Identification of Fatty Acid Esters of Pregnenolone and Allopregnanolone from Bovine Corpora Lutea*

Transcription

1 THE JOURNAL OP BIOLOGICAL CHEMISTRY Vol. 255, No. 22. Iswe of November 25, pp Prlnted ZR [ I S.A. Identifiction of Ftty Acid Esters of Pregnenolone nd Allopregnnolone from Bovine Corpor Lute* Dniel H. Albert$, Lur Ponticorvo, nd Seymour Liebermn (Received for publiction, April 23,198) From the Deprtments of Biochemistry nd of Obstetrics nd Gynecology nd the Interntionl lnstitute the for Study of Humn Reproduction, The College of Physicins nd Surgeons, Columbi University, New York, New York 132 The lipoidl derivtives of steroids present in bovine te (3). None of the nturlly occurring lipoidl derivtives of corpor lute hve been identified nd quntified. Non- pregnenolone present in drenls hs been identified. polr steroid derivtiyes were extrcted from the tissue In this pper, the isoltion nd chrcteriztion of steroidl with mixture of chloroform nd methnol. The extrct derivtives present in bovine corpor lute re described. ws subjected to column nd high performnce liquid Lyophilized tissue ws extrcted exhustively with mixture chromtogrphy in order to remove phospholipids, of CHCL nd methnol (2:l). The extrcted wxy mteril cholesterol, triglycerides, cholesteryl esters, nd unes- ws purified by chromtogrphy on silic gel, monitoring the terified steroids. A portion of the purified nonpolr elutes for rdioctivity derived from previously dded mteril ws subjected to methnolysis. By gs-liquid trcer mount of synthetic [3H]pregnenolone olete. The chromtogrphic nlysis, the products were shown to elutes contining tritium were further purified by high presconsist of the steroids 3~-hydroxy-6-pregnnn-2-one (8%) nd pregnenolone (2%), nd the methyl esters of plmitic (34%), steric (27%), oleic (22%), linoleic (7%), rchidonic (7%), plmitoleic (3%), nd eicostrienoic (1%) cids. Mss spectrometric nlysis of the intct lipoidl derivtives nd of their methoximes confirmed the presence of seven esters of llopregnnolone nd pregnenolone: llopregnnolone plmitte, pregneno- lone plmitte, llopregnnolone sterte, llopregnnolone olete, llopregnnolone linolete, llopregnnolone eicostrienote, nd pregnenolone rchidonte. The mss spectrl dt suggested tht four other esters were lso present: pregnenolone sterte, pregnenolone olete, pregnenolone linolete, nd llopregnnolone rchidonte. This pper reports on the nture of some lipoidl derivtives of 3P-hydroxy-5-pregnen-2-one (pregnenolone) nd 3P-hydroxy-5-pregnn-2-one (dopregnnolone) present in bovine corpor lute. e were led to serch for lipoidl derivtives of steroids in this steroidogenic tissue becuse we hd shown previously tht nonpolr derivtives of pregnenolone (1, 2), 17-hydroxypregnenolone (2), nd dehydroisondrosterone (2) could be isolted from nother steroidogenic tissue, bovine drenl glnds. Although chromtogrphic evidence suggested the presence of different clsses of derivtives of pregnenolone in drenl tissue (2), the only lipophilic compounds so fr identified re ftty cid esters of pregnenolone which were biosynthesized by incubting the steroid with lyophilized mitochondri from bovine drenls (3). Five esters were isolted nd identified, using chemicl, chromtogrphic, rdioisotopic, nd mss spectrometric techniques. They re: pregnenolone rchidonte, pregnenolone linolete, pregnenolone olete, pregnenolone plmitte, nd pregnenolone ster- * This work ws supported by Grnts AM 11, AM 2846, HD 761, nd HD 577 from the United Sttes Public Helth Service, Ntionl Institutes of Helth. The costs of publiction of this rticle were defryed in prt by the pyment of pge chrges. This rticle must therefore be hereby mrked dvertisement in ccordnce with 18 U.S.C. Section 1734 solely to indicte this fct. $ Present ddress, Deprtment of Medicine, New York University School of Medicine, 55 First Avenue, New York, N. Y sure liquid chromtogrphy. Even though the rdioctive smples recovered by this mens probbly contined lrge mount of contminnts, the unlbeled, nturlly occurring steroidl derivtives present therein could be identified. By methnolysis, these derivtives were converted into the corresponding free steroids nd the methyl esters of the ssocited ftty cids. The steroids were identified by mss spectrometry s pregnenolone nd llopregnnolone. The methyl esters of the ftty cids were identified by their retention times on GC nlysis. The ftty cids identified were: plmitic, steric, oleic, linoleic, rchidonte, plmitoleic, nd eicostrienoic. The intct steroidl ftty cid esters nd their methoximes were lso nlyzed by mss spectroscopy. Seven esters were identified llopregnnolone plmitte, pregnenolone plmitte, llopregnnolone sterte, llopregnnolone olete, llopregnnolone linolete, llopregnnolone eicostrienote, nd pregnenolone rchidonte. The presence of four dditionl esters ws suggested by the mss spectrl dt: pregnenolone sterte, pregnenolone olete, pregnenolone linolete, nd llopregnnolone rchidonte. EXPERIMENTAL PROCEDURES MteriL~-[7-~H]Pregnenolone (17.2 Ci/mmol) ws purchsed from New Englnd Nucler Corp. nd purified by prtition chromtogrphy on Celite (4). Unlbeled steroids were obtined from Sterloids, Inc., ilton, N. H. Esters of pregnenolone, 3/3-hydroxy-5p:egnn-2-one, s well s [7-3H]pregnenolone with plmitic, steric, oleic, linoleic, nd rchidonic cid, were synthesized from the pproprite steroid nd ftty cyl chloride by the procedure described previously (3). Tissue Extrction-Corpor lute dissected from bovine ovries were homogenized in miniml volume of wter in ring Blendor. The homogente ws dried by lyophiliztion. A suspension of 5 g of the dry powder (obtined from 6 g of corpor lute) ws stirred for 6 h in 75 ml of ch1oroform:methnol (2:L v/ solution. The insoluble residue ws seprted by fdtrtion, resuspended in 3 ml of the chloroformxnethnol solution, nd stirred for n dditionl 3 h. After removl of the solid mteril, the filtrtes were combined. Addition of 2 ml of wter cused the combined filtrtes to seprte into two phses. The queous phse ws discrded nd the lower phse ws tken to dryness under reduced pressure. The residue ws 1618 _~. ~ ~.~ The bbrevitions used re: GC, gs chromtogrphy; MS, mss spectrometry; HPLC, high performnce liquid chromtogrphy; Me:&, trimethylslyl.

2 Lipoidl Derivtives of Steroids in Bovine Corpor Lute wxy solid nd constituted the source of of the lipoidl derivtives. Quntifiction of the Lipoidl Derivtives of Pregnenolone-Extrcts contining the lipoidl derivtives of pregnenolone were ssyed by the rdiochemicl procedure described previously (1). To ech smple, negligible weight of [3H]pregnenolone contining 1. x lo6 cpm ws dded nd the mixture ws prtitioned between n equl volume of 9% methnol nd isooctne. To ensure the removl of free steroids, the isooctne extrct ws wshed twice more with the 9% methnol solution. The hydrocrbon extrct, now devoid of rdioctivity, ws tken to dryness nd the residue ws treted with lkli to relese the steroids from their lipoidl derivtives (1). After the ddition of 1 x lo6 cpm of tritited pregnenolone s n internl stndrd, further purifiction ws chieved by dsorption chromtogrphy on silic gel nd then by prtition chromtogrphy on Celite (1 g) using the solvent system isooctne:methnol:hzo (1:9:1, v/v/ (1). The tritited trcer ws found in the fourth nd fifth holdbck volumes. Finl purifiction ws chieved by high pressure liquid chromtogrphy on Cle-Corsil column (ters Assocites, Milford, Mss.) using s eluent cetonitrile:hpo (55:45, v/. Trils with uthentic compounds hve shown tht this procedure effectively seprtes pregnenolone from llopregnnolone. The purified pregnenolone ws cetylted with [14C]cetic nhydride, purified, nd recrystllized s described previously (1). Quntifiction of pregnenolone ws bsed upon the 3H/ 4C rtio present in the finl product obtined fter three or four recrystlliztions using the eqution jig = /&d, where = dpm of tritited internl stndrd; b = 3H/ 4C rtio of recrystllized pregnenolone cette; c = 19,5, the specific ctivity of [ YJlcetic nhydride in dpm/w of nrenenolone (determined on known mount-of pregnenolonej; nd d= the frction of the smple to which the tritited internl stndrd hd been dded. Isoltion of Lipoidl Derivtives of Steroids-In order to id in monitoring the purifiction of lipoidl derivtives nd to provide bsis for determining procedurl losses, weightless smples contining 1,6 cpm of echof the following were dded to the wxy residue extrcted from 2 g of bovine corpor lute: Il- 4Cltrinhnitin. f26- C]cholesteryl ole& [C C]pregnenolone, nd [7- Hjpregnenokme olete. Initil purifiction ws chieved by chromtogrphic nlysis on silic gel (Silic oelm 3-2) column (5 x 9.5 cm). Elution ws crried out with l-liter portions of the following: isooctne; isooctne: benzene (5:5, v/; benzene; nd ethyl cette. The lipoidl derivtives trced by the presence of [3H]pregnenolone olete were recovered in the frction eluted by benzene. These derivtives were further purified by high performnce liquid chromtogrphy using column (7 (inner dimeter) x 9 mm) pcked with Porcil C (ters Assocites). The elution scheme ws s follows: () 2 ml of isooctne:benzene (5:5, v/; (b) 4 ml of isooctne:benzene (2575); (c) 4 ml of benzene; nd (d) 2 ml of ethyl cette. The isooctne: benzene (25:75) mixture (Frction b) eluted both the t&mm-lbeled pregnenolone olete nd the unlbeled lipoidl derivtives of steroids occurring nturlly. This frction ws devoid of C, indicting tht the procedure hd seprted triglycerides, cholesteryl esters, nd free pregnenolone from the lipoidl derivtives. Although the smple of lipoidl derivtives obtined in this mnner contined contminnts of unknown structure, it ws possible to identify the steroids therein without dditionl chromtogrphy. This ws ccomplished by subjecting liquots to the sodium methoxide methnolysis procedure described by Tuckey nd Stevenson (5). The products, methyl esters of ftty cids nd steroids s the free lcohols, were seprted on.5-g silicic cid columns (.5 x.5 cm) from which the methyl esters were eluted with benzene nd the steroids were eluted with ethyl cette. The free steroids obtined by this procedure were nlyzed by GC nlysis fter being converted into their trimethylsilyl (Me,Si) derivtives with N,O-bis-trimethylsilylcetmide (Pierce Chemicl Co., Rockford, Ill.). Gs Chromtogrphic nd Mss Spectrl Anlysis of Clevge Products-Anlysis of ftty cid methyl esters obtined from the methnolysis process ws chieved on Perkin-Elmer 392 gs cbromtogrph using flme ioniztion detector nd column (2 mm x 2 m) pcked with 1% SP-233 on Supelcoport (1/12) (Supelco, Bellefonte, P.). The temperture ws progrmmed from 165 to 235 C t 2 C/min. The injector ws mintined t 25O C, nd the detector ws mintined t 29 C. The flow rte of the crrier ns (helium) ws 3 ml/min. Peks were identitled by comprison of their retention times with uthentic stndrds. The trimethvlsilvl derivtives of steroids were chromtogrphed on column (2 mm x 4 m) pcked with 1% SP-1 on 1/12 Sunelconort t 2 C. The effluent ws split so tht one-third went to flme ioniztion detector nd two-thirds ws diverted through jet moleculr seprtor into the source of the mss spectrometer. The trnsfer line, jet seprtor, nd source temperture were held t 23 C. The voltge used for ioniztion ws 75 ev. Spectr were collected by computer with repetitive mgnetic scnning t 4 s/decde. Spectr nd retention times were compred with those of uthentic steroids treted in the sme wy. GC Anlysis of the Nturlly Occurring Lipoidl Derivtives- Aliquots of HPLC Frction b were nlyzed bv gs chromtogrphy on column (2 mm x 1 m) pcked with 3% SP-21 on Supelcoport 1/12 (&Delco) mintined t 315 C. The iniector nd detector of the instrument, Brber-Colmn model 565 equipped with n effluent strem splitter, were mintined t 35 C. The column effluent ws collected in n inverted Psteur pipette (.3 x 5 cm) filled with glss wool. Two-thirds of this frction ws used for further nlysis by mss spectrometry (see below). One-third of ech of the collected smples ws treted by the methnolysis procedure, fter which the free steroids nd methyl esters of the ftty cids were identified in the mnner described bove. In ddition, smples of syntheticlly prepred steroid esters were nlyzed by gs chromtogrphy nd their retention times were compred with those of the smples of nturl origin. Mss Spectrl Anlysis of the Lipoidl Derivtives-One-third of ech pek of the intct steroid esters collected from the GC effluent ws subjected to mss spectrl nlysis by direct probe insertion into the source of the mss spectrometer. Chemicl ioniztion ws used with methne s the regent gs. The source temperture ws held t 22 C. Spectr were recorded by computer while scnning the mgnetic field t 4s/decde s the probe temperture ws slowly incresed from 5 to 4 C. Another third of ech of the intct steroid esters collected from the GC effluent ws converted into the methoxime derivtives s described previously (3). The products were nlyzed by direct probe insertion under the sme conditions s those described bove for steroid esters. In one cse, electron impct ioniztion (75 ev) ws used. Syntheticlly prepred esters of pregnenolone nd llopregnnolone were nlyzed in n identicl fshion. RESULTS Quntifiction of Lipoidl Derivtives of Pregnenolone in Bovine Corpor Lute On three different occsions, btches of bovine corpor lute were obtined from the slughterhouse. Ech ws processed in the mnner described bove nd the quntities of pregnenolone estimted to be present in the three extrcts s lipoidl derivtives re recorded in Tble I. These vlues were determined by the double isotope dilution method (1). The qulittive identifiction of pregnenolone ws estblished by the fct tht both the 3H/ 4C rtios nd the specific ctivities with respect to both 3H nd 14C (dt not shown) of the products reched constnt vlues fter three or four recrystuiztions. After Chrcteriztion of the Lipoidl Derivtives of Steroids Present in Bovine Corpor Lute lkfethnolysk Methnolysis of the mteril in Frction b from the HPLC nlysis yielded methyl esters of ftty cids nd free steroids. Both mterils were nlyzed by GC. The trcing of the GC nlysis of the methyl esters of the ftty cids is shown in Fig. 1. The methyl esters were identified by their retention times s phnitic (16:), 33.7%; stek (NO), 27.% oleic (l&l), 22.2%; linoleic (18:2), 7.%; nd rchidonic (2:4), 7.2%. PImitoleic (16:l) nd eicostrienoic esters (2:3) were present in lesser mounts, 2.6 nd 1.% respectively. The free steroids were converted into their Me&i derivtives which were then nlyzed by GC-MS techniques. Fig. 2 shows the trcing of the GC nlysis. The presence of two components ws reveled. The compound lbeled A ws identified s the MeSi derivtive of 3/3-hydroxy-5-pregnn-2-

3 162 Lipoidl Derivtives of Steroids in Bovine Corpor Lute TABLE I Rdiochemicl nlysis of the concentrtion of lipoidl derivtives ofpregnenolone in three smples of bovine corpor lute Crystlline dt of pregnenolone cette" Smple - X, I MLI I X2 I ML I X, I MLz 1 X, I M 4 'H/"C rtio pg/kg tissue X,, product of the nth crystlliztion; ML,, residue left in the mother liquor of the nth crystlliztion. The solvents used for crystlliztionswere: XI, methnol; Xz, chloroform:hexne; X, ethyl cette:ligroin, b.p. 6-9'C; nd X.,, cetone:petroleum ether, b.p. 3-4 C. w z IJJ n 89 The retention times of the three peks corresponded respectively to those of uthentic stndrds of 16-, l-, nd 2- crbon ftty cids esterified to either llopregnnolone or pregnenolone. To substntite the identity of the mteril in ech pek, portion of the contents of ech ws subjected to methnolysis, nd the relesed free steroids (s MeSi derivtives) nd ftty cids (s methyl esters) were nlyzed by m z m n I I I I I I I A TIME (minutes) FIG. 1. Gs chromtogrphic nlysis of the methyl esters of the ftty cids from Frction b. Conditions: glss column (2 mm X 2 m) pcked with 1% SP-233 on 1/12 Supelcoport; crrier gs, helium t 3 ml/min, injector temperture, 25 C; detector temperture, 29 C; column temperture, progrmmed 'C t 2"C/ min. one (llopregnnolone) by comprison of its retention time (24.5 min) nd its mss spectrum with those of n uthentic smple. By the sme mens, the compound lbeled B ws identified s the MeSi derivtive of 3P-hydroxy-5-pregnen- 2-one (pregnenolone). Authentic smples of the other three ring A isomers of llopregnnolone were lso exmined by GC-MS nlysis. The mss spectr of their MeBSi derivtives were significntly different from tht of 3P-hydroxy-5-pregnn-2O-one. Their retention times on the column used were lso different: 3phydroxy-5p-pregnn-2O-one, 19.6 min, S-hydroxy-5P-pregnn-2-one, 19.6 min; nd 3-hydroxy-5-pregnn-2-one, 17.2 min. By GC Anlysis of Intct Steroid Esters The results obtined from nlysis of the products of methnolysis suggested the presence of ftty esters of llopregnnolone nd of pregnenolone. Verifiction ws sought by subjecting the lipoidl steroid frction (HPLC Frction b) itself to preprtive high temperture gs chromtogrphy. On the column used, steroid esters hving cyl'groups of the sme crbon chin length but differing in the degree of unsturtion in the ftty cid moiety cnnot be seprted from ech other. Likewise, esters of sturted steroids cnnot be distinguished from those of their 5,6-unsturted counterprts. Gs chromtogrphy resolved the mteril into three frctions (Fig. 3). TIME (minutes) FIG. 2. Gs chromtogrphic nlysis of the steroids from Frction b s the trimethylsily derivtives. Conditions:glss column (2 mm X 4 m) pcked with 1% SP-lo on 1/12 Supelcoport; crrier gs, helium t 25 ml/min; injector temperture, 26'C; detector temperture, 29 C; column temperture, 2"C, the trnsfer linetothemssspectrometernd thejetseprtorndsource temperture, 23OC; ioniztion voltge, 75 ev. z n I I I I I 1 I TIME (minutes) FIG. 3. Gs chromtogrphic profile of the steroid esters in Frction b. Conditions: glss column (2 mm X 1 m) pcked with 3% SP-21 mintined t 315 C; injector nd detector temperture, 35OC; crrier gs, helium t 35 ml/min.

4 Lipoidl Derivtives gs chromtogrphy. In both Peks 1 nd 2, llopregnnolone nd pregnenolone were found in the rtio 4:l. Pek 1 contined predominntly esters of plmitic cid (75%of the totl), wheres Pek 2 consisted primrily of 18-crbon ftty cid esters (32% 18:,45% 18:1, nd 13% 18:2). Most of the contents of Pek 3 ws reserved for mss spectrl nlysis. However, smll portion ws subjected to the methnolysis procedure. Neither ftty cid methyl esters nor steroids were detected by GC nlysis. This filure is ttributed to the poor recovery from high temperture gs-liquid chromtogrphic nlysis of steroid esters of polyunsturted ftty cids. By Mss Spectrl Anlysis of the Intct, Nturlly Occurring Steroid Esters The mterils in the three peks could not be nlyzed by GC-MS directly, becuse the steroid esters contined in them did not survive the trnsport t high temperture from the GC column to the source of the mss spectrometer. Insted, MS nlysis ws chieved by direct insertion probe. By this method, interpretble spectr were usully obtined despite the fct tht the three peks contined pprecible quntities of contminnts, some probbly due to column bleed. ith some bdly contminted smples, steroidl esters were identified from their mss spectr fter subtrcting bckground by mens of the computer. The following interprettions of the spectr re bsed upon comprisons with those of uthentic steroid ftty cid esters nd their methoximes, nlyzed using identicl conditions. Pek 1-The chemicl ioniztion mss spectrum of the contents of this pek (Fig. 4A) showed cluster of ions t m/e 557 (corresponding to (M + l)+), t m/e 556 (corresponding to ), nd t m/e 555 (corresponding to (M- I)+) for llopregnnolone plmitte (M, = 556). However, the ion t m/e 555 could lso be due to the (M + 1)+ ion for pregnenolone plmitte (MI = 554). The pek t m/e 539 ppers to result from the loss of wter from the intense pseudomoleculr ion t m/e 557 (A )+. The pek t m/e 31 represents the steroid frgment left fter the loss of the ftty cid moiety (M RCOOH)+ from llopregnnolone plmitte. The ion t m/e 299 cn represent the steroidl frgment rising from 1s of the ftty cid moiety from either llopregnnolone phi- tte (M- 1 - RCOOH) or tht from pregnenolone plmitte (M RCOOH). The pek t m/e 297 is steroidl frgment tht could rise only from pregnenolone plmitte '"1 A 5 i I m/e FIG. 4. Chemicl ioniztion mss spectr of Pek I. A, spectrum of the mixture of constituents of Pek I. B, spectrum of the mixture of the methoxime derivtives of these constituents Both spectr were recorded using methne s the regent gs, source temperture of 2OC, nd probe temperture between 32 nd 38 C. of Steroids Bovine Corpor Lute 1621 by loss of the ftty cid moiety from the (M - 1) ion, (M RCOOH). The chemicl ioniztion spectr of uthentic llopregnnolone plmitte nd of pregnenolone plmitte both show (M + l), (M - l), nd (M + 1) - 18 ions. Ions from llopregnnolone esters re much more intense thn re those from pregnenolone esters. Thus, in the nlysis of mixture of these two esters (especilly where the concentrtion of the llopregnnolone species predomintes), the spectr of pregnenolone esters re less evident thn re those of llopregnnolone esters. The (M + 1) - 18 frgment is lwys more intense when the ftty cid moiety is sturted. Fig. 4B dpicts the chemicl ioniztion mss spectrum of the methoxime of the mteril in Pek 1. Peks t m/e 586, 585, nd 584 correspond to the (M + l)+, M', nd (M - 1)' pseudomoleculr species of the methoxime derivtive of llopregnnolone plmitte (Mr = 585). However, the pek t m/e 584 could lso represent the pseudomoleculr ion (M + 1)' for pregnenolone plmitte methoxime (M, = 583). In fct, the pek t m/e 582 clerly indictes the presence of pregnenolone plmitte methoxime, since this ion cn rise only by loss of hydrogen tom from tht ester. This ion nd the steroidl frgment t m/e 33 (M RCOOH)' (s well s the ion t m/e 328 for pregnenolone plmitte methoxime) re lrger by 29 tomic mss units thn the corresponding peks shown in Fig. 44, differences tht re due to the chnge in reltive moleculr mss resulting from the conversion of the Cm-keto group to the =C=N--OCH3. The pek t m/e 554 (M- OCH3)+ is lso chrcteristic of the methoxime. In the spectr of uthentic llopregnnolone ftty cid esters (not shown) m/e 299 ((M- 1) - RCOOH) hs n intensity 6 to 8% of the bse pek. Likewise, the uthentic methoximes of llopregnnolone ftty cid esters give rise to spectr where the intensity of m/e 328 ((M- 1) - RCOOH) is of the order of 4 to 5% of the bse pek. On the other hnd, in spectr of pregnenolone esters nd their methoximes, m/e 299 nd 328 re the bse peks. In the spectr depicted in Fig. 4, these two mss frgments (299 nd 328) hve intensities equl to 2 to 25% of the bse peks, which, for the underivtized esters ppers t m/e 31, nd for the methohes occurs t m/e 33. This is the result tht would be expected from the contribution to the peks t 299 nd 328by the presence in Pek 1 of smll mounts of pregnenolone esters. Furthermore, while the pseudomoleculr ion (m/e 555) my come from either llopregnnolone plmitte or pregnenolone plmitte, the ion t m/e 582 (M- 1) in the spectrum of the methoxime derivtive (Fig. 4B) cn come only from pregnenolone plmitte methoxime (Mr = 583). This evidence, together with the identifiction of the clevge products s methyl plmitte nd the steroids s llopregnnolone nd pregnenolone (s Me&i derivtives), estblished with resonble certinty the presence in Pek 1 of llopregnnolone plmitte nd pregnenolone plmitte. Pek 2-The chemicl ioniztion spectrum of the mteril in this pek is depicted in Fig. 5A. It shows cluster of ions from m/e 578 to 585, corresponding to moleculr ions () nd pseudomoleculr ions (M + 1)+ or (M- 1)+ for mixture of llopregnnolone sterte (MI = 584), llopregnnolone olete (MI = 582), nd llopregnnolone linolete (M, = 58), together with the corresponding pregnenolone esters which re two tomic mss units less, 582, 58, nd 578. The cluster of peks (m/e 563 to 567) origintes from the loss of wter from the respective (M + 1) pseudomoleculr ions. The bse pek t m/e 31 nd the frgment t m/e 299 represent the llopregnnolone nd pregnenolone moieties, respectively, fter the loss of the ftty cids (M RCOOH)'. Fig. 5B shows the chemicl ioniztion spectrum of the methoxime derivtives of the components in Pek 2. The

5 ~ Lipoidl Derivtives L loo] A I I IO rn /e FIG. 5. Chemicl ioniztion mss spectr of Pek 2. A, spectrum of the mixture of constituents of Pek 2. B, spectrum of the mixture of methoxime derivtives of these constituents. Conditions: sme s those used for spectr shown in Fig. 4. expected increse of 29 tomic mss units in the moleculr weight over the vlues for the underivtized compounds (Fig. 5A) is evident. The qusimoleculr ions (M- 1)' nd (M + I)+ t m/e 68 nd 61 correspond to the methoxime derivtive of both llopregnnolone olete nd pregnenolone sterte (Mr = 611); those t m/e 612 nd 614 correspond to llopregnnolone sterte methoxime (Mr = 613). Peks t m/e 578, 58, nd 582 (M- OCH,) re lso chrcteristic of the methoximes. The bse pek t m/e 33 nd the pek t m/e 328 correspond to the methoximted steroid frgments of llopregnnolone nd pregnenolone, respectively, rising from the loss of the ftty cid moieties (M RCOOH)+. Thus, from the dt shown in Fig. 5, the presence of llopregnnolone sterte (Mr = 584) is clerly indicted by the pseudomoleculr ions t m/e 583 (M - 1) nd 585 (M + 1) in Fig. 5A nd by ions t m/e 612 nd 614 in Fig. 5B (for llopregnnolone sterte methoxime, M, = 613). The bse pek t m/e 31 in Fig. 5A nd t m/e 33 in Fig. 5B is strong evidence for the llopregnnolone moiety. The presence of llopregnnolone olete (Mr = 582) is suggested by the ions t m/e 581 nd 583 in the spectrum shown in Fig. 5A. The ions t m/e 579 nd 581 in this spectrum could represent either llopregnnolone linolete (Mr = 58) or pregnenolone olete (Mr = 58). If the pregnenolone esters were the most bundnt species, lrge pek t m/e 299 would be expected. However, the intensity of this pek is only slightly lrger thn it would be if only llopregnnolone esters were present. Thus, it ppers tht esters of pregnenolone re minor constituents of Pek 2. This conclusion is consistent with the observtion mde on the frgment t m/e 328 tht ppers in the spectrum of the methoxime derivtives (Fig. 5B). The intensity of this pek is lso only slightly lrger thn it would be if only esters of the sturted Czl-steroid were present. These results, tken together with those obtined by methnolysis, which proved tht the rtio of llopregnno1one:pregnenolone in this pek ws 4:l nd tht steric, oleic, nd linoleic cid esters re present, constitute strong evidence for the presence of llopregnnolone sterte, llopregnnolone olete, nd llopregnnolone linolete in Pek 2. The evidence for the occurrence of smll quntities of pregnenolone esters with some or ll of these cids is suggested from the intensity dt. Pek 3-The chemicl ioniztion mss spectrum of the constituents in this pek is shown in Fig. 6A. Peks t m/e 61 (M - l)+ nd 63 (M + 1)+ could correspond to pregnenolone rchidonte (Mr = 62); those t m/e 63 (M - I)+ nd 65 (M + 1)+ re indictive of llopregnnolone rchidonte of Steroids Bovine Corpor Lute (Mr = 64); those t m/e 65 (M - 1)+ nd 67 (M + 1)+ could represent llopregnnolone eicostrienote (Mr = 66). Becuse of the high bckground in the region round m/e 6 (6 to 65) ssocited with this smple, there is some uncertinty concerning the identity of the qusimoleculr ions observed in the methne chemicl ioniztion spectrum (Fig. 6A). Ordinrily, the ions t m/e 65 (M - 1) nd 67 (M + 1) nd the bse pek t m/e 31 would be sufficient to identify llopregnnolone eicostrienote (Mr = 66). However, ions in the region just bove mss 6 incresed in intensity when the temperture of the probe ws rised bove tht necessry for voltiliztion of the steroid esters. Thus, it ppers tht the nlysis ws confused by the presence in this smple of contminnts tht gve rise to ions of these msses. Evidence for the identity of the components present in Pek 3 is best estblished by the electron impct spectrum of the methoximes shown in Fig. 6B. The moleculr ions hve shifted the expected 29 tomic mss units to m/e 631, 633, nd 635, where they no longer overlp with the ions contributed by the contminnts. In this spectrum, where moleculr weights re obtined directly, the ion t m/e 635 cn correspond only to llopregnnolone eicostrienote methoxime (Mr = 635); tht t m/e 633 cn represent either llopregnnolone rchidonte methoxime or pregnenolone eicostrienote methoxime (Mr = 633). The ion t m/e 631 cn correspond only to pregnenolone rchidonte methoxime (Mr = 631). The peks t m/e 33 nd 328 represent the methoximted steroidl frgments of llopregnnolone nd of pregnenolone originting by loss of the ftty cid moieties from the moleculr ions (M- RCOO). The frgments t m/e 33 nd 34 lend further support to the conclusion tht Pek 3 contined esters of rchidonic cid, since the frgments correspond to the mss of this cid. Unfortuntely, the M - OCH3 frgments (m/e 6 to 64) which re chrcteristic of spectr of the methoxime derivtives of these steroid esters were obliterted from this spectrum when the bckground ws subtrcted. From this dt, from the retention times of the intct steroid esters on GC nlysis, nd from the fct tht the methyl esters of rchidonic cid nd eicostrienoic cid were obtined s methnolysis products of the HPLC Frction b, the presence of llopregnnolone eicostrienote nd pregnenolone rchidonte were unequivoclly identified s constituents of Pek 3. Becuse no single mss ion cn be ttributed solely to llopregnnolone rchidonte, the identity of this ester hs not been estblished with certinty. However, the evidence suggests tht it is present in Pek 3 in substntil mounts. z ~ 5 IO 425 A d ; > f o z w s B 1 d. 61 1,m I Id.il",.. L (. IO m /e FIG. 6. Mss spectr of Pek 3. A, chemicl ioniztion spectrum of the mixture of constituents of Pek 3. B, electron impct spectrum of the mixture of methoxime derivtives of these constituents. ( I

6 Lipoidl Derivtives DISCUSSION This report describes for the first time the chrcteriztion of nturlly occurring lipoidl derivtives of steroids from steroidogenic endocrine glnd. Although the presence in bovine corpor lute of seven steroidl esters of ftty cids ws estblished nd the presence of four others ws suggested by the mss spectrl dt, none of the esters were isolted s pure compounds. Identifiction of the steroidl esters depended upon mss spectrl nlysis which provided identities of the individul components of the still unresolved mixtures. Becuse the spectr of some of the esters nd their methoximes disply m/e peks tht re the sme s those of relted compounds, the identity of four esters could not be estblished rigorously, even though t lest one of these, llopregnnolone rchidonte, ws probbly present in sensible mounts. The identities of seven other esters were clerly estblished becuse the spectr of the intct esters or their methoximes contined m/e peks tht were chrcteristic for ech of the seven. It is likely tht the isoltion methods used in this study did not succeed in reveling ll the lipoidl derivtives of steroids tht exist in bovine corpor lute. As is evident from the experimentl detils, only selected frctions were nlyzed by MS nlysis. The components nlyzed by mss spectroscopy were those hving chromtogrphic chrcteristics similr to the dded trcer, [3H]pregnenolone olete. Other frctions from the silic gel chromtogrm s well s mterils other thn Frction b from the HPLC nlysis remin to be exmined. If other lipoidl derivtives with different properties re present in this tissue, the methods used would not hve detected them. The procedures, however, were dequte to detect nd isolte pregnenolone s well s llopregnnolone, which ws the most bundnt steroid present in the frctions exmined. The discovery of esters of llopregnnolone in corpor lutel tissue is not surprising, since the free Cnl-hydroxyketone nd progesterone were mong the first steroids to be isolted from nturl sources. In 1934, four groups of investigtors (6-9) were serching for the progesttionl hormone from ovries. All four reported the isoltion of both 3P-hydroxy-5-pregnn- 2-one nd progesterone. Since then, others hve lso isolted this substnce from the sme source (1, 11). Quntittive of Steroids Bovine Corpor Lute 1623 determintions reported in 1975 (12) indicted tht the concentrtions of llopregnnolone (its esters were not mesured) prlleled the vritions of those of progesterone during the estrous cycle. At ll times, the concentrtion of llopregnnolone ws lmost equl to tht of the hormone. The significnce of these reltionships is unknown. Allopregnnolone is generlly considered to be inctive s clssicl progesttionl gent, nd in this respect it differs from dihydrotestosterone. This C19-sturted ketone does not possess the chrcteristic of n,,b-unsturted ketone function, but it nevertheless is potent ndrogen (in some tissues) like its reltive, the mle sex hormone, testosterone. Thus, it is not prudent to rest with the ssumption tht the bsence of n,p-unsturted crbonyl function in llopregnnolone ensures tht this Czl- steroid hs no physiologicl function. Its bundnce in corpor lute suggests tht its presence is not fortuitous. This pper reports the presence of esters of llopregnnolone nd pregnenolone in corpor lute, the concentrtion of the former predominting. The physiologicl significnce of these findings is, t present, unknown. In fct, the role of ny of the ftty cid esters of steroids tht hve been found in steroidogenic tissues remins to be determined. REFERENCES 1. Hochberg, R. B., Bndy, L., Ponticorvo, L., nd Liebermn, S. (1977) Proc. Ntl. Acd. Sci. U. S. A. 74, Hochberg, R., Bndy, L., Ponticorvo, L., elch, M., nd Liebermn, S. (1979) J. Steroid Biochem. 11, Mellon-Nussbum, S., Ponticorvo, L., nd Liebermn, S. (1979) J. Biol. Chem. 254, Siiteri, P. K. (1963) Steroids 2, Tuckey, R. C., nd Stevenson, P. M. (1979) Anl. Biochem. 94, Butenndt, A., estphl, U., nd Hohlweg,. (1934) Hoppe- Seyler s Z. Physiol. Chem. 227, Slott, K. H., Ruschig, H., nd Fels, E. (1934) Ber. Dt. Chem. Ges. 67, intersteiner, O., nd Allen,. M. (1934) J. Biol. Chem. 17, Hrtmnn, M., nd ettstein, A. (1934) Helu. Chim. Act 17, Herd, R. D. H. (1978) J. Am. Chem. SOC. 6, Prelog, V., nd Meister, P. (1949) Hel Chim. Act 32, Axelson, M., Schumcher, G., Sjovd, J., Gustfsson, B., nd Lindell, J.. (1975) Act Endocrinol