cytochromes P-450 reconstituted in phospholipid vesicles:

Transcription

1 Proc. NtL Acd. Sci. USA Vol. 79, pp , April 1982 Medicl Sciences Reductive metbolism of crbon tetrchloride by humn cytochromes P-450 reconstituted in phospholipid vesicles: Mss spectrl identifiction of trichloromethyl rdicl bound to dioleoyl phosphtidylcholine (liver necrosis/free rdicls/lipid peroxidtion/spin trpping/humn proteins) JAMES R. TRUDELL*, BERNHARD BOSTERLNG*, AND ANTHONY J. TREVORt *Deprtment of Anesthesi, Stnford University School of Medicine, Stnford, Cliforni 94305; nd tdeprtment of Phrmcology, University of Cliforni, Sn Frncisco, Cliforni Communicted by Jmes P. Collmn, Jnury 18, 1982 ABSTRACT t hs been proposed tht covlent binding of rective metbolites to liver membrne constituents my be responsible for the heptoxicity of crbon tetrchloride. This study demonstrtes tht trichloromethyl free rdicl is the mjor reductive metbolite of crbon tetrchloride by cytochrome P-450 nd tht this free rdicl is cpble ofbinding to double bonds of ftty cyl chins of the phospholipids in the membrne surrounding cytochrome P-450. The structurl identifiction of the rective free rdicl metbolite nd the product of its ddition to phospholipids ws ccomplished by use of reconstituted system of humn cytochromes P-450, NADPH-cytochrome P-450 reductse, nd cytochrome b5 in phospholipid vesicles. The reconstituted vesicles contined mixture of dioleoyl phosphtidylcholine nd egg phosphtidylethnolmine tht served s both structurl components nd trgets for trichloromethyl free rdicl binding. After incubtion of these vesicles under N2 tmosphere in the presence of NADPH with '4CC14, the phospholipids were extrcted nd then seprted by high-pressure liquid chromtogrphy. The dioleoyl phosphtidylcholine frction ws trnsesterified nd the resulting single "4C-lbeled ftty cid methyl ester ws purified by reversephse chromtogrphy. Desorption chemicl ioniztion mss spectrometry with mmoni s regent gs s well s desorption electron-impct mss spectrometry permitted identifiction of the moleculr structure s mixture of 9- nd 1O-(trichloromethyl)sterte methyl esters. Crbon tetrchloride hs been known for over 40 yers to cuse cute centrilobulr heptic necrosis in mmmls (1). n 1961 unique form of reductive metbolism of crbon tetrchloride ws described tht involved chloroform production vi homolytic fission of crbon-chlorine bond (2). The trichloromethyl free rdicl ws postulted s n intermedite in this rection nd, on the bsis ofits potentil rectivity, ws suggested to be involved in crbon tetrchloride heptotoxicity (2-5). Evidence for the formtion of crbon-centered free rdicls from metbolism of crbon tetrchloride hs lso come from electron prmgnetic resonnce studies of spin-trp dducts (6, 7). However, difficulties in the interprettion of hyperfine splitting in EPR spectr hve mde definitive structurl identifiction difficult (8). Moreover, the mechnism by which these free rdicls bind to constituents ofbiologicl membrnes hs not been previously demonstrted. n ddition, the existence of rective dichlorocrbene hs been proposed (5) on the bsis of spectrl studies of complex formtion between hlocrbon metbolites nd cytochrome P-450. The observed formtion ofcrbon monoxide from crbon tetrchloride is in ccord with two suc- The publiction costs ofthis rticle were defryed in prt by pge chrge pyment. This rticle must therefore be hereby mrked "dvertisement" in ccordnce with 18 U. S. C solely to indicte this fct. cessive one-electron reductions ctlyzed by cytochrome P-450 (9). Products ofthe metbolism ofcrbon tetrchloride re known to be cpble of covlent binding to both lipids nd proteins (3, 4, 10). However, the moleculr structures of such rective metbolites hve not been determined nd there hs been no chrcteriztion of dducts formed or their mechnism of binding to tissue molecules. Pst ttempts to isolte dducts formed fter metbolism of 14C-lbeled crbon tetrchloride hve been unsuccessful due to the heterogeneity of rdioctive products (11). To circumvent the problem of heterogeneity of microsoml phospholipids, we hve reconstituted purified cytochrome P- 450 from humn liver, NADPH-cytochrome P-450 reductse, nd cytochrome b5 in phospholipid vesicles tht contined dioleoyl phosphtidylcholine (Ole2-PtdCho) s the sole phosphtidylcholine (2, 12-14). n this study, mixture of humn cytochromes P-450 ws used in order to encompss ll the different possible metbolic pthwys tht my result from the mny isoenzymes of cytochrome P-450. Humn cytochromes P-450 were used to increse the relevnce to humn heptotoxicity in tht mjor species differences in pthwys of hlocrbon metbolism hve been reported (15). Cytochrome b5ws included in the reconstituted system in the sme molr rtio s it occurs in the endoplsmic reticulum becuse it hs been shown to prticipte in electron trnsfer from NADPH-cytochrome P-450 reductse to cytochrome P-450 (16, 17). The Ole2- PtdCho ws shown to ct s structurl component of the vesicle, to support ctlytic ctivity of the reconstituted enzyme system, nd to serve s n effective trget for rective metbolites tht my bind covlently to the double bonds in ftty cyl chins. The most simple component to dd to reconstituted system s trget for rdicl or crbene ddition would be stright-chin hydrocrbon or ftty cid with single double bond. However, simple mono-unsturted smll molecules such s hexene or methyl olete re themselves good substrtes for cytochrome P-450 nd re therefore unsuitble. For this reson we used Ole2-PtdCho s the only phosphtidylcholine in the reconstituted system becuse the single double bond in the oleoyl chins results in the most simple mixture of dducts fter free rdicl ddition (9- or 10- substitution). The reconstituted system ws fully chrcterized s to protein nd phospholipid content, metbolic ctivity towrd vriety of substrtes, nd dependence on NADPH for ctivity (12, 18). We hve used the reconstituted vesicle system described bove in n extensive study of the reductive metbolism of the 2678 Abbrevitions: Ole2-PtdCho, dioleoyl phosphtidylcholine; egg PtdEtn, egg phosphtidylethnolmine; ODS, octdecylsilic.

2 Medicl Sciences: Trudell et L inhltion nesthetic hlothne (2-bromo-2-chloro-1, 1, 1-trifluoroethne). We hve shown tht only in the presence ofnadph do 14C-lbeled metbolites bind to phospholipids in reconstituted vesicles in mnner similr to tht in microsomes. We were ble to demonstrte tht 1-chloro-2,2,2-trifluoroethyl rdicl is the mjor reductive metbolite of hlothne nd tht it binds to the double bond of the ftty cyl chin of Ole2-PtdCho (14, 18). n seprte study we demonstrted tht the hlothne free rdicl dduct formed metboliclly in the reconstituted system is identicl to tht formed by free rdicls creted by ultrviolet irrdition of hlothne in the presence of methyl olete (19). MATERALS AND METHODS Humn livers were obtined from hert donors in the Stnford Hert Trnsplnttion progrm within 15 min fter cesstion of blood flow. Humn cytochromes P-450 were purified 12-fold from liver microsomes by DEAE-cellulose nd hydroxylptite chromtogrphy to specific content of4.8 nmol/mg ofprotein (20). This preprtion is mixture of the mjor forms of humn cytochromes P-450 (13). NADPH-cytochrome P450 reductse ws purified from liver microsomes ofphenobrbitl-pretreted rbbits to n ctivity of 32 Jkmol/min per mg of protein (21). Cytochrome b5 ws purified from the sme microsomes s the reductse to purity of over 90% (22). Ole2-PtdCho ws obtined from Sigm, nd egg phosphtidylethnolmine (egg PtdEtn) ws prepred from fresh eggs under n rgon tmosphere by the method of Singleton (23). The lipids were repurified by HPLC on Lichrosorb Si-100 prior to use. 14CC14 with greter thn 99% purity ws purchsed from New Englnd Nucler. Reconstitution of the three purified proteins into phospholipid vesicles ws chieved by modifiction of the slow cholte dilysis method (12, 13). The mixture of humn cytochromes P-450 in 0.3 M potssium phosphte buffer, ph 7.5, contining 20% (vol/vol) glycerol ws monomerized by the ddition of 0.1% zwitterionic detergent 3-[(3-cholmidopropyl)dimethylmmonio]-l-propnesulfonte (CHAPS) (24) followed by stnding t 22 C for 2 hr. To solution of 2.4 mg of humn cytochrome P-450, solutions of0.4 mg of cytochrome b5 nd 0.8 mg ofcytochrome P-450 reductse were dded to yield molr rtio of 1:0.5:0.2. After ddition of 24 mg of Ole2-PtdCho nd 12 mg of egg PtdEtn dissolved in 4% sodium cholte to the solution of the proteins, the finl volume ws 14 ml nd the sodium cholte concentrtion ws 2%. An equilibrtion time of 16 hr t 4 C ws llowed for complete formtion of mixed micelles (12). Dithiothreitol ws present t 1 mm for further protection ginst peroxidtion. Dilysis of the mixture ws performed in dilysis bg of 0.5 cm dimeter suspended in 1-cm inside dimeter column with continuous flow of 1 liter per dy of N2- sturted 20 mm potssium phosphte buffer, ph 7.5, contining 20% glycerol nd 0.5 mm dithiothreitol for 3 dys followed by 1 dy of the sme buffer without dithiothreitol. Over 90% ofcytochrome P-450 ws recovered in the reconstituted vesicles nd less thn 6% cytochrome P-420 ws observed. Before incubtion with 14CC14, the vesicle suspension ws deoxygented by strem of N2 for 15 min followed by 30 min of gentle stirring t 30 C fter the ddition of 5 mm D-glucose 6-phosphte, glucose oxidse (ctivity 115,umol/min per mg) t 20,ug/ml, nd thymol-free ctlse (ctivity 125,umol/min per mg) t 10,ug/ml. Then n NADPH-generting system ws dded through septum to yield finl concentrtion of0.5 mm NADP, 5 mm D-glucose 6-phosphte, nd glucose-6-phosphte dehydrogense t 1 interntionl unit/ml. Finlly, '4CC14 [diluted with CC14 to specific ctivity of ACi/1l., 70.1,uCi/ mmol (1 Ci = 3.7 x 1010 becquerels)] ws dded to give 0.4 tl1/ Proc. Ntl. Acd. Sci. USA 79 (1982) 2679 ml, nd the mixture ws stirred for 1 hr t 30'C. Excess unrected 14CC14 ws removed by repeted extrctions with ethyl cette. The phospholipids were extrcted with 80 ml ofchc1j CH30H, 2:1 (vol/vol). The solvents were evported, nd the phospholipids were dissolved in. hexne/isopropyl lcohol/ wter, 6:8:1 (vol/vol), nd then pplied to 1 X 25 cm Lichrosorb Si-100 HPLC column. The PtdEtn frction ws eluted t flow rte of 2.5 ml/min with hexne/isopropyl lcohol/ wter, 6:8:1 (vol/vol); t 14 min the solvent system ws chnged to hexne/isopropyl lcohol/wter, 6:8:1.8 (vol/vol), to elute the Ole2-PtdCho frction. Elution of mteril from the column ws monitored by refrctive index nd bsorbnce t 208 nm. The phosphtidylcholine frction from the Lichrosorb Si-100 column ws vcuum dried nd then subjected to trnsesterifiction with 2 ml of BCl3 in methnol t 450C for 2 hr. The resulting methyl esters were extrcted into 5 ml of hexne tht ws bckwshed with 4 ml of wter. The hexne extrct ws subjected to HPLC on 1 x 25 cm Lichrosorb reverse-phse C-18 column eluted with methnol/wter, 96:4 (vol/vol), t flow rte of 2.5 ml/min. The mjor rdioctive frction, which eluted from this column t 41 min (Fig. 1), ws tken to dryness under rgon nd rechromtogrphed on 0.46 x 25 cm reversephse Lichrosorb C-8 HPLC column eluted with methnol/ wter, 90:10 (vol/vol). The mjor rdioctive frction tht eluted from the Lichrosorb C-8 HPLC column ws further purified on 0.46 x 25 cm reverse-phse Spherosorb octdecylsilic (ODS) (Altex, Berkeley, CA) column by elution with methnol/wter, 96:4 (vol/vol), t flow rte of 0.6 ml/min. The single rdioctive pek ws subjected to nlysis by mss spectrometry. Direct inlet electron impct mss spectr were mesured on Vrin CH-7 mss spectrometer t 20 ev. Desorption electron impct mss spectr were mesured on Ribermg R10-lOB by desorption of the smple from tungsten coil tht ws heted by incresing the current in the coil t 10 ma/sec from 50 ma to 500 ma (Fig. 2). The source temperture ws mintined t 130 C; the filment current ws 0.2 ma. The desorption chemicl ioniztion mss spectr were mesured on the Ribermg mss spectrometer, using 0.1 torr (13 P) of mmoni s the regent gs with the sme heting rte ofthe tungsten coil. RESULTS After nerobic incubtion of the reconstituted vesicles with 14CC14 nd NADPH the phospholipids were extrcted nd subjected to HPLC. As shown in Tble 1, rective metbolites de- Tble 1. Binding of 14C-lbeled metbolites to phospholipid frctions from reconstituted vesicles incubted with 14CC04 Totl 14Cmetbolite nmol bound per 14C-lbeled % of totl Phospholipid frction per nmol phospholipid frction dpm,g cyt. P-450 lbeled Egg PtdEtn 20, Ole2-PtdCho 19, (3.5) 0.7 (0.7) Reconstituted vesicles were incubted under rgon for 1 hr t 300C with NADPH nd 14CCl4. The phospholipids were extrcted nd pplied to Lichrosorb Si-100 HPLC column to seprte egg PtdEtn from Ole2-PtdCho. The quntity of metbolite-bound phospholipids, the nmol of 14C-contining metbolite formed per nmol of cytochrome P- 450, nd the percentge of metbolite-bound phospholipids in the vesicdes were clculted from the specific ctivity of The numbers in prentheses re results of repetition of the experiment with seprtely reconstituted vesicles mde from the sme stock of proteins nd phospholipids.

3 2680 Medicl Sciences: Trudell et l. F Co cq ) 0 o o.! ' Time, min 1 3c 0 x 2 $L_ FG. 1. HPLC profile of ftty cid methyl esters from 0le2-PtdCho frction. An liquot of the 0le2-PtdCho frction obtined by preprtive HPLC (Tble 1) ws subjected to trnsesterifiction. The resulting ftty cid methyl esters were seprted on 1 x 25 cm reverse-phse C-18 column with methnol/wter, 96:4 (vol/vol), t 2.5 ml/min. The rdioctivity in ech frction is shown; the bsorbnce ws monitored continuously t 208 nm. Only one mjor rdioctive pek is seen, t 41 min, well seprted from the unchnged methyl olete pek t 28 min. rived from the 14CC14 bound in similr quntities to both phospholipid frctions. A repetition ofthe experiment with seprtely reconstituted vesicles from the sme stock ofproteins nd phospholipids gve the nerly identicl vlues in prentheses. This suggests tht the rective intermedite from metbolism of 14CC14 rects to similr extent with the mono-unsturted cyl chins of Ole2-PtdCho s with the polyunsturted cyl chins of egg PtdEtn. From the specific ctivity of 14CC14, the totl quntity of bound rdioctive mteril is clculted to be 260 jig, which is equivlent to 7.2 nmol of metbolite bound per nmol of cytochrome P-450 in 60 min. This corresponds to lbeling of pproximtely 2% of the totl phospholipids present in the rection mixture. No binding of metbolites to the methyl olete frction ws observed when the incubtion ws crried out in the bsence of NADPH (18). The 0e2-PtdCho frction ws trnsesterified nd subjected to chromtogrphy on Lichrosorb C-18 reverse-phse column >-4 s 0-4 : Proc. Ntl. Acd. Sci. USA 79 (1982) Tble 2. Binding of "4C-lbeled metbolites of '4CC14 to methyl esters of ftty cids derived from Ole2-PtdCho Totl 14C metbolite bound Chromtogrphic per frction % of frction nmol Ag totl Originl Ole2-PtdCho Trnsesterified Ole2-PtdCho Lichrosorb C-18 pek Lichrosorb C-8 pek Spherosorb ODS pek Reconstituted vesicles were incubted with NADPH nd 14CCl4 under rgon t 300C for 1 hr. After extrction of phospholipids, the Ole2- PtdCho frction ws isolted by HPLC, trnsesterified with BC13/ MeOH, nd chromtogrphed on Lichrosorb C-18 column. The isolted rdioctive mteril ws rechromtogrphed on Lichrosorb C- 8 nd then Spherosorb ODS column. From the specific ctivity of '4CCl4 the binding of rdioctive metbolite to the seprted mteril could be quntitted, permitting estimtion of recovery t ech chromtogrphic step. Fig. 1 shows the elution of single pek of rdioctive mteril t 41 min distinct from methyl olete nd other UV-bsorbing components. This rdioctive pek ws rechromtogrphed, first on Lichrosorb C-8 column nd then on Spherosorb ODS reverse-phse column. The percentge recoveries ofrdioctive mteril t ech chromtogrphic step together with clculted quntities of bound metbolites re shown in Tble 2. The recovery of rdioctive mteril t ech chromtogrphic step ws pproximtely 70%. The rdioctive methyl ester frction purified by HPLC ws subjected to mss spectrometric nlyses by three techniques. Direct inlet electron-impct ioniztion on Vrin CH-7 mss spectrometer yielded spectrum with only low-mss frgment ions. Fig. 2 shows the desorption electron-impct mss spectrum on Ribermg R10-lOB mss spectrometer. This spectrum is lmost identicl to tht obtined on the direct probe nlysis on the Vrin CH-7. The lrge pek t m/e 297 corresponds to loss of trichloromethyl substituent, possibility nticipted from our studies on phospholipid binding of reductive metbolites ofnother hlocrbon, the inhltion nesthetic hlothne (2-bromo-2-chloro-1,1,1-trifluoroethne) (14, 18, 19). :11,,.qJ1~ir..qiqi1, 1l.11111l l.., ,...., g.[qlil x20 X mjkiit t! hilih. v m/e FG. 2. The Ole2-PtdCho frction isolted in Fig. 1 ws trnsesterified nd the resulting '4C-contining ftty cid methyl esters were purified on three successive reverse-phse HPLC columns. The single rdioctive frction obtined from the third HPLC column ws subjected to desorption electroir impct mss spectrometry on RibermgR10-lOB mss spectrometer. The mss spectrum is plotted s percent reltive bundnce of the pek t mss-to-chrge rtio m/e 74. The slnted lines indicte the positions t which 'the reltive bundnce ws multiplied by the indicted mount to mke the pek more esily seen. No moleculr ion pek t m/e 414 is observed.

4 Medicl Sciences: Trudell et L Proc. Ntl. Acd. Sci. USA 79 (1982) 2681 ru c)q l._ co FG. 3. The Olez-PtdCho frction isolted in Fig. 1 ws trnsesterified nd the resulting 14C-contining ftty cid methyl esters were purified on three successive reverse-phse HPLC columns. The single rdioctive frction obtined from the third HPLC col ; umn ws subjected to desorption chemicl ioniztion mss spectrometry using mmoni s regent gs in Ribermg R10-lOB mss spectrometer. The mss spectrum is plotted s percent reltive bundnce of the lrgest pek. A pseudomoleculr ion ws observed t m/e 432 (M + NH'j), which is the expected ddition product of molecule of moleculr weight 414 under conditions of chemicl ioniztion with mmoni s le _ouonw 1 _,. regent gs. The isotope -- pttern t m/e 432,434, nd 436 is con sistent with molecule contining m/e three chlorine toms.,./ t is often difficult to observe moleculr ion (M) in the electron-impct mss spectr of polychlorinted orgnic molecules (25). However, the use of desorption chemicl ioniztion mss spectrometry with mmoni s regent gs in the Ribermg R10-lOB gve pseudomoleculr ion pek t m/e 432 (M + NH+) (Fig. 3). This pseudomoleculr ion is expected for molecule of moleculr weight 414 under conditions ofchemicl ioniztion mss spectrometry with mmoni s regent gs. The rtios of reltive bundnce of ions t m/e 432, 434, nd 436 re those expected from molecule contining three chlorine toms s consequence of the nturl bundnce of 3C to 37C1 being 3:1 (26). The spectrum in Fig. 3 is consistent with mixture of 9- nd 10-(trichloromethyl)sterte methyl esters. This product mixture would be expected from ddition of trichloromethyl rdicl to either tom of the double bond of oleic cid followed by bstrction of hydrogen rdicl from neighboring molecule. Fig. 4 shows the intensities of the pseudomoleculr ion (M + NH+) t m/e 432 s well s the totl ion current s function Scn FG. 4. The highly purified "4C-metbolite bound to ftty cid ws pplied to tungsten desorption coil for mesurement of chemicl ioniztion mss spectrum s described for Fig. 3. As the heting current through the coil ws incresed from 50 to 500 ma t 10 ma/sec, series of 125 mss spectr were recorded. n the upper mss chromtogrm the bundnce of the pseudomoleculr ions t m/e 432 is displyed nd compred to the bundnce of the totl ion current in the lower trce. The close similrity of the bundnce of the pseudomoleculr ions to the totl ioncurrent suggests tht the smple ws reltively homogeneous. of time during heting of the desorption coil. The similrity between the profiles of the mss chromtogrm t m/e 432 nd the totl ion current suggests tht the smple is homogeneous. DSCUSSON The present identifiction of the moleculr structure of the dduct formed vi metbolite binding to the ftty cyl chin of 0le2-PtdCho confirms the ssignment of the rective intermedite s the trichloromethyl rdicl s proposed by mny investigtors (6, 7, 27, 28). A mechnism for production of the observed mixture of 9- nd 10-(trichloromethyl)sterte methyl esters is proposed in Fig. 5. Binding of crbon tetrchloride to cytochrome P-450 s substrte is followed by one-electron reduction to form the trichloromethyl rdicl (B). n the bsence of oxygen this rdicl leves the substrte binding site of the cytochrome nd diffuses into the phospholipid bilyer. f the rdicl bstrcts hydrogen rdicl from neighboring phospholipid, it will form chloroform (2) nd free rdicl on the phospholipid which my then be subject to peroxidtion or diene conjugtion rections. Alterntively, the -CC13 rdicl my dd to one crbon of double bond in ftty cyl chin to produce free rdicl on the djcent crbon tom (C). The resultnt ftty cyl free rdicl my undergo diene conjugtion if it is polyunsturted or lipoperoxidtion if it intercts with oxygen, or it my form sturted cyl chin by bstrction of hydrogen tom from neighboring molecule (28) to form substituted, sturted cyl chin (D). n our experiments, the 9- nd 10-(trichloromethyl)sterte methyl esters (E) were formed by trnsesterifiction of the substituted phospholipid (D) Ȧlthough this study demonstrtes tht the trichloromethyl free rdicl is mjor product of the reductive metbolism of crbon tetrchloride, it does not rule out formtion of cytochrome P-450-dichlorocrbene complex (5, 9) nd its subsequent dissocition to yield smll mounts of rective dichlorocrbene intermedites. From considertions of the rtes of crbon monoxide versus chloroform formtion during reductive metbolism of crbon tetrchloride (9), the quntities of ny dichlorocrbene formed nd possibly bound to phospholipids in our vesicle studies would be predicted to be less thn 5% of the bound free rdicls.

5 2682 Medicl Sciences: Trudell et l. Proc. Ntl. Acd. Sci. USA 79 (1982) CH2-CH2-N(CH3) Cl C cl A NADPH REDUCTASE CYT. b5 CYT. P-450 NDP+H C -c - c-c: Cl B Ole PtdCho ' HC J X RH 00 H2C - CH-0H2 0oJ Lo0 ) BC3 *X MeOH CH- 0 0< OCH3 h C FG. 5. Possible mechnism for formtion of 9- or 10-substituted trichloromethylsterte methyl esters from CC14 nd Ole2-PtdCho. Under nerobic conditions, in the presence of NADPH nd cytochrome P-450, CC14 is subject to one-electron reduction to the trichloromethyl rdicl (B). Addition of this rdicl~to one crbon of double bond in ftty cyl chin of the phospholipid cn produce free rdicl on the djcent crbon tom (C). Abstrction of hydrogen tom from neighboring molecule results in formtion of substituted sturted cyl chin (D). Trnsesterifiction produces mixture of 9- nd 10-(trichloromethyl)sterte methyl esters (E). D E Rection of trichloromethyl free rdicls with phospholipid molecules provides possible mechnism for the observed covlent binding of crbon tetrchloride metbolites to heptic microsoml lipids nd proteins (4, 9-11). Under the present experimentl conditions, pproximtely 2% of the totl phospholipids in the vesicles were lbeled fter incubtion with 4CC14. n the cse of microsomes, where the protein-to-phospholipid rtio is 10 times tht of the reconstituted vesicles, the sme rte of production nd. binding of -CC13 rdicls would result in correspondingly higher percentge of the phospholipids being ttcked. We thnk Ms. Annemrie Wegmnn nd Dr. Crl Djerssi for mking the Ribermg desorption chemicl ioniztion mss spectrometer vilble to us through Ntionl nstitutes of Helth Grnt GM to their mss spectrometry lbortory nd to Ms. Annemrie Wegmnn for mesuring the mss spectr. This study would hve been impossible with conventionl electron impct mss spectrometer. We lso thnk Ms. MrieBendix for expert technicl ssistnce, Ms. Audrey Stevens for editoril ssistnce, nd the Stnford Crdic Anesthesi nd Crdic Trnsplnttion tems for their help in obtining the humn liver specimens. This study ws supported by Grnt OH from the Ntionl nstitute of Occuptionl Sfety nd Helth nd stipend to B.B. from the Alexnder von Humbolt-Stiftung. 1. Cmeron, G. R. & Krunrtue, W. A. E. (1936)J. Pthot BcterioL 42, Butler, T. C. (1961)J. Phrmcol. Exp. Ther. 134, Reynolds, E. S. (1967)J. Phrncot Exp. Ther. 155, Uehleke, H., Hellmer, K. H. & Tbrelli, S. (1973) Xenobiotic 3, Wolf, C. R., Mnsuy, D., Nstinczyk, W., Deutschmnn, G. & Ullrich, V. (1977) MoL PhrmcoL 13, Li, E. K., McCy, P. B., Noguchi, T. & Fong, K. L. (1979) Biochem. PhrmcoL 28, Poyer, J. L., McCy, P. B., Li, E. K., Jnzen, E. G. & Dvis, E. R. (1980) Biochem. Biophys. Res. Commun. 94, Klynrmn, B., Mson, R. P., Perez-Reyes, E., Chignell, C. F., Wolf, G. R. & Philpot, R. M. (1979) Biochem. Biophys. Res. Commun. 89, Ahr, H. J., King, L. J., Nstinczyk, W. & Ullrich, V. (1930) Biochem. Phrmcol. 29, Sipes, G.., Krishn, G. & Gillette, J. R. (1977) Life Sci. 20, Benedetti, A., Csini, A. F., Ferrli, M. & Comporti, M. (1977) Chem. Biol nterct. 17, Bbsterling, B., Stier, A., Hildebrndt, A. G., Dwson, J. H. & Trudell, J. R. (1979) Mol Phrmcol 16, Bosterling, B. & Trudell, J. R. (1980) in Microsomes, Drug Oxidtions, nd Chemicl Crcinogenesis, eds. Coon, M. J., Conney, A. H., Estbrook, R. W., Gelboin, H. V., Gillette, J. R. & O'Brien, P. J. (Acdemic, New York), Vol. 1, pp Trudell, J. R., Bbsterling, B. & Trevor, A. J. (1981) Biochem. Biophys. Res. Commun. 102, Cohen, E. N., Trudell, J. R., Edmunds, H. N. & Wtson, E. (1975) Anesthesiology 43, Bbsterling, B., Trudell, J. R., Trevor, A. J. & Bendix, M. (1982) J. Biol Chem. 257, in press. 17. Bonfils, C., Blny, C. & Mruel, P. (1981) J. Biol Chem. 256, Trudell, J. R., Bosterling, B. & Trevor, A. J., Mol Phrmcol., in press. 19. BNsterling, B., Trevor, A. J. & Trudell, J. R. (1982) Anesthesiology 56, Erickson, S. K. & Bbsterling, B. (1981)J. Lipid Res. 22, Ysukochi, Y. & Msters, B. S. S. (1976) J. Biol. Chem. 251, Sptz, L. & Strittmtter, P. (1971) Proc. Ntl Acd. Sci. USA 68, Singleton, W. S., Gry, M. S., Brown, M. L. & White, J. L. (1965) J. Am. Oil Chem. Soc. 42, Hjelmelnd, L. M. (1980) Proc. Ntl. Acd. Sci. USA 77, Budzikiewicz, H., Djerssi, C. & Willims, D. H. (1967) Mss Spectrometry of Orgnic Compounds (Holden-Dy, Sn Frncisco), p McLfferty, F. W. (1973) nterprettions of Mss Spectr (Benjmin, Reding, MA), p Wolf, C. R., Hrrelson, W. G., Nstinczyk, W. M., Philpot, R. M., Klynrmn, B. & Mson, R. P. (1980) Mol Phrmcol 18, Slter, T. F. (1978) Biochemicl Mechnism of Tissue njury (Acdemic, London), pp