] was supplied by Aldrich (Beerse, Belgium) Octadecyl-2-OAcglyceryl 3-phosphorylcholine (; n = 16), 1-0-octadecyl-rac-glycerol (; n = 16), and its isopropylidene derivative (V; n = 16) were gifts of J. J. Godfroid (Universit6 Paris V). Release of PAF-Acether and Lyso-PAF-Acether. Purification of hog blood leukocytes and extraction of the released lipids were detailed elsewhere (2, 9, 10). Briefly, leukocytes were purified by differential centrifugations and washings and then incubated for 18 hr at ph 9.5. The chloroform-soluble lipids extracted from leukocyte supernatants were then fractionated by silicic acid column chromatography and by highpressure liquid chromatography (HPLC) (9, 10). The publication costs of this article were defrayed in part by page Abbreviations: PAF, platelet-activating factor; lyso-, devoid of an acyl charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate chromatography; C, chemical ionization; E, electron impact; amu, group; HPLC, high-pressure liquid chromatography; TLC, thin-layer this fact. atomic mass units; PtdCho, phosphatidylcholine. 7019

2 7020 Biochemistry: Polonsky et al. Bioassay. Assay for platelet aggregation was performed on washed rabbit platelets as described (9, 10). PAF activity was expressed in arbitrary units; 1 unit is the amount, in Al, of medium necessary for 50% of the maximum aggregation induced by thrombin at 0.1 unit/ml. Chromatographic, Spectroscopic, and Chemical Procedures. Thin-layer chromatography (TLC) of phospholipids was performed on silica gel 60 F 254 plates (rck, Darmstadt, Federal Republic of Germany) in chloroform/methanol/water, 70:35:7 (vol/vol); TLC of other products was performed on silica gel F 1500 LS 254 plates (Schleicher & Schuell, Dassel, Federal Republic of Germany) which first were washed with ethanol, acetone, and diethyl ether and heated for 1 hr at 600C. Detection was by use of iodine vapor, Dittmer or Dragendorff reagents, or spraying with 50% sulfuric acid followed by heating. HPLC was conducted on a Varian liquid chromatograph, model 8500, equipped with a differential refractometer; Micropak Si-5 columns (Varian) 25 cm X 12.7 mm (8 mm inside diameter) were used and elution was performed with chloroform/methanol/water, 66:50:5 (vol/vol), at a flow rate of 200 ml/hr [at 100 bars (107 Pa)]. nfrared spectra were measured in chloroform solutions with a Perkin-Elmer 297 spectrophotometer. 1H NMR spectra were obtained with a Cameca spectrometer, and chemical shifts are given in ppm with respect to internal 4Si. Electron impact (E) mass spectra were recorded on an AE MS50 instrument. C mass spectra were measured at 'C and a gas (isobutane) pressure of torr ( Pa) in an AE MS9 spectrometer equipped with a C source (14). Reduction with RED-Al: The reagent (0.5 ml) was added to 5 mg of the natural phospholipid () dissolved in 1.5 ml of benzene. The solution was heated at 370C for 1 hr; products were recovered by extraction with diethyl ether after addition CH2 -R "0 x/e CH2-O P-OCH2- CH2N- 2 : :Oe0 of diluted sulfuric acid to the cooled solution. was purified by preparative TLC in methylene dichloride/methanol, 95:5 (vol/vol), and eluted from the silica gel with methylene dichloride/ethanol, 90/10 (vol/vol). (n = 14) w'as prepared in the same manner from 9.3 mg of (n = 14). The isopropylidene derivatives (V) were prepared by treatment of the corresponding 1-O-alkylglycerols with 2,2-dimethoxypropane and p-toluenesulfonic acid in benzene for 1.5 hr. The products were isolated by usual work-up and purified by preparative TLC in methylene dichloride/methanol, 96:4 (vol/vol). Mild acidic hydrolysis was carried out by treatment with 10% (wt/vol) trichloracetic acid for 30 min at 370C as described by Dawson (15). Catalytic hydrogenation was performed in 95% ethanol with platinum oxide as catalyst, for 3 hr, at a hydrogen pressure of 3 bars. Acetylation: Combined fractions bi and b2 of phospholipid (3.5 mg) were mixed with acetic anhydride (0.3 ml) and pyridine (0.3 ml) and kept at 220C for 18 hr. The reagents were removed under vacuum and the crude product was purified by HPLC. (n = 14) was prepared in the same manner from synthetic (n = 14) and had a retention time of 20 min on HPLC. RESULTS C Mass Spectrometry of Phospholipids. Failure to obtain interpretable mass spectra of glycerophospholipids under El prompted us to undertake an investigation of the use of C mass spectrometry for the determination of structure of phospholipids. Analysis of a large number of nonderivatized glycerophospholipids and also of sphingomyelin by Cl provided a substantial amount of structural information. n general, it showed MH+ of low abundance but displayed characteristic R.-CH2'.(CH2)j- CH3 R =-CO (CH2)ri CH3 Proc. Nati. Acad. Sci. USA 77 (1980) b a ion A: cleavage a + 2H ion B: A-H20 or cleavage b _ CH2 - O-CH2 (CH2) nch3 0 CH2-O- POCH2 CH2NM O ad.1 HO - CH2 CH2 N H M/ 90 HS FG. 1. Scheme. s CH2 =CH- N H m/ 72 CH2 - O-CH2 O / CH2-O=P-OCH2CH2N % C CH2 - O-CH2 (CH2)n CH3 0 (B 11 CH2-O =P O D

3 Biochemistry: Polonsky et al. fragment ion peaks of significant diagnostic value. The spectra of glycerophospholipids having the 2-hydroxy group free showed an intense peak corresponding to ion A and a weak peak due to ion B (Fig. 1); the intensity profile is modified in the spectra of phospholipids without a free hydroxyl at position 2. n the case of natural phospholipids, these ions are accompanied by homologous ions that differ by 28, 56, and 84 atomic mass units (amu), reflecting the homologous pattern of the fatty acid (or ether, amide) composition. The choline-containing phospholipids exhibited strong peaks at~m/z 90 and 72 in their C spectra. Peaks due to MH+-32 (ion C) and MH+-89 (ion D) were also observed in the spectra of choline-containing glycerophospholipids, possibly from the loss of methanol and N,N-dimethylethanolamine, respectively. These ions may arise by the transposition of one of the N-methyls to the phosphate group prior to fragmentation. showed peaks due to the same ions (C and D) originating from the additional loss of the elements of ketene (42 amu) from the acetyl group. Lyso-PAF-Acether (1-O-Alkylglyceryl 3-Phosphoryicholine). As described (9, 10), PAF-active fractions were eluted from silicic acid columns between sphingomyelin and a phospholipid isopolar with lyso-2-phosphatidylcholine (lyso-2- PtdCho). The lipids (35 mg from about 90 liters of hog blood), eluted subsequent to the PAF-active fractions, were purified, in several runs, by HPLC. The elution profile (Fig. 2) showed peaks with retention times identical to those of two species of sphingomyelin (al and a2) and to those of lyso-ptdcho (b, and b2). PAF-acether itself was eluted between a2 and b, when PAF-active fractions were chromatographed under the same conditions. The phospholipid fractions eluted in b, (4.7 mg) and in b2 (6.3 mg) were devoid of any PAF-acether activity, displayed identical R spectra, and had the same activity after acetylation (see below). Chemical and spectral studies of these fractions showed that they contained (ether analog of lyso- PtdCho) as the major component and that the two elution peaks (b, and b2) corresponded to different populations with respect to the alkyl-ether composition. Proc. Natl. Acad. Sci. USA 77 (1980) 7021 CH2-O-CH2-(CH2) -CH3 RO-CH 0 D /CH3 CH2-O- P--OCH2- CH2-N-CH3 CH3 : R = L n = 14,15,16,17... : R = COCH3 CH2 0- CH2 (CH2)= CH3 CH2-O- CHZ (CH2);7CH3 OHCH-o CH20H CH20 CH3 V CH2-0-CH2-(CH2-CH3 a CH-O zc CH3 CH2-0 V CH=ON %CH3 Cs CH20 CH3 V m/z lol Phospholipid migrated on TLC coincident with lyso- PtdCho and could be detected by the Dragendorff method and by the phosphorus-specific reagent. ts infrared spectrum showed no evidence of carbonyl function and displayed OH and P-O-choline absorptions (3350, 1082, 1040, and 965 cm'1). The presence of a choline polar head was further supported by the 250 MHz 1H NMR spectrum'which revealed a broad singlet (9H) at 3.33 ppm assigned to the -N+(CH3)3, group (16). The C spectrum (Fig. 3 Lower) of synthetic (n = 14) showed a weak MH+ peak at m/z 482 and significant fragment ions A, B, C, and D at m/z 317, 299, 450, and 393, respectively. njection FG. 2. -, HPLC profile of lipids eluted from the silicic acid column subsequent to the PAF-active fractions;..., HPLCprofileof acetylated b1 + b2 fractions.

4 7022 Biochemistry: Polonsky et al. luu. ns () Proc. Nati. Acad. Sci. USA 77 (1980) L) S i.n EEH N EEL jill WJ 1Alol UElR llbillill 100 U (s) 4"CM v ff ] (ML 4o (A) (D) 45S(C) l Al. SS(s) (MH*) '' iO FG. 3. (Lower) C mass spectrum of 1-0-hexadecyl-rac-glycerol 3-phosphorylcholine (synthetic ; n = 14). (Upper) C mass spectrum of phospholipid from fraction b2. The C spectrum (Fig. 3 Upper) of phospholipid from peak b was similar to that of (n = 14) and revealed the presence of a C16:0 alkyl group as the predominant homolog whereas that of fraction b, showed additional fragment ions 26 and 28 amu higher, reflecting the presence of C18:1 and C18:0 alkyl groups. The presence of the choline group was manifested by the occurrence of peaks at m/z 90 and 72 (see Fig. 1). That the phospholipid under investigation was was further substantiated by the preparation of the following derivatives. Reduction of (fractions b, and b2 combined) with RED-Al as described by Snyder et al. (17) produced. ts sensitivity to periodic acid supported the presence of vicinal hydroxyl groups and its infrared spectrum showed C-O-C absorption at 1110 cm 1 (18). The RF value of on silica gel TLC was similar to that of synthetic (n 16), and its C mass spectrum showed a strong MH+ peak at m/z 317, less intense peaks at m/z 343 and 345, and weak peaks at m/z 357 and 359 corresponding to C16:0, C18:1, C18:0, C19:1, and C19:0 alkyl chains. Treatment of with 2,2-dimethoxypropane afforded the isopropylidene derivative V which was compared with the derivatives V prepared from synthetic 1-O-octadecyl glycerol and 1-O-hexadecyl glycerol. All the three had similar RF values on silica gel TLC. The E mass spectra of the synthetic derivatives revealed the presence of the characteristic M-15 oxonium ion peaks at m/z 341 (V) (n = 14) and 369 (V) (n = 16) and also a peak at m/e 101 due to ion V. The spectrum of the compound presumed to be V derived from the natural phospholipid displayed the same peaks, indicating the presence of C16:0 (predominant) and C18:O alkyl ethers in the molecule. Acetylation of. Acetylation of (fractions b, and b2 combined) gave. HPLC showed it to be eluted as a sharp peak (Fig. 2) with a retention time of 18 min, similar to that of PAF-acether. The presence of the apetoxy group was confirmed by its infrared and 1H NMR spectra. The C mass spectrum of (Fig. 4) showed the expected MH+ at m/z 524 corresponding to the predominant homolog C16:0 in the O-alkyl chain. This was accompanied by peaks at m/z 552 and 550 due to the MH+ corresponding to C18:0 and C18:1 species, respectively. Characteristic fragment ion peaks were also observed at m/z 359 (C16), 387 (C18:0), and LL AbX 11 FG. 4. C mass spectrum of obtained by acetylation of (fractions b1 and b2 combined). 524 (MH-) t-

Biochemistry: Polonsky et al. (C18:1) due to ion A and also at m/z 341 (C16), 369 (C18:0), and 367 (C18:1) due to ion B. The peaks corresponding to ions C and D were also observed.

5 Biochemistry: Polonsky et al. (C18:1) due to ion A and also at m/z 341 (C16), 369 (C18:0), and 367 (C18:1) due to ion B. The peaks corresponding to ions C and D were also observed. This interpretation was confirmed by comparison with the C mass spectra of the synthetic (n = 14) and (n = 16) which showed the corresponding MH+ peaks at m/z 524 and 552 along with all the expected fragment ion peaks consistent with their structures. The acetylated derivative () possessed the biological properties of PAF-acether. When assayed on rabbit platelets, it displayed a powerful aggregating activity. This activity was not inhibited by the presence of indomethacin, aspirin, and adenosine diphosphate scavengers, but it was completely destroyed after treatment with phospholipase A2, thereby meeting the criteria for differentiating PAF from other aggregating agents (8, 10). ts specific activity, 18 units/ng (,t0. 1 nm) was of the same magnitude as that of the most purified native PAF preparations (7 units/ng) (10) and synthetic (n = 14) (7 units/ng). As in the case of native PAF-acether, the aggregating activity of the acetylated compound () was not affected by mild acid hydrolysis or by catalytic hydrogenation. Proc. Natl. Acad. Sci. USA 77 (1980) 7023 DSCUSSON Hog leukocytes release along with PAF-acether. The structure of this phospholipid, designated lyso-paf-acether, was determined by chemical and spectral means, particularly by C mass spectrometry. Whereas ethanolamine plasmalogen has been detected in human leukocytes (19), the presence of saturated choline glyceryl ethers has not yet been reported. The saturated C16 and C18 ether chains seemed to be the principal homologs in our preparations. Acetylation of lyso-paf-acether gave a compound that had chromatographical behavior and biological properties identical with those of PAF-acether. Results obtained from mild acidic hydrolysis and from catalytic hydrogenation suggested that the presence of a double bond on the ether chain was not a prerequisite for PAF-acether activity. The "yso" compound was shown not to be produced through saponification of PAF-acether during preparation and extraction steps. The PAF-acether activity present in the leukocyte supernatants was almost completely recovered in the extracted lipids (10). t also has been shown (10) that, at 22 C and ph 9.5, the release of PAF reaches a maximum after 2 hr of cell incubation and no further change is observed during the next 16 hr. Furthermore, incubation of natural PAF-acether and of the synthetic compound (; n = 16) at ph 9.0 and 10.6 for 22 hr did not affect the PAF activity. Recent results indicate that PAF-acether and its lyso counterpart are both actively released by ph 7.35 by various cell systems activated by different agonists (unpublished data). The active release of lyso-paf-acether suggests its possible role in PAF-acether metabolism. t might be that activation of cells, triggering acylation/deacylation of phospholipid constituents of the cell membrane (20), yields high quantities of this substance which therefore are available as a precursor for PAF-acether. On the other hand, one may consider that the release of this lyso compound results from PAF-acether deacylation, under the influence of cellular phospholipase A2 (or related enzymes), thereby contributing to the control of this potentially harmful mediator. Whatever the final interpretation of the present findings in metabolic terms, they may represent a step leading to a better understanding of the formation, release, and degradation of PAF-acether and therefore of the numerous physiological and pathological situations in which this mediator seems to play a predominant role (21). Whereas glycero-phospholipids have been examined by field desorption mass spectrometry (22), the possibility offered by C mass spectrometry for their structural analysis has so far remained unexplored. The results described here clearly demonstrate the successful application of C mass spectrometry to phospholipid structure determination. We thank Ms. C. Boullet, Ms. J. Bidault, and Mr. J. P. Le Couedic for excellent technical assistance and Mr. C. rienne for H NMR measurements at 250 MHz. 1. Siraganian, R. P. & Osler, A. G. (1971) J. mmunol. 106, Benveniste, J., Cochrane, C. G. & Henson, P. M. (1972) J. Exp. d. 136, Benveniste, J. (1974) Nature (London) 249, Fesfis, L., Csaba, B. & Muszbek, L. (1977) Clin. Exp. mmunol. 27, ncia-huerta, J. M. & Benveniste, J. (1979) Eur. J. mmunol. 9, Lynch, J. M., Lotner, G. Z., Betz, S. J. & Henson, P. M. (1979) J. mmunol. 123, Pinckard, R. M., Farr, R. S. & Hanahan, D. J. (1979) J. mmunol. 123, Chignard, M., Le Couedic, J. P., Tence, M., Vargaftig, B. B. & Benveniste, J. (1979) Nature (London) 279, Benveniste, J., Le Couedic, J. P., Polonsky, J. & Tence, M. (1977) Nature (London) 269, Tence, M., Polonsky, J., Le Couedic, J. P. & Benveniste, J. (1980) Biochimwe 62, Benveniste, J., Tence, M., Bidault, J., Boullet, C., Varenne, P. & Polonsky, J. (1979) C. R. Acad. Sci. Ser. D 289, Demopoulos, C. A., Pinckard, R. N. & Hanahan, D. J. (1979) J. Biol. Chem. 254, Blank, M. L., Snyder, F., Byers, L. W., Brooks, B. & Muirhead, E. E. (1979) Biochem. Biophys. Res. Commun. 90, Varenne, P., Bardey, B., Longevialle, P. & Das, B. C. (1977) Bull. Soc. Chim., Dawson, R. M. C. (1960) Biochemistry 75, Chapman, D. & Morrison, A. (1966) J. Biol. Chem. 241, Snyder, F., Blank, M. L. & Wykle, R. L. (1971) J. Biol. Chem. 246, Palameta, B. & Kates, M. (1966) Biochemistry 5, Gottfried, E. L. (1967) J. Lipid Res. 8, Hirata, F., Corcoran, B. A., Venkatasubramanian, K., Schiffmann, E. & Axelrod, J. (1979) Proc. Natl. Acad. Sci. USA 76, Benveniste, J. (1980) in Advances in Allergology and mmunology ed. Oehling, A. (Pergamon, Oxford) pp Wood, G. W., Lau, P. Y., Morrow, G., Rao, G. N. S., Schmidt, D. E. & Tuebner, J. (1977) Chem. Phys. Lipids 18,

Ammonia chemical ionization mass spectrometry of intact diacyl phosphatidylcholine

Ammonia chemical ionization mass spectrometry of intact diacyl phosphatidylcholine C. G. Crawford and R. D. Plattner Northern Regional Research Center, Agricultural Research Service, United States Department