SEVERAL. of atherosclerotic human aorta. Long-chain bases in the sphingolipids

Transcription

1 Long-chin bses in the sphingolipids of therosclerotic humn ort R. V. PANGANAMALA, JACK C. GEER, nd DAVID G. CORNELL Deprtments of Physiologicl Chemistry nd Pthology, The Ohio Stte University, Columbus, Ohio ABSTRACT Long-chin bses were prepred from humn ort sphingomyelin by combined enzymtic hydrolysislkline hydrolysis procedure nd these bses were isolted by thin-lyer chromtogrphy. Aldehydes, obtined from the long-chin bses by periodte oxidtion, were converted to 1,3- dioxolne derivtives. Dioxolnes were identified nd quntified by gs-liquid chromtogrphy before nd fter ctlytic hydrogention, nd before nd fter seprtion into sturted, monoene, nd diene dioxolne frctions. The monoene dioxolnes were converted to ldehydes by reductive ozonolysis with dimethyl sulfide nd these ldehydes were isolted nd identified s dioxolne derivtives. The double bond positions in the mjor diene component were estblished by reductive ozonolysis nd permngnte-periodte oxidtion. Sphingenines in the cerebroside-sulftide nd sulftide frctions of ort were converted to ldehydes by the reductive ozonolysis of intct sphingolipids nd these ldehydes were nlyzed s the dioxolnes. Humn ort sphingomyelin contined significnt mounts of 4-hexdecsphingenine, 4-heptdecsphingenine, sphingnine, 4-sphingenine, nd 4,~14-~phingdienine. Smll mounts of hexdecsphingnine, 4-tetrdecsphingenine, sphingdienine isomer, n unknown sphingnine, nd two unknown diene long-chin bses were lso found in sphingomyelin. The presence of brnched-chin 4-sphingenine ws tenttively estblished nd the possible presence of sphingenine isomer ws suggested. The mjor sphingenines were the sme in the sphingomyelin, sulftide, nd cerebroside-sulftide frctions of humn ort. SUPPLEMENTARY KEY ORDS sphingomyelin. sulftide. cerebrosidesulftide. enzymtic hydrolysis. lkline hydrolysis. periodte oxidtion. reductive ozonolysis. permngnte-periodte oxidtion. 1,3-dioxolne. thin-lyer. gs-liquid chromtogrphy. sphingnine. sphingenine sphingdienine. brnched-chin sphingenine Abbrevitions: LCB, long-chin bse or bses; TLC, thin-lyer chromtogrphy; GLC, gs-liquid chromtogrphy; DMS, dimethyl sulfide; PTS, p-toluenesulfonic cid; EGS, ethylene glycol succinte polyester. SEVERAL METHODS hve been described for the chrcteriztion of long-chin bses (LCB) in sphingolipids. Sweeley nd Mosctelli (1) nlyzed ldehydes prepred from LCB by periodte oxidtion. Krlsson (2) prepred the 2,4-dinitrophenyl derivtives of LCB nd seprted these derivtives by thin-lyer chromtogrphy (TLC). Gver nd Sweeley (3), Polito, Akit, nd Sweeley (4), nd Crter nd Gver (5) prepred the trimethylsilyl derivtives of LCB nd nlyzed these derivtives by gs-liquid chromtogrphy (GLC) nd mss spectrometry (4). Smuelsson nd Smuelsson (6) recently nlyzed trimethylsilyl derivtives of sphingnine nd 4-sphingenine cermides by GLC. The sphingdienine components of the LCB hve been chrcterized by permngnte-periodte oxidtion (2, 4), ozonolysis (7), nd mss spectrometry of trimethylsilyl derivtives (4). In the present investigtion (Fig. l), ldehydes obtined from LCB by periodte oxidtion were nlyzed s their 1,3-dioxolne derivtives (8) nd double bonds were locted by reductive ozonolysis (9) or permngnte-periodte oxidtion. Approximtely one-fifth of the totl phospholipid of norml humn ort is sphingolipid. As therosclerosis increses in extent nd severity in the ort, the proportion of sphingolipids increses to pproximtely twice tht found in the norml ort, primrily through n increse in sphingomyelin. The high content of sphingomyelin in humn therom ws first reported by einhouse nd Hirsch (10) nd since then this observtion hs been confirmed in number of studies (11-14). Nomenclture is in ccordnce with the tenttive rules of the IUPAC-IUB Commission : the older term dihydrosphingosine is clled sphingnine; sphingosine is clled 4-sphingenine; higher nd lower homologues re identified by prefixes; phytosphingosine is clled 4D-hydroxysphingnine; unknown geometry is specified by the prefix x- before the number tht indictes the position of the double bond. JOURNAL OF LIPID RESEARCH VOLUME 10,

2 SPHINGOMYELIN ALDEHYDES I,3-DIOXOLANES 1 Mthonol -KOH CERAMIDES / I,3-Proponediol PTS I,3-DIOXOLANES LCB J Nolo4 ALDEHYDES TLC (A9N03) I t i SATURATED MONOENE DIENE J 1 ALDEHYDES DIALDEHYDES DICARBOXYLIC ACIDS 1.3 -Proponediol 1.3 -Propondiol PTS PTS ~y~~~~~ I,3-DIOXOLANES I,3-DIOXOLANES METHYL ESTERS FIG. 1. Isoltion of LCB nd the preprtion of derivtives for nlysis. sulfonic cid. The significnce of sphingomyelin in the pthogenesis of therosclerosis is unknown. Although the ftty cid composition of humn ort sphingomyelins hs been reported (14), the LCB composition of humn ort sphingomyelins hs not been investigted. In the present study, only orts with extensive nd severe therosclerosis were nlyzed in order tht sufficient lipid be vilble for vlidtion of the methods used in the isoltion nd chrcteriztion of LCB. The LCB compositions of humn orts representing incresing ge nd severity of lesions will be compred in subsequent investigtion. Mterils MATERIALS AND METHODS All regents were nlyticl grde. Commercil hexne ws wshed three times with concentrted sulfuric cid, once with wter, nd finlly with 10% sodium crbonte solution until neutrl. The wshed hexne ws distilled t 68 C. Pltinum oxide ws purchsed from Bker Ctlyst, Inc. (Newrk, N. J.). 1,3-Propnediol nd dimethyl sulfide (DMS) were purchsed from Mtheson, Colemn nd Bell (Cincinnti, Ohio). p- Toluenesulfonic cid (PTS) ws purchsed from Estmn Orgnic Chemicls (Rochester, N. Y.). Silicic cid (Unisil) ws purchsed from Clrkson Chemicl Co. (illimsport, P.). Silic Gel H (Brinkmnn Instruments, Inc., estbury, N. Y.) ws extrcted with cetone nd then with chloroform-methnol 2 : 1 (v/, dried t room temperture, nd ctivted t 110 C for 24 hr. Beef brin sulftides nd cermides, ethylene glycol succinte polyester (Hi-EFF-ZBP), mesh Gs- Chroin P, nd the methyl esters of dibsic cids were purchsed from Applied Science Lbortories, Inc. (Stte College, P.). Beef brin sphingomyelin ws pur- 446 JOURNAL OF LIPID RESEARCH VOLUME 10, 1969

3 chsed from Generl Biochemicls, Inc. (Chgrin Flls, Ohio). 1,7-0ctdiene nd 1,g-decdiene were purchsed from Aldrich Chemicl Co. (Milwukee, is.). Phospholipse C (Cl. welchii, type-1) ws purchsed from Sigm Chemicl Co. (St. Louis, Mo.). Cerebrosides, purified gngliosides, nd pure LCB ( mixture of sphingnine nd 4-spingenine) were kindly supplied by Dr. R. H. McCluer. Octdecnl, 9-octdecenl, nd 9,12-octdecdienl were synthesized from the corresponding ftty cids (8). Five orts were obtined t necropsy from persons between 57 nd 76 yr old. These orts were selected becuse of extensive nd dvnced lesions of therosclerosis. By terminology recommended by the orld Helth Orgniztion (15) the orts were t stge I11 of therosclerosis. The ortic intim, from the level of the first intercostl vessels of the descending thorcic segment to the ilic bifurction, ws removed by blunt dissection. Isoltion of Sphingomyelin The intiml tissue, g, ws homogenized in Sorvll Omni-Mixer in 10 volumes of chloroformmethnol 2 : 1. The filtered residue ws homogenized twice more in 10 volumes of chloroform-methnol 1 : 1 nd the filtered residue from this step ws then homogenized in 10 volumes of bsolute methnol. The combined extrcts were evported under reduced pressure in Rinco flsh evportor. The crude lipids were dissolved in chloroform-methnol 2:l nd wshed by the Folch prtition method (16). The chloroform phse ws then evported under reduced pressure. The lipids were dissolved in smll volume of chloroform nd plced on 60 g Unisil column (3.5 cm o.d.). Neutrl lipids were eluted with 600 ml of chloroform nd polr lipids were eluted with 600 ml of methnol. hen the polr lipid frction contined less thn 500 mg, sphingomyelin ws isolted by TLC. Pltes were developed with chloroform-methnol-wter 65 : 25 : 4 nd bnds were viewed under UV rdition fter the pltes hd been spryed with 2 ',7 '-dichlorofluorescein. The sphingomyelin frction (R, 0.23) ws extrcted from the Silic Gel H with methnol. Sphingomyelin ws isolted from lrger polr lipid frctions ( mg) by different method. Aldehydes were liberted from plsmlogens by mild cid hydrolysis (17) nd the hydrolyste ws then plced on 20 g Unisil column. Aldehydes were eluted from the column with 100 ml of chloroform nd polr lipids were eluted with 250 ml of methnol. The cid-stble lipids were then subjected to mild lkline hydrolysis (1 8) nd the lkli-stble frction ws extrcted into chloroform-methnol 2 : 1. The frction ws plced on 20 g Unisil column nd lipids were eluted successively with 100 ml of chloroform, 200 ml of cetone, nd 200 ml of methnol. The methnol elute contined only sphingomyelin s judged by TLC. Isoltion of Sulftides About 500 mg of the polr lipid frction isolted from ortic intim ws pplied to TLC pltes. Pltes were developed with chloroform-methnol-wter 65 : 25 : 4 nd then spryed with 2',7 '-dichlorofluorescein ; the overlpping choline phosphoglyceride (R, 0.28) nd sulftide (R, 0.34) bnd ws extrcted with methnol. This frction ws incubted with phospholipse C nd then plced on 20 g Unisil column. The diglycerides obtined from the choline phosphoglycerides were eluted with 100 ml of chloroform nd the sulftides were eluted with 250 ml of methnol. Removl of choline phosphoglycerides ws judged by the bsence of phosphoruscontining spot (molybdic cid spry) on TLC. Isoltion of Cerebroside-Sulftide (Glycolipid) Frction The polr lipid frction isolted from ortic intim ws pplied to 30 g column of ctivted Florisil. The column ws pcked with Florisil suspended in chloroform-methnol 70 : 30 nd the glycolipids were eluted with 200 nil of the sme solvent by the method of Rouser, Kritchevsky, nd Ymmoto (19) except tht 2,3-dimethoxypropne ws omitted from the solvent. No glycolipids were detected on TLC when n dditionl 100 ml of chloroform-methnol 70:30 ws pssed through the Florisil column. Homogeneity ws judged by the bsence of phosphorus-contining spots (molybdic cid spry) on TLC when the plte ws developed with chloroform-methnol-wter 65 : 25 : 4. Glycolipids becme visible when the pltes hd been spryed with orcinol-sulfuric cid nd wrmed. The frction contined two cerebrosides (R, 0.81 nd 0.72) nd sulftide (R, 0.34). The sme glycolipids were detected in the totl polr lipid mixture by TLC. Isoltion of LCB Approximtely 60 mg of sphingomyelin ws plced in mixture of 4 ml of diethyl ether nd 4 ml of 0.15 M Tris buffer (ph 7.3) contining 25 mg of phospholipse C. The mixture ws stirred t 37 C for 3 hr. Cermides were extrcted from the rection mixture with chloroform. The chloroform extrct ws wshed with 10% queous sodium chloride nd the solvent ws removed under reduced pressure. Cermides were then refluxed with 1.0 N KOH in methnol for 18 hr, nd the LCB frction from this hydrolyste ws extrcted into chloroform or ether. The extrct ws wshed three times with 10% queous sodium chloride nd then pssed through nhydrous sodium sulfte. The solvent w removed under reduced pressure. LCB were purified by TLC in chloroform-methnol-wter 65 : 25 : 4. In n PANGANAMALA, GEER, AND CORNELL Long-Chin Bses in Aort SDhinooliDids d41

4 lterntive isoltion procedure, the chloroform or ether extrct of the cermide hydrolyste ws pplied to 20 g Unisil column. Any ftty cids tht were present in the extrct were eluted with 100 ml of chloroform nd the LCB were eluted with 200 ml of methnol. The homogeneity of the LCB frction ws judged by TLC in chloroform-methnol-wter 65 : 25 : 4 or chloroformmethnol-mmoni 40 : 10 : 1 (20). Preprtion of 1,b-Dwxolnes The LCB frction ws oxidized with 0.2 M queous sodium metperiodte (1). Aldehydes were purified by elution from 20 g Unisil column with 150 nil of chloroform nd then converted to 1,3-dioxolnes (8). The 1,3-dioxolnes were purified by TLC in hexneether 90:lO (R, 0.46). Seprtion of Sturted nd Unsturted 1,3-Dioxolnes Sturted nd unsturted dioxolne frctions were isolted by TLC. Silic Gel H pltes were impregnted with AgN03 (21) nd developed with hexne-ether 85:15. Bnds were visible under UV rdition fter the pltes hd been spryed with 2,7 -dichlorofluorescein. Bnds were identified by compring their R, vlues with the R, vlues of reference compounds synthesized from ftty ldehydes. The dioxolnes were extrcted from TLC pltes with chloroform-hexne 1 : 1. Hydrogention The 1,3-dioxolnes were hydrogented by the procedure of Ro, Rmchndrn, nd Cornwell (8) except tht pyridine ws not used in the hydrogention procedure. Clevge The unsturted 1,3-dioxolnes were cleved by reductive ozonolysis procedure which employed DMS (9). Aldehydes were extrcted into hexne, wshed twice with smll mounts of wter, nd converted to dioxolnes by the methods referred to bove. Dioxolnes were purified by TLC on Silic Gel H. In n lterntive procedure (see Fig. l), dioxolnes in the diene frction were cleved by permngnte-periodte oxidtion nd the resulting dicrboxylic cids were converted to methyl esters (22). In the reductive ozonolysis of intct sphingolipids (Fig. l), the lipid ws dissolved in 3 ml of chloroformmethnol 2 : 1 nd this solution ws dded to 15 ml of pentne or methnol sturted with ozone. DMS ws dded, pentne or methnol ws evported nd ldehydes were extrcted into hexne. The hexne solution ws wshed twice with smll mounts of wter. The ldehydes were then converted to dioxolnes nd purified by TLC. Gs-Liquid Chromtogrphy Dioxolnes nd methyl esters were nlyzed in Aerogrph 200 nd Aerogrph 1200 chromtogrphs equipped with flme ioniztion detectors. 10-ft (3.3 m) stinless steel columns, /8 inch (3.2 mm) o.d., contining 20% ethylene glycol succinte polyester (EGS) on mesh Gs-Chrom P, were used for seprtions on polr phse. 5-ft (1.65 m) stinless steel columns, /8 inch (3.2 mm) o.d., contining 50/, SE-30 (methylpolysiloxy gum) on mesh Gs-Chroni, were purchsed (Vrin Aerogrph, lnut Creek, Clif.) for chromtogrphic seprtions on nonpolr phse. Peks were identified by reltive retention volumes (( R.18 vlues ), clculted by dividing the retention volume of the pek by the retention volume of methyl sterte. Pek res were mesured by tringultion. Operting conditions re listed with the pproprite figures nd tbles. Isoltion of LCB RESULTS hen cermide ws prepred by the enzymtic hydrolysis of sphingomyelin with phospholipse C, the hydrolysis product showed only one spot on TLC in chloroform-methnol-wter 65 : 25 : 4. The R, vlue for this spot, 0.86, corresponded to the R, vlue of commercil cermide smple. No unrected sphingomyelin or other phosphorus-contining spot ws detected. LCB ws prepred by lkline hydrolysis of the cermide frction. The product gve one spot (R, 0.48) on TLC in chloroform-methnol-wter 65 : 25 : 4 which corresponded to the LCB isolted from brin gngliosides (20). The product gve two spots on TLC in chloroformmethnol-mmoni 40 : 10 : 1 nd these spots hd the sme R, vlues s 4-sphingenine nd sphingnine frctions isolted from brin gngliosides (20). No cermide, 4D-hydroxysphingnine or 0-methyl-Csphingenine were detected on TLC with either developing solvent. Seprtion of Sturted nd Unsturted 1,S-Dioxolnes The sturted monoene nd diene components of the LCB frction yielded sturted, monoene, nd diene ldehydes on periodte oxidtion. These ldehydes were converted to their corresponding dioxolnes nd the dioxolnes were seprted by TLC on pltes impregnted with AgN03 (Fig. 2). The R, vlue of the sturted frction, 0.69, ws the sme for dioxolnes prepred from LCB nd dioxolnes prepred from tetrdecnl. The R, vlues for the monoene frctions were different for dioxolnes prepred from LCB where the double bond ws in the,@ position (R, 0.53) nd dioxolnes prepred from 9-octdecenl (R, 0.45). Similr differ- 448 JOURNAL OF LIPID RESEARCH VOLUME 10, 1969

5 FIG. 3. GLC nlysis of 1,3-dioxolnes prepred from the sphingomyrlin IXR frrtion of humn ort by prriodtr oxidtion: A, wturtrd; B, monocne; C, diene; D, totl mixture. Trcings were obtinrd from n EGS column in n Aerogrph 200 equipprd with llmr ioniztion detector. Opcrtinq tempertures were: injector 280 C, column 18ZoC, detector 280 C. lielium ws the crrier qs nd the flow rtr ws 30 ml/min. FIG. 2. Seprtion of 1,3-diosolnrs on.igno-tix: I:, sohvmt front; S, sturted;.4f, monocnc; 11, diene; 0, oriyin. Chromtogrm ws drvelopcd in hexnr-cthm 85 : 15 nd chrrrd with sulfuric cid. Right: dioxolnes from tetrdecnl, 9-octdccenl, nd 3,12-octdccdien1. Lrft: dioxolnes from LCR in which onr double bond is, B to the dioxolne?roup. ences were seen in dioxolnc I?, vlues for the diene frctions obtined from LCR (I?, 0.26) nd 9,12-octdecdienl (R, 0.16). GLC A n1g.vi.v of I,.5'-Dioxolnc,v The GLC nlysis of 1,3-dioxolnes prepred from the LCR frction of humn ort by periodte oxidtion is shown in Fig. 3D. The Rlx vlues for mny dioxolne peks did not correspond to the Rls vlues of dioxolnes prepred from known ftty ldehydes. Sincc reference compounds contining,p-double bonds were not \.ilble, the GLC nlysis of sturted nd unstiirted dioxolne frctions nd hydroqention nd reductive ozonolysis dt were required for the identifiction of peks. GLC trcinqs for sturted, monoene, nd diene dioxolne frctions re shown in Fig. 3A, R, nd C. The R lx \.llies obtined from these GLC nlyses re siinimrized in Tble 1. These dt nd log RIR dt were used for the tenttive identifiction of 1,3-dioxolnes in the originl mixture nd these tenttive pek ssiqnmcnts were then confirmed by ctlytic hydroyention. Thus 14 : 1 nd 15 : 1 dioxolnes were converted to 14 : 0 nd 15 :0 dioxolnes, nd 16 : 1 nd 16 :2 dioxolnes were converted to 16 : 0 dioxolne, by ctlytic hydrogention (Fiq. 4, Tbles 1 nd 2). A sinl1 pek in the hydroqented mixture with retention tirnc qreter thn tht of 16:O dioxolne (Fiq. 4) ws not identified. The RI~ for this pek did not correspond to 17 :O or 18:0 dioxolne. No other peks, except 16:lbr pek described below, were obscrved consistently or in mesurble niounts during. GLC fter ctlytic hydroqention. Rcductive ozonolysis of,@-double bonds in the monoene dioxolne frction yielded sturted ldehydes, which were converted to dioxolnes. The mjor corn- PNcNA\rAl..i. GEER, AND CORNELI. Long-chin Bses in Aort Sphingolipids,449

6 TABLE 1 RELATIVE S FOR 1,3-DIOXOLANES OF STANDARD ALDEHYDES AND ALDEHYDES PREPARED FROM LONG-CHAIN BASES IN THE SPHINGOMYELIN OF HUMAN AORTA Rlst t OC TLC with AgN03 Totl 1,3-Di- Stn- Stu- Hydrooxolne* drd Totl rted Monoene Diene gented 12:o :O Unknown :O :l o 16:Obr 1.2 Unknownb :O : Unknown, :l :lbr :l :2, ~2, :2, * The crbon number: double bond nomenclture refers to the prent ldehyde. Different unknown peks nd 16:2 isomers re indicted by subscripts. t Reltive retention time with respect to methyl sterte. The retention volume of methyl sterte t 182OC ws 633 ml. Operting conditions re summrized in Fig. 3. w 2 x w D 0 B 16:O FIG. 4. GLC nlysis of the totl 1,3-dioxolne mixture (Fig. 3D) fter ctlytic hydrogention. Operting conditions re summrized in Fig. 3. ponents of this mixture hd two fewer crbon toms nd with the exception of 13 : 0 were in the sme reltive concentrtions (Fig. 5) s the dioxolnes in the monoene frction (Fig. 3B). Unrected ozonides from,@ unsturted dioxolnes were not detected by TLC fter reductive ozonolysis with DMS lthough some unrected ozonide ws detected fter the reductive ozonolysis of 9-octdecenyl-l,3-dioxolne. The conversion of this ozonide to ldehydes ws improved nd ozonides were not detected when methnol ws substituted for pentne in the ozonolysis rection. A 16:lbr dioxolne with n $-double bond ws tenttively identified s minor component from the following dt. The pek occurred between the 15 : 1 nd 16:l peks in the totl mixture nd the monoene frction (Tble 1). A smll pek with the sme reltive concentrtion ws found between the 15 : 0 nd 16 : 0 dioxolnes obtined fter hydrogention (Tble 2 nd Fig. 4). Finlly, smll pek ws found between the 13 : 0 nd 15 : 0 dioxolnes obtined fter reductive ozonolysis (Fig. 5). The totl dioxolne mixture contined three unknown minor components. One of these unknown peks ws found in the sturted frction nd the other two unknown peks were found in the diene frction (Tble 1). Hydrogention nd reductive ozonolysis did not provide ny dditionl dt for the identifiction of these peks. One pek in the diene frction, 16:2,, hd the sme R18 z 2 n 8 FIG. 5. GLC nlysis of dioxolnes from the monoene 1,3-dioxolne frction (Fig. 3B) fter reductive ozonolysis. Operting conditions re summrized in Fig. 3. vlue s the 16:l dioxolne. Since the 16:l dioxolne ws the mjor component of the totl mixture, this diene probbly represented monoene contmintion in the diene frction rther thn 16 :2 positionl isomer. The 16:2b nd 16:2, peks probbly do represent positionl isomers. GLC peks which corresponded to 14 L 450 JOURNAL OF LIPID RESEARCH VOLUME 10, 1969

7 'rable 2 COMPOSITION OF ~~IXTURE OF LONG-CHAIN BASES IN THE SPHINGOMYELIN OF HUMAN AORTA* Long-chin Bse 1,3-Dioxolne Totl t TLC with AgNO Totl Sturted Monoene Diene Hydrogented$ Tetrdecsphingnine Hexdecsphingnine Unknown, 4-Tetr decsp hingenine Unknownb Sphingnine 4-Hexdecsphingenine Unknown, 4-Heptdecsphingenine [Sphingenine(iso)] 4-Sphingenine (br ) 4-Sphingenine Sphingdienine, Sphingdienineb Sphingdienine, 12:o 14:O 15:O 12: 1 16:Obr 16:O 14: 1 15:l [16:l(iso)] 16:lbr 16:l 16:2. 16:2b 16:2, re % 0.6 f f f f f f f 1.9 [4'315 \ L f 0.6 r i63.2 f 1.4 I 1.0 f f 2.3 re % re yo , , , , j * Dioxolne nomenclture is described in Tble 1. Operting conditions re summrized in Fig. 3. t Men i SD for sphingomyelins from five different orts. Lipids from two orts were combined. $ Hydrogention dt from two dioxolne preprtions. 5 See text for the tenttive identifiction of sphingenine(is0) nd the ssumptions used in the quntifiction of this compound. minor dioxolne components were not obtined when blnk Silic Gel H pltes nd blnk Silic Gel H pltes impregnted with AgNO were extrcted with chloroform-methnol 2 : 1. LCB Composition of Sphingomyelin From Hu.mn Aort The reltive composition of the LCB moieties in the sphingomyelin of humn ort ws obtined from the GLC nlysis of the totl dioxolne mixture before nd fter hydrogention nd the GLC nlysis of the sturted, monoene, nd diene frctions (Tble 2). Aldehydes nlyzed by GLC on n EGS column prior to their conversion to dioxolnes hd the sme composition s the totl dioxolne mixture. The principl LCB in the sturted frction ws sphingnine. 4-Sphingenine nd sphingdienine were the principl LCB components of the monoene nd diene frctions. The sturted frction contined smll mount of tetrdecsphingnine nd the diene frction contined smll mount of n unknown component, unknowno; neither of these components ws detected in the totl mixture. Composition dt suggested tht the 15 : 1 dioxolne pek contined both the expected 15 : 1 dioxolne with the double bond in the,/3 position nd second component in which the double bond ws not in the,p position. The evidence for the identity of this dioxolne ws obtined from hydrogention nd reductive ozonolysis experiments. Thus the 15 : 1 pek, 8.8% of the totl mixture, ws lrger thn the 15:O pek, 4.3y0 of the hydrogented mixture (Tble 2) nd it ppered tht the 15 : 1 dioxolne pek contined n dditionl dioxolne which did not yield 15 : 0 product on hydrogention. The composition of the dioxolne mixture from the monoene frction fter reductive ozonolysis ws: 12 : 0, 10.6%; 13:0, 5.9%; 14:Obr, 1.3%; 14:0, 82.1% (dt from Fig. 5). Since the reltive concentrtion of the 15:l dioxolne in the monoene frction ws 13.3% (Tble 2), it ppered tht the 15:l dioxolne pek contined n dditionl dioxolne which did not yield 13:O dioxolne on reductive ozonolysis. These dt would be explined by the presence of 16 : 1 positionl isomer which hd the sme retention volume s the 15 : 1 dioxolne with the double bond in the,/3 position. Dioxolnes with,fl-double bonds which were prepred from LCB hd lrger retention volumes thn dioxolnes prepred from ftty ldehydes (8) nd it is possible tht 16 : 1 isomer nd the 15 : 1 dioxolne from n LCB hd the sme retention volume. Furthermore, the sum of the 16:O + 16:l + 16:2 peks in the originl mixture, 81.7% (Tble 2), ws less thn the 16:O content of the hydrogented mixture, 88.4% (Tble 2), nd these dt lso suggested tht the 15:l pek my hve contined 16 : 1 positionl isomer which ppered s prt of the 16 : 0 component fter hydrogention. 4-Heptdecsphingenine ws, therefore, estimted from the 15 :O content of the hydrogented mixture. The unknown component, which my be sphingenine isomer, ws estimted s the difference between the concentrtion of the 15 : 1 pek in the originl dioxolne mixture nd the concentrtion of the 15 : 0 pek in the hydrogented dioxolne mixture (Tble 2). PANGANAMALA, GEER, AND CORN ELL Long-chin. Bses in Aort Sphingolipidp 451

8 Structure of Sphingdienine From Humn Aort Sphingomyelin The 1,3-dioxolnes isolted in the diene frction by TLC were cleved by reductive ozonolysis nd the dildehyde products were converted to their,w-didioxolne derivtives. Reference CS, CS, nd C,,w-didioxolnes were synthesized from 1,7-0ctdiene, 1,9-decdiene, nd 9- octdecenl, respectively. Dioxolnes were chromtogrphed on SE-30 nd EGS columns (Fig. 6) nd the mjor pek ws identified s derivtive of the (2x0 dildehyde from log Rt dt. Smll mounts of C9, CS, nd C7 didioxolnes were lso identified. The dibsic cids, which were obtined by periodte-permngnte oxidtion, were nlyzed s their methyl esters nd the mjor pek hd the sme retention time s the methyl ester of Clo dibsic cid on SE-30 nd EGS columns (Fig. 7). These dt showed tht the two double bonds were seprted by 10 crbon toms nd the dt suggested strongly tht the mjor diene LCB ws 4,x14- sphingdienine since this LCB hs been identified s the mjor diene LCB in humn plsm (4). The minor C9, Cg, nd C, peks probbly included both overoxidtion rtifcts nd sphingdienine isomers. It is interesting tht periodte-permngnte oxidtion yielded lrger quntities of the Cg nd C8 derivtives (Fig. 7) thn did reductive ozonolysis (Fig. 6). Reductie Ozonolysis of Intct Sphingolzpids The reductive ozonolysis of intct sphingolipids yielded long-chin ldehydes from their sphingenine moieties nd short-chin ldehydes such s the 9 : 0 dioxolne from their unsturted ftty cid moieties. GLC of the dioxolnes prepred from brin gngliosides is illustrted in Fig. 8. In contrst to cerebrosides, gngliosides contined lrge mount of 4-eicossphingenine which ws identified s the 2-hexdecyl-l,3-dioxolne derivtive. Direct reductive ozonolysis thus confirmed other studies on the LCB components of brin gngliosides nd cerebrosides (20). No 4-eicossphingenine ws detected by the reductive ozonolysis of sphingomyelin, sulftide, or cerebroside-sulftide frctions from humn ort (Fig. 9). The 12:O nd 13:O dioxolne peks which hd been detected fter the reductive ozonolysis of the monoene dioxolne frction (Fig. 5) were lso detected fter the reductive ozonolysis of intct sphingolipids. DISCUSSION Severl investigtors hve described the formtion of cermide by the enzymtic hydrolysis of sphingomyelin (2, 23) nd the formtion of LCB by the lkline hydrolysis of cermide (2). The dvntges of these hydrolysis FIG. 6. GLC nlysis of or,wdidioxolnes prepred from the sphingdienine dioxolne frction of sphingomyelin fter reductive ozonolysis: A, SE-30 column t 172'C; B, EGS column t 174'C. Other operting conditions rc summrized in Fig JOURNAL OF LIPID RESEARCH VOLUME 10,

9 ~ ~ z 0 n 0 8 FIG. 7. GLC nlysis of dibsic cid methyl esters prepred from the sphmgdienine dioxolne frction of sphingomyelin fter periodte-permngnte oxidtion: A, EGS column t 170OC; B, SE-30 column t 105 'C. Other operting conditions re summrized in Fig. 3. L FIG. 8. GLC nlysis of 1,3-dioxolnes prepred from purified brin gngliosides fter ozonolysis-reduction. Operting conditions re summrized in Fig. 3. procedures were confirmed in the present study. Both enzymtic nd lkline hydrolysis rections were complete s judged by the bsence of rectnts on TLC. No side-products such s 0-methyl-4-sphingenine were detected by either TLC or GLC. No 4D-hydroxysphingnine ws detected in the LCB from ort sphingomyelin by TLC (20). Periodte oxidtion of LCB is well-estblished procedure (1). Aldehydes re redily identified by GLC (1, 2); however, ldehydes re unstble compounds nd they tend to decompose nd polymerize on stnding (8). The 1,3-dioxolne derivtives of ldehydes were stble for long periods of time nd these derivtives were well suited for the seprtion of sturted, monoene, nd diene frctions on TLC pltes impregnted with AgN08. Unsturted LCB components yielded,&unsturted ldehydes nd the dioxolne derivtives of these compounds hd somewht higher R, vlues thn reference dioxolnes synthesized from ftty ldehydes in which the double bond ws in the 9,lO-position. Gunstone, Ismil, nd Jie (24) found tht the position of the double bond hd the sme effect on R, vlues in homologous series of methyl octdecenotes. Dioxolnes were redily identified nd quntified by GLC before nd fter ctlytic hydrogention. The position of the,p-double bond ws estblished by reductive ozonolysis nd the GLC nlysis of the ldehyde product s the dioxolne derivtive. The Rls vlues for unsturted dioxolnes prepred from LCB were much greter thn the R18 vlues for unsturted dioxolnes prepred from ftty ldehydes. For exmple, the 16 : 1 dioxolne from LCB hd the sme RIB vlue, 2.6, s the dioxolne from 9-octdecenl (8). Methyl octdecenote with trns double bond t the 2,3-position hs n PANGANAMALA, GEER, AND CORNELL Long-Chin Bses in Aort Sphingolipids 453

10 m z B m LT K n c 0 Y K L FIG. 9. GLC nlysis of 1,3-dioxolnes prepred from intct ort sphingolipids nd n LCB frction fter ozonolysis reduction: A, sphingomyelin; B, sulftides; C, LCB from sphingomyelin; D, cerebroside-sulftide frction. Operting conditions re summrized in Fig. 3. nomlous crbon number which is much higher thn the corresponding czs compound (24). The RIB dt for unsturted dioxolnes from LCB my be explined by the presence of trns double bond in the,o-position. The loction of double bonds in sphingdienine ws indicted both by reductive ozonolysis with GLC nlysis of the,@-didioxolne derivtives nd by permngnte-periodte oxidtion with GLC nlysis of the methyl esters of the dibsic cid derivtives. The reductive ozonolysis procedure hd no dvntge over the permngnte-periodte oxidtion procedure described by Chng nd Sweeley (22) except tht smller mounts of overoxidtion rtifcts my hve been formed (Figs. 6 nd 7). The mjor LCB components of sphingolipids re homologues of 4-sphingenines nd these components re converted to ldehydes by the reductive ozonolysis of intct sphingolipids. These sturted ldehydes were redily identified nd quntified s dioxolnes. The presence of 4-sphingenine nd 4-eicossphingenine in brin gngliosides ws demonstrted by this procedure. c C D Reductive ozonolysis of intct ort sphingolipids showed tht neither the cerebrosidesulftide frction nor the sulftide frction contined identifible mounts of 4-eicossphingenine. The reltive composition of the LCB from humn ort sphingomyelin ws similr but not identicl to the reltive composition of LCB from humn plsm sphingomyelin. Krlsson (2) found more 4-hexdecsphingenine nd less sphingnine in humn plsm. Aort sphingomyelin contined no heptdecsphingnine or 4D-hydroxysphingnine since these LCB components would hve yielded 15:O ldehyde by periodte oxidtion. A smll mount of heptdecsphingnine hs been identified in plsm (2). Trce mounts of tetrdecsphingnine nd 4-tetrdecsphingenine were found in ort sphingomyelin nd these LCB hve not been reported in humn plsm sphingomyelin. Aort sphingomyelin lso contined trce mounts of n unknown sphingnine nd two unknown sphingdienines which hve not been reported in humn plsm. The double bonds in the mjor sphingdienine from ort were seprted by 10 crbon tonis nd it is very likely tht this sphingdienine ws identicl with the mjor sphingdienine, 4,~14-~phingdienine, humn plsm (2, 4). Smll mounts of second isomer, sphingdienineb, were identified by 16 : 2b dioxolne peks in both the totl mixture nd the diene frction seprted on TLC. This isomer my correspond to the 4~12-sphingdienine tenttively identified in humn plsm sphingomyelin by Krlsson (25) or the 4,x13- sphingdienine found in bovine hert sphingomyelin by PopoviE (7). Polito et l. (4) hve suggested tht the Cg nd CB clevge products they obtined from humn plsm sphingomyelin were overoxidtion rtifcts. The concentrtion of the 16 : 2b dioxolne ws too low for us to distinguish between overoxidtion nd the presence of sphingdienine isomers. The third diene isomer tht ws seprted s dioxolne derivtive on TLC, 16:2,, hd the sme Rls s the mjor 16:l dioxolne nd this LCB derivtive my merely represent the incomplete seprtion of monoene nd diene dioxolne frctions. Dioxolnes from the LCB frction of humn ort sphingomyelin contined smll pek which ws tenttively identified s the 16 : 1 br derivtive of brnchedchin 4-sphingenine. This LCB component my correspond to the brnched-chin 4-sphingenine obtined from bovine kidney sphingomyelin by Crter nd Hirschberg (26). Hydrogention nd reductive ozonolysis dt suggested tht the 15 : 1 dioxolne pek contined second component, which did not yield the 15 : 0 dioxolne fter hydrogention or the 13 : 0 dioxolne fter reductjvc ozonolysis. These dt would be explined by the presence of n isomer of sphingenine in which the double 454 JOURNAL OF LIPID RESEARCH VOLUME 10, 1969

11 bond ws not t the 4,5-position. Additionl studies re required before the presence of this suggested isomer is estblished. e re indebted to Doctor R. H. McCluer for suggestions nd comments nd to Doctor D.. Buntine for the collection of ort tissue. This study ws supported in prt by PHS Reserch Grnts HE nd GM-09506, grnt from the United Helth Foundtions, Inc., nd postdoctorl fellowship (Doctor Pngnml) from the Centrl Ohio Hert Assocition. Mnuscript received 27 December 1968; ccepted I4 April REFERENCES 1. Sweeley, C. C., nd E. A. Mosctelli J. Lipid Res. 1: Krlsson, K. A Act Chem. Scnd. 21: Gver, R. C., nd C. C. Sweeley J. Amer. Oil Chem. SOC. 42: Polito, A. J., T. Akit, nd C. C. Sweeley Biochemistry. 7: Crter, H. E., nd R. C. Gver J. Lipid Res. 8: Smuelsson, B., nd K. Smuelsson Biochim. Biophys. Acto. 164: Popoviz, M Bull. Sci. Conseil Acd. RSF Yougoslovie. 11: Ro, P. V., S. Rmchndrn, nd D. G. Cornwell J. Lipid Res. 8: Rmchndrn, S., P. V. Ro, nd D. G. Cornwell J. Lipid Res. 9: einhouse, S., nd E. F. Hirsch A.M.A. Arch. Pothol. 29: Buck, R. C., nd R. J. Rossiter A.M.A. Arch. Pthol. 51: Steele, J. M., nd H. J. Kyden Trns. Assoc. Amer. Physicins Phildelphi. 68: Pries, C., A. Aumont, nd C. J. F. Bottcher Biochim. Biophys. Act. 125: 277, 14. Bijttcher, C. J. F., nd C. M. vn Gent J. Atheroscler. Res. 1: Bottcher, C. J. F Proc. Roy. SOC. Med. 57: Folch, J., M. Lees, nd G. H. Slone Stnley J. Biol. Chem. 226: Gry, G. M Biochem. J. 70: Dwson, R. M. C Biochem. J. 75: Rouser, G., G. Kritchevsky, nd A. Ymmoto In Lipid Chromtogrphic Anlysis. G. V. Mrinetti, editor. Mrcel Dekker, Inc., New York Smbsivro, K., nd R. H. McCluer J. Lipid Res. 4: Morris, L. J J. Lipid Res. 4: Chng, T. L., nd C. C. Sweeley J. Lipid Res. 3: Michlec, C., nd 2. Kolmn J. Chromtogr. 31: Gunstone, F. D., I. A. Ismil, nd M. L. K. Jie Chem. Phys. Lipids. 1: Krlsson, K. A Act Chem. Scnd. 18: Crter, H. E., nd C. B. Hirschberg Biochemistry. 7: PANGANAMALA, GEER, AND CORNELL Long-Chin Bses in Aort Sphingolipids 455