Identification and quantitation of free ceramides in human platelets

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Identification and quantitation of free ceramides in human platelets illiam Krivit* and Sven HammarstrSmS Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota 5555, and

1 Identification and quantitation of free ceramides in human platelets illiam Krivit* and Sven HammarstrSmS Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota 5555, and Department of Medical Chemistry, Royal Veterinary College, Fack, S-5, Stockholm 50, Sweden Abstract Free ceramides were isolated from human plate- tive evidence for the formation of ceramide from [l-"c]- lets. Their structures were unequivocally determined by gas- acetate in platelets was reported (10). This report deliquid chromatography-mass spectrometry of the trimethyl- scribes the identification and quantitative determination silyl ether derivatives. The major components were N-(palmiof endogenous platelet ceramides using recently detoyl) sphingosine, N-(stearoyl) sphingosine, N-(eicosanoyl) veloped methods for GLC and GLC-MS of trimethylsphingosine, N-(docosanoyl) sphingosine, N-(tetracosanoyl) silyl ether derivatives of ceramides (7, 11-1). sphingosine, and N-(tetracosenoyl) sphingosine. Sphinganineand sphingadienine-containing ceramides as well as ceramides containing other unsaturated acids were also present. The amount of ceramides was determined by quantitative gas-liquid chromatography, using radioactive ceramide as internal stan- MATERIALS AND METHODS dard and synthetic crystalline ceramides for comparison of peak Preparation of platelets (1) areas. The concentration of ceramides was found to be 1.1 pg/lo9 platelets or 0.7 pg/mg of platelet protein. hole blood was obtained from normal human donors in Blood Central Laboratory, Sodersjukhuset (Stockholm, Sweden), by Dr. Jan van Mellen and his staff, Supplementary key words gas-liquid chromatography-mass spectrometry trimethylsilyl ethers using acid-citrate-dextrose (ACD, NIH formula A) anticoagulant and siliconized glassware. Two units of blood (each 500 ml) were carefully drawn into separate containers and centrifuged at 90 g for 15 min. The CERAMIDES appear to be intermediates in the bio- platelet-rich plasma was removed and centrifuged at synthesis (1-) and catabolism (1) of sphingolipids such 2000 g for 15 min and the sedimented platelets were as cerebrosides, ceramide-oligosaccharides, sulfatides, washed three times by suspending them in 0.5 M Tris gangliosides, and sphingomyelin. Previous investigations buffer, ph 7.5, and repeated centrifugation. The washed have demonstrated the presence of free ceramides in platelets were suspended in Tris buffer, counted in duliver (), spleen (5), lung (6), plasma (7), and brain (8). Earlier reports on platelet lipids have mainly dealt with the phospholipid composition (9). Recently, tentaplicates, and made up to a concentration of lo9 platelets/ ml. The suspension was quickly homogenized using a Potter-Elvehjem-type homogenizer. The platelet preparation, examined by stained smear, indicated the pres- Abbreviations: GLC-MS, gas-liquid chromatography-mass ence of only one red blood cell per 0 platelets and spectrometry; LCB, long-chain base; LCB, sphing--enine even fewer white cells. (D-t7~fh~O-1,-dihydroxy-2-a~no--tru~~-octadecene); LCB 18: 2, sphinga-,1-dienine (~-crythro-l,-dihydroxy-2-amino--trum-l- cis-octadecadiene) ; LCB18 : 1-20 : 0, N-(eicosanoyl) sphing-- Lipid extraction (15) enine; LCB -18 : 0, N-(stearoyl)hexadecasphing--enine, etc.; TLC, thin-layer chromatography; TMSi, trimethylsilyl. 2 ml of homogenized platelet suspension was added * Present address: Department of Pediatrics, University of dropwise to swirling chloroform-methanol 2 : 1 (v/v) in a Minnesota Medical Center, Minneapolis, Minn (the flask on a magnetic stirrer and the extraction was conaddress to which requests for reprints should be sent). 1 Present address: Department of Chemistry 11, Karolinska tinued for 15 min. N- [l~-1c]oleoyl-rac-sphinganine Institutet, Fack, S-1, Stockholm 60, Sweden. (0.05 PCi; 0.1 Pg) was then added as an internal stan- Journal of Lipid Research Volume 1,

$Silicic acid chromatography (7, 16) The lipids were first fractionated on a 10 X 0.25 cm I.D. column containing 2.5 g of silicic acid (Mallinckrodt, mesh, activated at 150 C for 2 hr).$

2 dard to each sample; the extract was filtered and then thoroughly mixed with 8 ml of water. The mixture was left overnight. After removing the upper phase, the lower phase was evaporated to dryness. Silicic acid chromatography (7, 16) The lipids were first fractionated on a 10 X 0.25 cm I.D. column containing 2.5 g of silicic acid (Mallinckrodt, mesh, activated at 150 C for 2 hr). 20 ml of chloroform eluted neutral lipids, and ceramides were eluted with chloroform-methanol 95 : 5 (v/v). The lipids in the latter eluate were chromatographed on a second column of silicic acid (0.5 g). The column was eluted with 12 ml of ethyl acetate-benzene 1 : 9 (v/v) followed by 12 ml of ethyl acetate-benzene 6: (v/v). The latter eluate was evaporated to dryness and the residue was subjected to TLC. Thin-layer chromatography (7, 12) Glass plates coated with silica gel G (E. Merck A.G., Darmstadt, est Germany) or silica gel G-AgNO 15 : 1 (w/w) were activated at 120 C for 1 hr. The ethyl acetate-benzene 6 : eluate was first purified on an ordinary plate, using chloroform-methanol-water 190 : 10 : 1 (v/v/v) as the developing solvent. The compounds were detected in UV light after spraying the plate with 0.2% (w/v) 2',7'-dichlorofluorescein in ethanol, and the purified ceramides were eluted from the gel with ethyl acetate. After conversion to the di-0-acetyl derivatives (treatment with acetic anhydride in pyridine [12]) these were rechromatographed on the AgNOs-impregnated plates (solvent system chloroform-benzene-methanol 80: 20: 1 [v/v/v]). The acetates were eluted from this plate and subjected to mild alkaline methanolysis as previously described (12). Scanning of thin-layer chromatograms for radioactivity was done with a Berthold Dunnschicht Scanner 11. Synthetic ceramides and ceramide acetates for reference purposes were generously supplied by Dr. Karin Samuelsson. Gas-liquid chromatography (11) Ceramides were converted to TMSi derivatives by treatment with 50 pl of freshly prepared hexamethyldisilazane-trimethylchlorosilane-pyridine 15 : 15 : 50 (v/ v/v). An aliquot of the mixture was injected into an F & M model 00 gas chromatograph equipped with a hydrogen flame ionization detector. The column ( cm X mm I.D.) contained 1% OV-I on mesh Supelcopxt (Supelco, Inc., Bellefonte, Pa.) at 275 C or OV-I on -120 mesh Gas-Chrom Q (170 cm X mm I.D.) at 10 C. The carrier gas was helium. Mass spectrometry (11) The pyridine solution of ceramide TMSi derivatives was evaporated to dryness in a stream of nitrogen and the derivatives were redissolved in hexane. An LKB model 9000 gas-liquid chromatograph-mass spectrometer (LKB Produkter, Bromma, Sweden) connected to an online digital computer system (PDP 8/I, Digital Equipment Corp., Maynard, Mass.) (17) was used. The computer effected background subtraction, conversion of ion intensities to relative abundances, and automatic plotting of mass spectra. The circular column (120 cm X 2.5 mm I.D.) containing 2% OV-1 on -120 mesh Gas- Chrom Q was maintained at 00 C for the analyses. The electron energy and the trap current of the mass spectrometer were set at 22.5 ev and 120 pa, respectively. Determination of ceramide concentration Quantitative GLC was carried out as described before (7) on platelet ceramides which had been,purified on ordinary, but not on AgNOs-impregnated, plates. Cholesterol as an internal standard was added to the platelets and to the reference ceramide prior to analysis. The reference ceramides were a mixture of accurately weighed synthetic ceramides containing rac-sphing-- enine and palmitic, stearic, arachidic, behenic, or lignoceric acid. Peak areas were determined by triangulation or planimetry, or both. The areas of platelet and reference ceramides were compared after correction according to the cholesterol areas. The mass of each GLC component originally present in the lipid extract was then calculated from the recovery of the radioactive ceramide which had been added prior to purification. The total ceramide concentration was expressed as pg of ceramide/ log platelets or as pg/mg of platelet protein. The latter was determined by the procedure of Lowry et al. (18), using bovine serum albumin as standard. RESULTS Fig. 1 shows a thin-layer chromatogram of platelet ceramides and the synthetic reference ceramides LCB -2:1, LCB -18:0, and LCB 18:1-1h:O (upper, middle, and lower spots, respectively). The platelet ceramides migrated with the same RF as LCB 18 : 1-2 : 1 (RF ). The recoveries of radioactive internal standard eluted from the plate were 72% and 62% in two instances. The ceramides were either acetylated and further separated into compounds containing saturated and unsaturated fatty acids by argentation TLC (Fig. 2) or directly converted to 0-TMSi derivatives and analyzed by quantitative GLC. Acetylated platelet ceramides yielded two spots on AgN0 TLC 526 Journal of Lipid Research Volume 1, 1972

3 Solvent front Free Ceramide Solvent front Ceramide Acetate 1 CB - 2: I CB - 18:o CB 18:I-Mh:O Origin Origin Standard Platelet Standard FIG. 1. Thin-layer chromatogram of ceramides isolated from human platelets. Solvent system, chloroform-methanol-water 190:lO:l (v/v/v). The reference ceramides are synthetic LCB -2:1, LCB -18:0, and LCB 18:1-1h:O (top to bottom spots). which had the same RF values as di-0-acetyl LCB - 2:O and LCB -2:l. Fig. shows gas-liquid chromatograms obtained for the separated ceramide acetates after methanolysis and conversion to 0-TMSi derivatives. % of the saturated (left curve) and 12% of the unsaturated (right curve) ceramide derivatives were used for the analysis. Mass spectra were recorded for most components of Fig. in a separate run. Mass spectrometry of TMSi derivatives of ceramides has been Platelet Standard FIG. 2. AgNOa TLC of 1,di-O-acetyl derivatives of human platelet ceramides. Solvent system, chloroform-benzenemethanol 80 :20 :1 (v/v/v). References, 1.di-0-acetyl derivatives of synthetic LCB -2:0, LCB -2:1, and LCB - (top to bottom spots). the subject of several papers from this laboratory (e.g., Refs. 11 and 1), and for a detailed description on how these molecules fragment during electron impact reference is made to one of these papers (11). Three informative ions of the spectra arc M - 15, M - u, and M - d, which indicate the molecular weight, the nature of the constituent acid, and the nature of the constituent LCB, respectively. Table 1 summarizes the m/e values and I -7 v) z 0 a v) IL (L: 2 V E 0 2a a i ln Z 2 m IL (L: E U I- 0 6a 8a Time (mi111 - J IL FIG.. Gas-liquid chromatograms of 1,-di-O-TMSi derivatives of human platelet ceramides, separated by AgNO, TLC as 1,di-O-acetyl derivatives (see Fig. 2). The left curve is for the upper and the right curve for the lower TLC component. Stationary phase, OV-1; column temperature, 10OC; carrier gas, helium. Mass spectra were recorded in a second run. The identification of the compounds is summarized in Table 1. The peak numbers indicate where the mass spectra were recorded. Krivit and Hammmstrom Free Ceramides in Human Platelets 527

4 ~~ TABLE 1. Relative abundancea and m/e values of ions in the mass spectra of human platelet ceramides (1,-di-O-TMSi derivatives) Retention M - u M - 15 Time M - d Peaks CrGCU) d e % LCB d e % Acid m/e % Molecular Species la : :O 666 LCB 18:1-16:0 6 16:l 98 6 LCB 16:1-18:0 8 LCB 16:2-18:0 2a a a 5a 6a 7a 8a 9a b b b 18 2b 8b 16:l 17: 1 17: 1 17:l :O = 20:o loo= 20:o 1 22:o 69c 20:O 12 22:o 22:o 2 2:O 5 2: :o 2 2:O 6 2: : 0 2: : b 5c 56 5b LCB - LCB - LCB 18:0-18:0 LCB 16:1-20:0 LCB 18:1-20:0 LCB 16:1-22:0 LCB 18:1-20:0 LCB 18:0-20:0 LCB 16:1-22:0 LCB 18:1-22:0 LCB 16:1-2:0 LCB 17 : 1-2: 0 LCB 18:1-22:0 LCB 18:0-22:0 LCB 16:1-2:0 LCB 17:1-2:0 LCB 18:1-2:0 LCB -2:O LCB 17:1-2:0 LCB 18:1-2:0 LCB 18:1-2:0 LCB 18:0-2:0 lb b 70 16:O lb LCB -16:0 LCB 18:1-16:0 2b b b LCB -18:0 LCB 18:1-18:1 b b 16:l c 20:l C 20 : : LCB -20:0 LCB 18:1-20:1 LCB16:1-22:1 b.o ) : 1 loob 22:o 2 2: LCB 18~2-22:O LCB18:1-22:1 LCB -2: 1 5b OOb 28b : :o 12 2: LCB 18:1-22:1 LCB -22:0 LCB -22:l LCB16:1-2:1 6b b 17: : 1 9b 2:O 1 2: LCB 18-2:l LCB -2:0 LCB 18:0-2:1 LCB17:1-2:1 7b b : 1 15* 2:O 776 LCB -2: 1 LCB -2:O 8b b 68b : 1 56b 2:O b LCB18:1-2:1 LCB -2:O LCB 18:0-2:1 a See Fig.. b Corrected for second isotope peak of the ion 2 mass units below. Corrected for the ion M - (b + l), which has the same m/e value. 528 Journal of Lipid Research Volume 1, 1972

~~ ~ ~~~~~ relative abundances of these ions for the platelet ceramides. The peak numbers refer to Fig.. The upper AgN0 TLC ceramides (Fig. 2) contained only saturated fatty acids.

5 ~~ ~ ~~~~~ relative abundances of these ions for the platelet ceramides. The peak numbers refer to Fig.. The upper AgN0 TLC ceramides (Fig. 2) contained only saturated fatty acids. The major long-chain bases were sphingosine, sphinganine, hexadecasphingosine, and heptadecasphingosine. The lower AgN08 TLC ceramides contained both saturated and monounsaturated acids. The unsaturated acids were combined with sphingosine, sphinganine, or hexadecasphingosine, whereas the saturated acids were combined with sphingadienine. 2-Hydroxy acid ceramides were not detected on inspection of thin-layer chromatograms nor by GLC-MS analysis of material eluted from the area beneath the platelet nonhydroxy acid ceramides (Fig. 1). Table 2 shows the results of the quantitative determination of platelet ceramides which had not been separated by AgN0 TLC. The main molecular species of each component are also indicated. The total ceramide content was 1.1 pg/109 platelets or 0.7 pg/mg of platelet protein. This corresponds to 0.5 pg of platelet ceramide/ml of whole blood (assuming a platelet concentration of - X IO8/ml). DISCUSSION The present report demonstrates the presence of ceramides in human platelets. Recently, a similar demonstration was reported for plasma ceramides (7). Comparison of the results reveals that the concentration of plasma ceramides is higher by a factor of about 6 when both are expressed as pg of ceramide/ml of blood. The qualitative compositions of platelet and plasma ceramides are similar; the major long-chain bases are LCB 18 : 1 and 18 : 0, the fatty acids have chain lengths between and (22, and no 2-hydroxy acids could be detected in either case. A quantitative difference in fatty acid chain length distribution was, however, apparent: the GLC fraction containing C2 acids predominated in plasma ceramides, whereas that containing CZZ acids was most abundant in platelets. TABLE 2. Quantitative determination of human platelet ceramides GLC Peak Main Constituent(s) pg/log Platelets la + lb LCB -16: f aI+ 2b LCB 18:1-18: f0.02 ( + )a + b LCB18:1-20: f 0.0 LCB 18:1-2:0 (8 + 9)a + (7 + 8)b { LCB 18:1-2:1 0.6 * 0.0 Total 1.22 f 0.05 a Means f SD; three determinations. A recent report on the incorporation of 1C-labeled acetate and palmitate into platelet lipids (10) indicated that ceramides were rapidly formed. The higher turnover rate suggested by these results is interesting and ought to be further studied by more specific techniques. The role of platelet lipids in blood coagulation has been reviewed by Marcus (9). About 15% of platelet dry weight is lipid, and of this approximately 80% is phosphatide. The major neutral lipid is cholesterol. From the present study it can be estimated that platelet ceramides constitute about 0.5% of platelet lipids. Although comprising only about 6% of the total lipids, phosphatidylserine was the most active platelet phosphatide in in vitro coagulation (9). hether ceramide is active in this respect is not known, and future work on abnormalities in platelet physiology ought to include specific measurements of ceramides. The technical assistance of Miss Linda Kern and Mrs. Majalena Granqvist is gratefully acknowledged. Mr. Dan Horlin, Mr. Ulf Israelsson, and Dr. Charles C. Sweeley assisted with computer operations and programming. A generousupply of ceramide standards was given by Dr. Karin Samuelsson.. K. was on sabbatical leave at the Royal Veterinary College and is indebted to a USPHS SpecialResearchFellowship (1-F0-HE-9, 2-01) and to an American Cancer Society Scholar Grant (PS-55). Supported by grantsfromusphs, nos. AM 170, CA 08101, and CA Manuscript received 2 August 1977; accepted 70 March REFERENCES 1. Mirtensson, E Glycosphingolipids of animal tissue. Progr. Chem. Fats Other Lipids. 10: Hammarstrom, S., and B. Samuelsson On the biosynthesis of cerebrosides containing 2-hydroxy acids. Massspectrometricevidencefortheceramidepathway. J. Biol. Chem. 27: Hammarstrom, S On the biosynthesis of cerebrosides containing non-hydroxy acids. 2. Mass spectrometric evidence for the ceramide pathway. Biochem. Biophys. Res. Commun. 5: Frankel, E., and F.Bielschowsky. 1!2. Untersuchungen uber die Lipoide der Saugetierleber. Uber Vorkommen das des Lignoceryl-sphingosins in der Schweinleber. Hoppc- Seyler s Z. Physiol. Chem. 21: Tropp, C., and V. iedersheim 19. Untersuchungen uber Lipoide tierischer Organe. Uber das Vorkommen des Lignoceryl-Sphingosins in der Rindermilz. HoppeSeyler s Z. Physiol. Chem. 222: Tropp, C Das Vorkommen von Lignoceryl-sphingosins in der Rinderlunge. Hoppe-Seyler s 2. Physiol. Chem. 27: Samuelsson, K Identification and quantitative determination of ceramides in human plasma. Scand. J. Clin. Lab. Invest. 27: Klenk, E., and R. T. C. Huang Zur Kenntnis der Gehirnceramide und der darin vorkommenden Sphingosinbasen. Hoppe-Styler s 2. Physiol. Chem. 9: Krivit and Hammarstrom Free Ceramides in Human Platelets 529

6 9. Marcus, A. J The role of lipids in blood coagulation. Aduan. Lipid Res. : Deykin, D., and R. K. Desser The incorporation of acetate and palmitate into lipids by human platelets. J. Clin. Invest. 7: Samuelsson, B., and K. Samuelsson Gas-liquid chromatography-mass spectrometry of synthetic ceramides. J. Lipid Res. 10: Samuelsson, B., and K. Samuelsson Separation and identification of ceramides derived from human plasma sphingomyelins. J. Lipid Res. 10: Hammarstrom, S., B. Samuelsson, and K. Samuelsson Gas-liquid chromatography-mass spectrometry of synthetic ceramides containing 2-hydroxy acids. J. Lipid Res. 11: Mourad, N A simple method for obtaining platelet concentrates free of aggregates. Transfusion. 8: Folch, J., M. Lees, and G. H. Sloane Stanley A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226: Hammarstrom, S A convenient procedure for the synthesis of ceramides. J. Lipid Res. 12: Sweeley, C. C., B. D. Ray,. I. ood, J. F. Holland, and M. I. Krichevsky On-line digital computer system for high-speed single focusing mass spectrometry. Anal. Chem. 2: Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Fohn phenol reagent. J. Biol. Chem. 19: Journal of Lipid Research Volume 1, 1972

Distribution of molecular species of sphingomyelins in different parts of bovine digestive tract

Distribution of molecular species of sphingomyelins in different parts of bovine digestive tract M. E. Breimer Membrane Biochemistry Group, Department of Medical Biochemistry, University of Giiteborg,