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Home , Dimethylethanolamine, Phosphorylcholine

Proc. Natil. Acad. Sci. USA Vol. 77, No. 12, pp. 7019-7023, December 1980 Biochemistry

Release of 1-0-alkylglyceryl 3-, O-deacetyl platelet-activating factor, from leukocytes: Chemical ionization mass spectrometry of (platelet aggregation/anaphylaxis mediator/) JUDITH POLONSKY*, MARTINE TENCE*t, PIERRE VARENNE*, BHUPESH C. DAS*, JEAN LUNELt, AND JACQUES BENVENISTEt *Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France; tInstitut National de la Sante et de la Recherche MWdicale, U. 131, 32 rue des Carnets, 92140 Clamart, France; and *Rh6ne-Poulenc Recherche et D6veloppement, Centre Nicolas Grillet 94400 Vitry, France Communicated by D. H. R. Barton, August 12, 1980

ABSTRACT Evidence is presented for the simultaneous physicochemical and biological properties of PAF. Because this release of platelet-activating factor (PAF-acether) and of its was obtained by successive methylation, hydrogenation, and deacetylated derivative (lyso-PAF-acether) from hog leukocytes. On the basis of spectroscopy and chemical reactions, the acetylation of the commercially available lyso- structure of 0-deacetyl-PAF is shown to be 1-aalkylglyceryl plasmalogen, the structure 1-O-alkyl-2-O-acetyl-sn-glyceryl 3-phosphorylcholine, an alkyl ether analog of lyso-phosphati- 3-phosphorylcholine and the name PAF-acether were proposed dylcholine. Acetylation of lyso-PAF yields a compound with (11). The same result has been reached by Hanahan's group (12) biological activity and chromatographical behavior indistin- for PAF and also by Snyder's group (13) for antihypertensive guishable from those of native PAF. Lyso-PAF may be consid- ered to be either the precursor or the enzymatic degradation polar renomedillary lipid (APRL) using plasmalogen product of PAF. The usefulness of chemical ionization mass as starting material. Recently, 1-O-octadecyl-2-O-acetyl-sn for structural determination of phospholipids is glyceryl 3-phosphorylcholine was synthesized and it showed biological properties identical with those of PAF-acether (un- published data). Platelet-activating factor (PAF) is. a mediator of anaphylaxis We herein report the isolation and structural elucidation of and inflammation discovered in the early 1970s (1, 2). It ag- the PAF-acether precursor, 1-O-alkylglyceryl 3-phosphoryl- gregates rabbit, rat, guinea pig, and human platelets and lib- choline, which is released along with PAF by hog leukocytes. erates their vasoactive . PAF is released by blood leu- Acetylation of this glyceryl ether phosphorylcholine, named kocytes from various mammalian species and by macrophages, a with under immunological and nonimmunological stimuli (1-7). lyso-PAF-acether, produced compound biological ac- PAF was also shown to originate from platelets themselves tivity and chromatogrAphical behavior indistinguishable from during aggregation provoked by the ionophore A 23187 (8). those of the native PAF-acether. We also demonstrate here the Structural analysis of purified PAF preparations led, in 1977, potential of chemical ionization (CI) mass spectrometry for to postulation of a phospholipidic structure for PAF, unique structural determination of phospholipids. among the mediators of anaphylaxis (9). The low availability of PAF precluded its structural study by the currently used MATERIALS AND METHODS methods. Some insight into the structure of PAF nevertheless Chemicals. 1-O-Hexadecyl-rac- 3-phosphoryl- was gained by using different lipases and specific chemical choline (I; n = 14) was purchased from Medmark (Grfinwald, treatments. The results obtained indicated that PAF was a 2- Munich). RED-Al [NaAlH2(OCH2-CH2-O-CH3)2, 70% in O-acylglyceryl phosphorylcholine not having an ester group benzene] was supplied by Aldrich (Beerse, Belgium). 1-0- at position 1 (9, 10). Octadecyl-2-OAcglyceryl 3-phosphorylcholine (II; n = 16), The absence of a hydroxyl group at position 1 was evidenced 1-0-octadecyl-rac-glycerol (III; n = 16), and its isopropylidene by lack of effect of attempted acetylation of highly purified derivative (IV; n = 16) were gifts of J. J. Godfroid (Universit6 PAF preparations on either the activity or the chromatogra- Paris VII). phical behavior of PAF (10). During these experiments it was Release of PAF-Acether and Lyso-PAF-Acether. Purifi- discovered that acetylation of partially purified PAF gave rise cation of hog blood leukocytes and extraction of the released to a significant increase in PAF activity and that the increased lipids were detailed elsewhere (2, 9, 10). Briefly, leukocytes activity was maintained after thorough purification. This ob- were purified by differential centrifugations and washings and servation suggested that a "precursor" of PAF was present in then incubated for 18 hr at pH 9.5. The chloroform-soluble this preparation and that the ester function present in PAF lipids extracted from leukocyte supernatants were then frac- might be an acetate group. This led to the partial synthesis of tionated by silicic acid column chromatography and by high- a highly active platelet-activating component possessing the pressure 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 "ad- group; HPLC, high-pressure liquid chromatography; TLC, thin-layer vertisement" in accordance with 18 U. S. C. §1734 solely to indicate chromatography; CI, chemical ionization; EI, electron impact; amu, this fact. atomic mass units; PtdCho, . 7019 Downloaded by guest on October 1, 2021 7020 Biochemistry: Polonsky et al. Proc. Nati. Acad. Sci. USA 77 (1980) Bioassay. Assay for platelet aggregation was performed on of diluted sulfuric acid to the cooled solution. III was purified washed rabbit platelets as described (9, 10). PAF activity was by preparative TLC in methylene dichloride/, 95:5 expressed in arbitrary units; 1 unit is the amount, in Al, of me- (vol/vol), and eluted from the silica gel with methylene di- dium necessary for 50% of the maximum aggregation induced chloride/, 90/10 (vol/vol). III (n = 14) w'as prepared by thrombin at 0.1 unit/ml. in the same manner from 9.3 mg of I (n = 14). The isopropyl- Chromatographic, Spectroscopic, and Chemical Proce- idene derivatives (IV) were prepared by treatment of the cor- dures. Thin-layer chromatography (TLC) of phospholipids was responding 1-O-alkylglycerols with 2,2-dimethoxypropane and performed on silica gel 60 F 254 plates (Merck, Darmstadt, p-toluenesulfonic acid in benzene for 1.5 hr. The products were Federal Republic of Germany) in chloroform/methanol/water, isolated by usual work-up and purified by preparative TLC in 70:35:7 (vol/vol); TLC of other products was performed on methylene dichloride/methanol, 96:4 (vol/vol). silica gel F 1500 LS 254 plates (Schleicher & Schuell, Dassel, Mild acidic hydrolysis was carried out by treatment with 10% Federal Republic of Germany) which first were washed with (wt/vol) trichloracetic acid for 30 min at 370C as described by ethanol, acetone, and diethyl ether and heated for 1 hr at 600C. Dawson (15). Catalytic hydrogenation was performed in 95% Detection was by use of vapor, Dittmer or Dragendorff ethanol with platinum oxide as catalyst, for 3 hr, at a hydrogen reagents, or spraying with 50% sulfuric acid followed by pressure of 3 bars. heating. HPLC was conducted on a Varian liquid chromato- Acetylation: Combined fractions bi and b2 of graph, model 8500, equipped with a differential refractometer; I (3.5 mg) were mixed with acetic anhydride (0.3 ml) and Micropak Si-5 columns (Varian) 25 cm X 12.7 mm (8 mm inside pyridine (0.3 ml) and kept at 220C for 18 hr. The reagents were diameter) were used and elution was performed with chloro- removed under vacuum and the crude product was purified form/methanol/water, 66:50:5 (vol/vol), at a flow rate of 200 by HPLC. II (n = 14) was prepared in the same manner from ml/hr [at 100 bars (107 Pa)]. synthetic I (n = 14) and had a retention time of 20 min on Infrared spectra were measured in chloroform solutions with HPLC. a Perkin-Elmer 297 spectrophotometer. 1H NMR spectra were obtained with a Cameca spectrometer, and chemical shifts are RESULTS given in ppm with respect to internal Me4Si. Electron impact (EI) mass spectra were recorded on an AEI MS50 instrument. CI Mass Spectrometry of Phospholipids. Failure to obtain CI mass spectra were measured at 200-260'C and a gas (iso- interpretable mass spectra of glycerophospholipids under El butane) pressure of 0.5-0.6 torr (66.6-80.0 Pa) in an AEI MS9 prompted us to undertake an investigation of the use of CI mass spectrometer equipped with a CI source (14). spectrometry for the determination of structure of phospho- Reduction with RED-Al: The reagent (0.5 ml) was added to lipids. Analysis of a large number of nonderivatized glycero- 5 mg of the natural phospholipid (I) dissolved in 1.5 ml of phospholipids and also of by Cl provided a benzene. The solution was heated at 370C for 1 hr; products substantial amount of structural information. In general, it were recovered by extraction with diethyl ether after addition showed MH+ of low abundance but displayed characteristic CH2 -R I CHOH I "0 x/e R .-CH2'.(CH2)j- CH3 CH2-O P-OCH2- CH2N-Me R =-CO (CH2)ri CH3 : I 2 :Oe0Me

b a

ion A: cleavage a + 2H ion B: A-H20 or cleavage b CH2 - O-CH2 (CH2)nCH3 CHOH @ O / Me CH2 - (CH2)nCH3 CH2-O=P-OCH2CH2N % O-CH2 Me C CHOH I HS I 0° Me CH2-O- POCH2 CH2NM CH2 -O-CH2 (CH2)n CH3 I OMe Me CHOH _ 0 (B CH2-O =P11 I OMe D s Me aD.1 Me - CH2 =CH- N HO CH2 CH2 N I Me H H M/I 90 m/ 72 FIG. 1. Scheme. Downloaded by guest on October 1, 2021 Biochemistry: Polonsky et al. Proc. Natl. Acad. Sci. USA 77 (1980) 7021 fragment ion peaks of significant diagnostic value. The spectra CH2-O-CH2-(CH2) -CH3 of glycerophospholipids having the 2-hydroxy group free showed an intense peak corresponding to ion A and a weak peak RO-CH 0 due to ion B (Fig. 1); the intensity profile is modified in the I II D /CH3 spectra of phospholipids without a free hydroxyl at position 2. CH2-O- P--OCH2- CH2-N-CH3 In the case of natural phospholipids, these ions are accompanied by homologous ions that differ by 28, 56, and 84 atomic mass CH3 units (amu), reflecting the homologous pattern of the fatty acid I: R = IL n = 14,15,16,17... (or ether, amide) composition. The choline-containing phos- II: R = COCH3 pholipids exhibited strong peaks at~m/z 90 and 72 in their CI spectra. Peaks due to MH+-32 (ion C) and MH+-89 (ion D) CH2 0- CH2 (CH2)= CH3 CH2-O- CHZ (CH2);7CH3 were also observed in the spectra of choline-containing gly- CHOH OHCH-o cerophospholipids, possibly from the loss of methanol and N,N-, respectively. These ions may arise CH20H CH20 CH3 by the transposition of one of the N-methyls to the phosphate group prior to fragmentation. II showed peaks due to the same III IV ions (C and D) originating from the additional loss of the ele- ments of ketene (42 amu) from the acetyl group. CH2-0-CH2-(CH2-CH3 Lyso-PAF-Acether (1-O-Alkylglyceryl 3-Phosphoryicho- I a CH=ON %CH3 were eluted CH-O line). As described (9, 10), PAF-active fractions | zC CH3 I Cs from silicic acid columns between sphingomyelin and a phos- CH2-0 CH20 CH3 pholipid isopolar with lyso-2-phosphatidylcholine (lyso-2- PtdCho). The lipids (35 mg from about 90 liters of hog blood), V VI m/z lol 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 Phospholipid I migrated on TLC coincident with lyso- sphingomyelin (al and a2) and to those of lyso-PtdCho (b, and PtdCho and could be detected by the Dragendorff method and b2). PAF-acether itself was eluted between a2 and b, when by the -specific reagent. Its infrared spectrum PAF-active fractions were chromatographed under the same showed no evidence of carbonyl function and displayed OH conditions. The phospholipid fractions eluted in b, (4.7 mg) and and P-O-choline absorptions (3350, 1082, 1040, and 965 in b2 (6.3 mg) were devoid of any PAF-acether activity, dis- cm'1). The presence of a choline polar head was further sup- played identical IR spectra, and had the same activity after ported by the 250 MHz 1H NMR spectrum'which revealed a acetylation (see below). Chemical and spectral studies of these broad singlet (9H) at 3.33 ppm assigned to the -N+(CH3)3, fractions showed that they contained I (ether analog of lyso- group (16). PtdCho) as the major component and that the two elution peaks The CI spectrum (Fig. 3 Lower) of synthetic I (n = 14) (b, and b2) corresponded to different populations with respect showed a weak MH+ peak at m/z 482 and significant fragment to the alkyl-ether composition. ions A, B, C, and D at m/z 317, 299, 450, and 393, respectively.

Injection FIG. 2. -, HPLC profile of lipids eluted from the silicic acid column subsequent to the PAF-active fractions;....., HPLCprofileof acetylated b1 + b2 fractions. Downloaded by guest on October 1, 2021 7022 Biochemistry: Polonsky et al. Proc. Nati. Acad. Sci. USA 77 (1980)

ns72 317() lUU. 4

50 393 IL) I

291(s) 4"ICM 379 Ii jill 1A I Illbillill 1 4 42(ML IS lol . 11 .. .IN EEHII N EEL I II .. .- . - .. II . IWJII UIIElRIII . II11 1 ] ff v 100 U0 300 360 4o0 460 500

100 I712 90

311 (IA)

50 313(D) 45S(C)

379

41. SS(s) I12 (MH*) l Al III . I I I 100 160 '' 250 30 3iO -450 50

FIG. 3. (Lower) CI mass spectrum of 1-0-hexadecyl-rac-glycerol 3-phosphorylcholine (synthetic I; n = 14). (Upper) CI mass spectrum of phospholipid I from fraction b2. The CI spectrum (Fig. 3 Upper) of phospholipid I from peak derivatives IV prepared from synthetic 1-O-octadecyl glycerol b was similar to that of I (n = 14) and revealed the presence and 1-O-hexadecyl glycerol. All the three had similar RF values of a C16:0 alkyl group as the predominant homolog whereas that on silica gel TLC. The EI mass spectra of the synthetic deriv- of fraction b, showed additional fragment ions 26 and 28 amu atives revealed the presence of the characteristic M-15 oxonium higher, reflecting the presence of C18:1 and C18:0 alkyl groups. ion peaks at m/z 341 (V) (n = 14) and 369 (V) (n = 16) and also The presence of the choline group was manifested by the oc- a peak at m/e 101 due to ion VI. The spectrum of the com- currence of peaks at m/z 90 and 72 (see Fig. 1). pound presumed to be IV derived from the natural phospho- That the phospholipid under investigation was I was further lipid I displayed the same peaks, indicating the presence of substantiated by the preparation of the following derivatives. C16:0 (predominant) and C18:O alkyl ethers in the molecule. Reduction of I (fractions b, and b2 combined) with RED-Al as Acetylation of I. Acetylation of I (fractions b, and b2 com- described by Snyder et al. (17) produced III. Its sensitivity to bined) gave II. HPLC showed it to be eluted as a sharp peak periodic acid supported the presence of vicinal hydroxyl groups (Fig. 2) with a retention time of 18 min, similar to that of and its infrared spectrum showed C-O-C absorption at 1110 PAF-acether. The presence of the apetoxy group was confirmed cm 1 (18). The RF value of III on silica gel TLC was similar by its infrared and 1H NMR spectra. to that of synthetic III (n 16), and its CI mass spectrum The CI mass spectrum of II (Fig. 4) showed the expected showed a strong MH+ peak at m/z 317, less intense peaks at MH+ at m/z 524 corresponding to the predominant homolog m/z 343 and 345, and weak peaks at m/z 357 and 359 corre- C16:0 in the O-alkyl chain. This was accompanied by peaks at sponding to C16:0, C18:1, C18:0, C19:1, and C19:0 alkyl chains. m/z 552 and 550 due to the MH+ corresponding to C18:0 and Treatment of III with 2,2-dimethoxypropane afforded the C18:1 species, respectively. Characteristic fragment ion peaks isopropylidene derivative IV which was compared with the were also observed at m/z 359 (C16), 387 (C18:0), and 385

132 122

90

524 (MH-) t- II 12 ILLAIbIX IIII 11

FIG. 4. CI mass spectrum of II obtained by acetylation of I (fractions b1 and b2 combined). Downloaded by guest on October 1, 2021 Biochemistry: Polonsky et al. Proc. Natl. Acad. Sci. USA 77 (1980) 7023 (C18:1) due to ion A and also at m/z 341 (C16), 369 (C18:0), and ylation, under the influence of cellular phospholipase A2 (or 367 (C18:1) due to ion B. The peaks corresponding to ions C and related enzymes), thereby contributing to the control of this D were also observed. This interpretation was confirmed by potentially harmful mediator. Whatever the final interpretation comparison with the CI mass spectra of the synthetic II (n = of the present findings in metabolic terms, they may represent 14) and II (n = 16) which showed the corresponding MH+ a step leading to a better understanding of the formation, re- peaks at m/z 524 and 552 along with all the expected fragment lease, and degradation of PAF-acether and therefore of the ion peaks consistent with their structures. numerous physiological and pathological situations in which The acetylated derivative (II) possessed the biological this mediator seems to play a predominant role (21). properties of PAF-acether. When assayed on rabbit platelets, Whereas glycero-phospholipids have been examined by field it displayed a powerful aggregating activity. This activity was desorption mass spectrometry (22), the possibility offered by not inhibited by the presence of indomethacin, aspirin, and CI mass spectrometry for their structural analysis has so far adenosine diphosphate scavengers, but it was completely de- remained unexplored. The results described here clearly stroyed after treatment with phospholipase A2, thereby meeting demonstrate the successful application of CI mass spectrometry the criteria for differentiating PAF from other aggregating to phospholipid structure determination. agents (8, 10). Its specific activity, 18 units/ng (,t0. 1 nM) was of the same magnitude as that of the most purified native PAF We thank Ms. C. Boullet, Ms. J. Bidault, and Mr. J. P. Le Couedic for excellent technical assistance and Mr. C. Merienne for NMR preparations (7 units/ng) (10) and synthetic II (n = 14) (7 IH measurements at 250 MHz. units/ng). As in the case of native PAF-acether, the aggregating activity of the acetylated compound (II) was not affected by 1. Siraganian, R. P. & Osler, A. G. (1971) J. Immunol. 106, mild acid hydrolysis or by catalytic hydrogenation. 1244-1251. 2. Benveniste, J., Cochrane, C. G. & Henson, P. M. (1972) J. Exp. DISCUSSION Med. 136, 1356-1377. Hog release I structure 3. Benveniste, J. (1974) Nature (London) 249, 581-582. leukocytes along with PAF-acether. The 4. Fesfis, L., Csaba, B. & Muszbek, L. (1977) Clin. Exp. Immunol. of this phospholipid, designated lyso-PAF-acether, was deter- 27,512-515. mined by chemical and spectral means, particularly by CI mass 5. Mencia-Huerta, J. M. & Benveniste, J. (1979) Eur. J. Immunol. spectrometry. Whereas ethanolamine plasmalogen has been 9,409-415. detected in human leukocytes (19), the presence of saturated 6. Lynch, J. M., Lotner, G. Z., Betz, S. J. & Henson, P. M. (1979) J. choline glyceryl ethers has not yet been reported. The saturated Immunol. 123, 1219-1226. C16 and C18 ether chains seemed to be the principal homologs 7. Pinckard, R. M., Farr, R. S. & Hanahan, D. J. (1979) J. Immunol. in our preparations. 123, 1847-1857. Acetylation of lyso-PAF-acether gave a compound that had 8. Chignard, M., Le Couedic, J. P., Tence, M., Vargaftig, B. B. & chromatographical behavior and biological properties identical Benveniste, J. (1979) Nature (London) 279, 799-800. 9. Benveniste, J., Le Couedic, J. P., Polonsky, J. & Tence, M. (1977) with those of PAF-acether. Results obtained from mild acidic Nature (London) 269, 170-171. hydrolysis and from catalytic hydrogenation suggested that the 10. Tence, M., Polonsky, J., Le Couedic, J. P. & Benveniste, J. (1980) presence of a double bond on the ether chain was not a pre- Biochimwe 62, 251-259. requisite for PAF-acether activity. 11. Benveniste, J., Tence, M., Bidault, J., Boullet, C., Varenne, P. & The "Iyso" compound was shown not to be produced through Polonsky, J. (1979) C. R. Acad. Sci. Ser. D 289, 1037-1040. saponification of PAF-acether during preparation and ex- 12. Demopoulos, C. A., Pinckard, R. N. & Hanahan, D. J. (1979) J. traction steps. The PAF-acether activity present in the leuko- Biol. Chem. 254, 9355-9358. cyte supernatants was almost completely recovered in the ex- 13. Blank, M. L., Snyder, F., Byers, L. W., Brooks, B. & Muirhead, tracted lipids (10). It also has been shown (10) that, at 22°C and E. E. (1979) Biochem. Biophys. Res. Commun. 90, 1194- 1200. pH 9.5, the release of PAF reaches a 2 maximum after hr of cell 14. Varenne, P., Bardey, B., Longevialle, P. & Das, B. C. (1977) Bull. incubation and no further change is observed during the next Soc. Chim., 886-892. 16 hr. Furthermore, incubation of natural PAF-acether and of 15. Dawson, R. M. C. (1960) Biochemistry 75,45-53. the synthetic compound (II; n = 16) at pH 9.0 and 10.6 for 22 16. Chapman, D. & Morrison, A. (1966) J. Biol. Chem. 241, hr did not affect the PAF activity. 5044-5052. Recent results indicate that PAF-acether and its lyso coun- 17. Snyder, F., Blank, M. L. & Wykle, R. L. (1971) J. Biol. Chem. terpart are both actively released by pH 7.35 by various cell 246,3639-3645. systems activated by different agonists (unpublished data). 18. Palameta, B. & Kates, M. (1966) Biochemistry 5, 618-625. The active release of lyso-PAF-acether suggests its possible 19. Gottfried, E. L. (1967) J. Lipid Res. 8, 321-327. role in PAF-acether metabolism. It might be that activation of 20. Hirata, F., Corcoran, B. A., Venkatasubramanian, K., Schiffmann, E. & Axelrod, J. (1979) Proc. Natl. Acad. Sci. USA 76, 2640- cells, triggering acylation/deacylation of phospholipid con- 2643. stituents of the cell membrane (20), yields high quantities of 21. Benveniste, J. (1980) in Advances in Allergology and Immu- this substance which therefore are available as a precursor for nology ed. Oehling, A. (Pergamon, Oxford) pp. 195-202. PAF-acether. On the other hand, one may consider that the 22. Wood, G. W., Lau, P. Y., Morrow, G., Rao, G. N. S., Schmidt, D. release of this lyso compound results from PAF-acether deac- E. & Tuebner, J. (1977) Chem. Phys. Lipids 18, 316-338. Downloaded by guest on October 1, 2021