Detection of Phospholipid Peroxides in Biological Samples

8/6/2019 Detection of Phospholipid Peroxides in Biological Samples

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Free Radical IJ iology & Medic ine , Vo l. 3, pp. 349-354. 1987 0891-5849/87 $3 .00+ 0OPrinted n the USA.All rights eserved. 1987Pergamon ournalsLtd.

D E T E C T I O N O F P H O S P H O L I P I D P E R O X I D E S I N B I O L O G I C A L S A M P L E S

FREDERIK J . G. M. VAN KUlJK and EDWARD A. DRATZ*Department of Chem istry, Montana State Un iversity, Bor.emun,MT 59717, USA

Abstract--Peroxidation of membrane l ipids has been hypothesized to play a key role in various types of t issuedegeneration and p athology. L ipid peroxides are formed when oxy gen reacts w ith an unsaturated fatty acid chain.Virtually all of the unsaturated fatty acids in biological systems are bound by ester linkages in phospholipids ortr iglycerides. P hospholipid and tr iglyceride peroxides are p r imary products o f l ipid pcroxidation and have rarelybeen m easured. M ost of the comm only use d methods for detection of l ip id peroxidation are ba sed on detectionof m alondialdehyde or other chem ical species that are derived f rom oxidized fatty acids. Th is review prese;~ts anoverview of recently developed m ethod s aimed at identifying and m easuring oxidized phospho lipids and tr iglyc-er ides which are direct evidence of the o ccurrence of l ipid peroxidation in viva.K ey w ord s--L ipid peroxidation, Lipid autoxidation, Phospholipid peroxides, Calorimetr ic assay, Enzymatic assay,Ultraviolet absorption spectra , Hig h performance l iquid chromatography, Gas chrom atography-mass spectrometry

INTRODUCTIONF o r m a t i o n o f o x y g e n r a d i c a l s a n d l i p i d p e r o x i d a t i o nh a v e b e e n s u g g e s t e d a s im p o r t a n t m e c h a n i s m s c a u s i n gc e ll d a m a g e i n m a n y t y p e s o f t i s s u e d e g e n e r a t i o n , i n -c lud ing he a r t d i s e a se , c a nc e r , a nd a g ing . ~ ' 2 The l i p idp e r o x i d a ti o n p r o c e s s i n v o l v e s t h e o x i d a t i o n o f p o l y u n -sa tu r a t e d f a t t y a c id s w h ic h a r e l a r ge ly e s t e r i f i e d i nm e m b r a n e p h o s p h o l i p i d s . I t h a s b e e n h y p o t h e s i z e d th a tt h e o x i d a t i o n o f m e m b r a n e l i p i d s c a u s e s a n i n c r e a s e dp e r m e a b i l i t y o f c e ll m e m b r a n e s . T h i s i s t h o u g h t t o l e adt o e d e m a , d i s t u r b a n c e i n e l e c t r o l y t e b a l a n c e s , a n d e l -e v a t i o n o f i n t r a c e l l u l a r c a l c i u m w h i c h c o n t r i b u t e t om a l f u n c t i o n o f t h e c e l l .

M a l o n d i a l d e h y d e d e t e c t i o n b y r e a c t i o n w i t h t h i o -b a r b i t u r i c a c i d ( T B A ) h a s b e e n t h e m o s t f r e q u e n t l yu s e d m e t h o d t o m e a s u r e l i p i d p e r o x i d a t io n i n v i v a f o rm a n y y e a r s . T h i s t e s t i s c o m l i c a t e d b y t h e b a c k g r o u n di n t e r fe r e n c e o f s e v e r a l o t h e r c o m m o n b i o l o g i c a l m o l e -c u l e s ( w h i c h r e a c t w i t h T B A ) . x 'e M e t h o d s s u c h a s i o-dom e t r i c t i t r a t i ons , 5.~ e nz ym e a s sa ys , 7 a nd u l t r a v io l e ta b s o r p t io n s p e c t r a s h a v e b e e n p r i m a r i l y a p p l i e d t oo x i d i z e d m o d e l f a t t y a c i d s o r t h e i r m e t h y l e s t e r d e -r i v a t i v e s . H o w e v e r , m e m b r a n e p h o s p h o l i p i d s a n d t r i -g l y c e r i d e s a r e t h o u g h t t o b e t h e p r i m a r y s i t e s o f t h el i p i d p e r o x i d a t i o n p r o c e s s s h i c e t h e y a r e t h e p r in c i p a ld e p o s i t s o f u n s a t u r a t e d f a t ty a c i d s .

*T o who m rep rint requests and correspodence should be addressed.

T h i s r e v i e w w i l l s u m m a r i z e m e t h o d s a i m e d a t d e -t e c t i o n o f p h o s p h o l i p i d a n d t r i g l y c e r i d e p e r o x i d e s .M e t h o d s w h i c h h a v e p r e v i o u s l y b e e n u s e d t o s t u d yo x i d a t i o n o f f r e e f a tt y a c i d s w e r e m o d i f ie d i n o r d e r t oa p p l y t h e m t o m e a s u r e m e n t s o f p h o s p h o l i p i d h y d r o -p e r o x i d e s . T h e n e w m e t h o d s w e r e d e v e l o p e d w i t h p u r -i f ie d pho spho l ip id subs t r a t e s t ha t w e r e p r e pa r e d byp h o t o o x i d a t i o n o f f at t y a c i d s c o v a l e n t l y b o u n d t o p h o s -p h a t i d y i c h o l i n e ) S y n t h e s is o f p h o s p h o l ip i d h y d r o p e r -o x i d e s t a n d a r d s w i l l b e d e s c r i b e d , f o l l o w e d b y o t h e ru s e f u l m e t h o d s s u c h a s c a l o r i m e t r i c re a c t i o n s , e n z y m ea s s a y s , u l l r av i o l e t s p e c t r a , a n d h i g h p e r f o r m a n c e l i q -u i d c h r o m a t o g r a p h y ( H P L C ) . F i n a l ly , p o w e r f u l n e wg a s c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y ( G C - M S )m e t h o d s d e v e l o p e d t o d e t e r m i n e l i p i d p e r o x i d a ti o n a r ed e s c r i b e d .

II . SYNT HESIS OF PHOSPHO LIPIDPER OX ID E STA N D A R D S

C o m m e r c i a l l y a v a i l a b l e l i p o x y g e n a s e c a n n o t b eu s e d f o r s y n t h e s i s o f p h o s p h o l i p i d h y d r o p e r o x i d e s ,s i n c e t h i s e n z y m e i s n o t a b l e t o u s e p h o s p h o l i p i d s a sa subs t r : i t e . ~ i n som e m ode l sy s t e m s , ox id i z e d phos -p h o l i p i d s w e r e o b t a i n e d b y a u t o o x i d a t i o n r e a c t i o n . Z tS u c h r e a c t io n s a r e s l o w , a n d a c o n s i d e r a b l e a m o u n t o ft h e d e s i r e d p r o d u c t s m a y d e c o m p o s e d u r i n g t h e i n -c u b a t i o n t i m e . T h e u n a v a i l a b i l i t y o f w e l l - c h a r a c t e r i z e dp h o s p h o l i p i d p e r o x i d e s t a n d a rd s h a s b e e n a n o b s t a c l et o t h e d e v e lo p m e n t o f i m p r o v e d d e f e ct i o n m e t h o d s .

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A procedure for synthes iz ing phosphol ipid perox-ides was develo ped in our labora tory, based on thephotosen si t ized product ion of s ingle t oxy gen. 9 Phos-phol ipid solut ions in methanol were i l luminated for 12h w ith rose bengal ph otosensi t izer and a ir us ing thebeam from the 150 Watt xenon arc in the sample com-partment of a spect1:ofluorometer.9 After i l lum inat ion,the phospho l ipid hydroperoxides were extrac ted intodichlorom ethane and the rose bengal par ti tioned intothe methanol ic phase. The p hosph ol ipid peroxides tandards were charac ter ized in a num ber of ways tobe desc ribed. Thc~ standards can be stored for severalmonths in d ich lorome thane a t -20 C . 9

The pho tooxidat ion products of a s imple phosph o-lipid, ( 16 :0 ) ( I 8: 2) p hosph at idylchol ine were s tudiedin some deta i l . Th e extent of ox idat ion was l imited bythe amoun t o f oxy gen available. 9 Apalysis o f the hy -droperoxide groups was carr ied out with a colo r im etr icassay (descr ibed be!ow) which ind ica ted that 2 0-2 5%of the double bonds formed hydrope roxides ind i f fe r -ent preparations. It is kno wn that sing let oxy gen re-ac ts w ith l inolea te to y ie ld a mixture of co njugatedand nonco njugated der iva t ives , whereas autooxidat ionyie lds exclus ively conjug ated der iva t ives .9 The char-ac ter iza t ion of the dif ferent isomers f rom oxidizedphospha t idylchol ine was on ly poss ib le wi th a G C-M Smethod tha t a l lowed ident i f ica t ion of the individualproducts by th eir specific frag men tation patterns. 9

I I I . COLO RIMET RIC DETERMINATIONSThe amount of hydroperoxide in a sample can be

quanti ta ted by co lor imetr ic de terminat ion of peroxidefunctional gro up s. 5.* Asakawa and M atsushita 6 re-por ted a techniqu e which we m odif ied as descr ibed indetail elsew her e. 12 Briefly, the pho spho lipid hy dro-peroxides were reduced with potass ium iodide in thepresence of an anhydrou s a luminum ch lor ide ca ta lys t ,hydrochlor ic ac id was added, and a s tarch solut ionyie lded a blue com plex with the iodine produced. Ab-sorbance was me asure d at 570 nm , and calibration waspe r formed wi th cumene hydrope roxide so lu t ions ofknown concent rat ions .

In order to apply this method to phosphol ipid hy-droperoxides , severa l modif ica t ions of the methodfrom Asakaw a and Matsushi ta were necessary. In the irmethod hexane was added a t the ini t ia l peroxide re-duct ion s tep to remo ve the hydroxy der iva t ives . Whenphosphol ip id pe roxides a re ana lyzed , the hexane w asom it ted s ince i t causes , turbidi ty tha t d ecreases there l iabi li ty of the absorbance measurem ents . Fur ther-more , a l l vo lumes w ere reduced tenfo ld to ga in a cor -responding increase in sensi t ivi ty. Addit ional minormodifications are de scrib ed elsewhere.12

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A standard curve obtained with stock solutions ofcum ene hydropero xide show ed tha t the absorbance in-creased no nl inear ly above 20 nm ol peroxid e/samp le , z~I t was a lso found tha t the degree of nonl inear i ty in-creased with larger total amounts of lipid present. Weproposed tha t some of the iodine produced in thereact ion with the peroxides par t{t ions with the dou-b le bonds of the phospho l ip ids . This depen dence onl ipid concentra t ion is a l imita t ion of the coior im etr icmeth od, w hich is most accura te a t low l ipid levels . Theenzyme assay descr ibed below does not appear to beinfluen ced by lipid leve ls and is therefore ihore reliableunde r circumstances where high am ounts of l ipids arepresent .

IV. ENZY ME ASSAYSThe ac t ivi ty of gluta thione peroxidase (GSH-Px)

was measured under conditions whei 'e the rate wasl imited by the amount of peroxide substra te presentaccording to the m ethod Heath and ' rappel 7 mod if iedto use pH 7.4, 0.75 mM glutathione, and ! . '-aMEDTA.~S This enzy me conv er ts hydrop eroxides to hy-droxy der iva t ives , producing s toichiometr ic amountsof oxidized gluta thione . The oxidized gluta thione isconv er ted back to the reduced form by gluta thionereductase with the s toichiometr ic consum ption ofNADPH . Free fa tty ac id hydroperox ides or cumen ehydroperoxides could be de termined direc t ly by thismetho d. How ever , i t was found tha t phosp hol ipid hy-droperoxides are extremely poor substra tes for GSH-Px. T herefore , an extra incubat ion s tep with phospho -lipase A2 was ad ded , in orde r to release the fatty acidhydrop eroxides which are ex cel lent substra tes forGSH-Px. js The essen t ia l role o f pho sphol ipase As inreduc tion o f pho sphol ip id hydrope roxides by GSH-Pxwas recent ly re view ed. ~3

The tota l ch ange i.n opt ica l densi ty a t 340 nm, causedby the consumption of NADPH in the GSH-Px per-oxide reduc t ion , a l lows de te rmina t ion of the n umberof nm oles of l ip id p~ oxid es . The to ta l amoun t of phos -phol ipid hydro peroxid es de tec ted nzym atica lly agreedwith the va lues fo und by color imetr ic de terminat ion towithin 1% under low l ipid levels condi t ions where thecolor imetr ic assay gives accura te resul ts . The en zym eassay can be used eff ic ient ly to de term ine peroxideva lues of phosphol ip id pe roxides in memb ranes a fte rphosphol ipase As incubat ion with a sens i t ivi ty of !nm ole , wh ereas the coior imetr ic assay is applicable formeasurement of pe roxide va lues of p hosphol ip id hy-dropero xides dissolved in organic solvents with aboutthe same sensi t ivi ty. A disadvantage of the enzym aticmethod is that the incfibation required for A2 actioncould poss ibly ch ange the peroxide value und er somecondit ions .


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Forum 351V, ULTRAVIOLETABSORPTION SPECTRA

Spectrophotometry has of ten been used for de ter-minat ion of conjugated diene products of l ipid per-oxidat ion a t 234 nm . 3.s These measu rements arecom plica ted by the high m olar absorption of m any or-ganic solv ents in the ultraviolet range. Measurementswe re carried out in spectral grade dich loro me than e incuvet tes with a pa th length of I ram. Spectra of oxi-dized ph ospho l ipids are very s imilar before and af terreduc tion of the hydrope roxide group. 9 This impl iesthat optical density at 234 nm measures total conju-gated dienes withou t dis t inguishing between hydroper-oxides , hyd roxy der iva t ives , and other decom posi t ionproducts conta ining conjugated dienes . In addi t ion, i tshould be noted that ultraviolet spectra are not suffi-c ient for quant i ta t ion of the oxidat ion products ob-ta ined f rom react ions with s ingle t oxy gen because thenonconjugated products produced by s ingle t oxygenhave essent ia l ly no absorbance a t 234 nm .

This direc t spectrophotom etr ic method has been ex-plored to m easure the conjugated diene content in t is -sues . However , ant ioxidants are required to avoidaddi t ional oxidat ion dur ing extrac t ion of the l ipids andmanipu la t ion of the samples , jz EDTA o r d esfera l a reusual ly used to inhibi t i ron promoted decomposi t ionof l ipid peroxides , and butyla ted hydroxy toluene(BHT) is used to inhibi t formation o f l ipid peroxidesdur ing such procedures . BHT introduces a problembecause the aromatic structure in this com po und ab-sorbs s t rongly in the ul t raviole t range. The B HT ab-sorpt ion interferes with the m easurement of dienes ,which l imits the accuracy of this method. So far wehave used th i s me thod only to show the pre sence ofdienes in phsopholipids tha t were photooxidized inmethano l . In such a m odel system we did no t emplo yant ioxidants and the s ingle t oxygen reac t ions and sam-ple preparations we re carried o ut in pure spectral gradesolvents .9

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VI. HIG H PERFORMANCE LIQUIDCHROMATOGRAPHY

Prev ious ly, Crawford et aI.14 reported a meth od forsepa ra t ion of ox id ized d i l ino leoyl phospha t idylchol inefrom the non oxidized precursor phosph ol ipid. TheHPLC sys tem employed a uBond apack C~s co lu m nwith aqueou s methanGl as a solvent . O ur meth od wasmodi f ied to use a CIS-J0 # reco lum n wi th 16% load-ing , and 100% methanol conta in ing O . 1% ammo niumaceta te as a mo bile phase. 9 I f amm onium aceta te isused, the hyd roperoxid es and hydroxy der iva t ives givemu ch sh arper peaks than p reviously repor ted. ~.~s How -ever , i t was not poss ible to separa te the hydrop eroxidesfrom the hyd roxy der iva t ives in this HPLC sy stem, asalso d escrib ed by Ursini et al. ~6 for a linear solv ent

gradient system. The use of an appropriate isocraticHPLC system, presented in more deta i l e lsewhere ,a l lows up to s ix analyses per hour . 9

W hen mixtures of oxid ized phosph ol ipids f rom t is-sues are an alyzed, the d if ferent oxidized products runas a part ia lly resolved group o f peaks on HPLC jus tbefore and af ter the photoo xidized ( I 6:0 )(18 : 2) phos-phat idylchol ine . Th erefore , H PLC can be used as anefficient, low resolution clean up step that do es not dis-c r imina te be tween hydrope roxides and hydroxy de r iv-a t ives . Since the sen si t ivi ty of the de tec t ion is l imitedto about 10 nm ole of oxidized l ipids , we do not usethis method for detection of small samples of tissue.Instead, we measure lipid peroxidation by identifica-t ion of the products with a m ass spectrometer , and thedifferen t ox idize d species are separated by gaschroma tography.

Vll. GAS CHROMATOGRAPHY-MASSSPECTROMETRY

A. New techniquesThe f ina l par t of this review deals with the m ethods

for direc t ident i f ica tion and measu remen t of l ipid per-oxides by GC -M S. Vir tua lly a l l of the GC -M S anal-ys is that hav e been carr ied out on oxid ized l ipids areonly appropriate for free fatty acids. O nly Hu ghes eta l. ~7 appl ied HPLC and G C-M S method s to measure-ment of mem brane phosphol ip id oxida t ion produc ts.However , the ir method was based on an enzymaticliberation of the oxidized fatty acids from the phos-pholipids, which required an additional purificationstep pr ior to methyla t ion with diazomethane . We re-cent ly presented novel GC -M S metho ds that a re basedon a on e-step transesterification in organic solven ts atroom temperautre , which l ibera te the oxidized fa t tyacids and s im ultaneously conver ts them to fa t ty ac idmethyl es ters9 or fatty acid pentafluorobenzyl (PFB)esters ~s for g as chro ma togra ph ic separation. Th e in-cubations in organic solvents prov ide high product re-covery as shown by s tudies with standard p hospho l ipidperoxides . Fur ther der iva t iza t ion was s imilar to tha trepor ted for model s tudies of fa t ty ac id methyl es teroxidat ion.

B. Transesterification to fo rm m ethyl estersTissue samples or phosphol ip id hydrope roxideswere ex t racted by the m e thod of B l igh and D yer 9 mod -

if ied to use dichioromethane ins tead of chloroform.The hydrope roxides were reduced wi th sodium boro-hydr ide in me thanol , and reext rac ted in to d ich ioro-ine thane . The extrac t was dr ied over sodium sulfa te ,evapora ted to a small volum e (ca. 50 ~ul) , 2 0 / t l oftransesterification reagent, 0.2 M m-(trifluoromethyl

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352 Forumphenyl ) t r ime thyl ammonium hydroxide in me thanolwas add ed, and the samples w ere incubated for 30 ra ina t room tempera ture . This procedure provides quan-t i ta t ive co nve rs ion of ox id ized and nonox id ized phos -

phol ipids or t r iglycer ides to fa t ty ac id meth yl es t ers ) 2Dry pyr idine and BSTFA were added to form TMSderivat ives of the a lcohol funct ions .

The techniq ue descr ibed above w as f i rs t appliedto photoox id ized (16:0 ) (18:2 ) phospha t idylcholine .9Equal am ounts of the conjuga ted and no nconjuga tedl inolea te peroxides were found af ter reac t ion of thephospholipid with s ingle t oxygen. This ra t io of con-jugated and nonco njugated products is in agreementwith Thomas and Pryor2~ and Terao and M atsushita. 22in contrast, Frank el et al. 23 and Cle me nts et al. 24 hav ereported tha t 66% conjugated and 33% nonco njugatedproducts w hen l inolea te is photoo xidized. The poss iblecauses o f this disagreement were discussed previously,and add itional experi.ments are required to fu lly resolvethis poin t. 9

The G C-M S me thod desc r ibed above uses e lec t ronioniza t ion and has a sens i t ivi ty of about 10 ng of ox-idat ion products on the colum n, 9 We have shown tha tthis sensitiv ity is sufficient to detec t and study th e lipidperoxide con tent of adipose t r iglycer ides in vi tamin Eand selen ium deficien t rats. 2~ Larg e amo unts o f ox i-dat ion.produ cts were found in the adipose of ant ioxi-dant def ic ient animais l whereas negl igible amountswere found in the supplem ented animals i t was a lsoshown tha t only conjugated l inolea te peroxides werepresent , which suggests tha t autooxidat ive processeshave taken place in these animals . In addi t ion, prac-t ica lly n o oxid ized olea te was found, e ven thougholea te was the predominant fa t ty ac id present . Thispotent ia l ly important f inding has been discussed insome deta i l e lsewh ere . ~

I t should be emphas ized tha t the GC -M S me thodas descr ibed is semiquant i ta t ive . For quant i ta t ion, is -otopica l ly labeled compo unds can be used for internalstandards. For example, oxygen-18 substitution at thefa t ty ac id carboxyl oxygen was used by Str i fe andMu rphy 26 and Leis et al.2~ for qu antitativ e "analysis ofl ipoxygenase metabol i tes of a rachidonic ac id. Boey-roans et al. 2s used octad euterated arachid onic a cid,and we invest iga ted the G C-M S fragmen ta t ion patternsof photooxid ized v inyl pe rdeutera ted docosahc~aenoicacid. 9

C. Transesterification to formpentafluorobenzyl esters.,

Since m ore sensi t ivi ty was des ired to analy ze traceamou nts of l ipid peroxidat ion products in small sam-ples , a m ore sensi t ive der iva t iza t ion approach was

sought . Str i fe and M urphy found tha t reac tion of f reefa tty ac ids with pentaf iuorobenzy lbromide prod ucedpentaf luorobenzyl es ters which provide an enhancedsens i tiv i ty when GC -M S i s emp loyed wi th nega t iveion che m ical ioniza tion (NICI). 26,29This derivatizationprocedure w ould require an addi t ional chemical or en-zym atic step in our assay to liberate the fatty acid estersfrom phospholipids . Therefore a one-s tep procedurewas deve loped which conve r t s phosphol ip ids d i rec tlyinto fatty acid PFB esters.

Prepara tion of pen taf luorobenzy les ter samples forGC -M S i s s imi la r to the m e thod desc r ibed for me thylesters. Tran sesterification is instead carried out in di-ch lorome thane wi th 20% ( v /v ) pentaf luorobenzyi a lco-hol and i % ( w / v ) sodium p entoxide in a tota l volum eo f about I00 ill. ~s Sam ples are incu bated for 30 m inat room temperature. It is useful to incubate at 60Cwhen t r iglycer ides are to be t ranses ter i f ied for analy-s is . We have shown tha t under these c ircumstances ,a comple te convers ion of phosphol ipids or t r iglyc-erides to fatty acid PFB esters occurred. This reactionhas broad utili ty and is also applicable for formationof other esters, such as benzyl esters that are usefulderiv atives for ana lysis o f fatty acid s by H PLC. 3

PFB es ters are much more s table der iva t ives forGC -M S analys is than methyl es ters. W hen analyzedas methy l es ters , there was a severe loss of oxidat ionproducts f rom 20 :4 and 22 :6 on the GC column asevidenced by lower r ecove r ie s on longe r co lumns .Sample los s d id not occur on longe r co lumns whenPFB es ter der iva t ives were formed of these com-pounds . The grea ter stabil ity of the PFB es ters of phos-phol ipid oxidat ion products a l lowed a more sensi t ivedetec t ion and NICI provided an addi t ional advantagefac tor of 20 -1 00 with PFB es ters , re la t ive to the mostopt imu m El condit ions .ZS A com binat ion of these fac-tors improves the ov era l l sens i t ivi ty about a thousand-fold to 10 pg o f oxidat ion product for the PFB es terson the gas ch romatog raph column.~S

D. Detection of aldehydes aspentafluorobenzyl oximesA rapid der iva t iza t ion techniqu e was em ploy ed for

analys is of a ldehydic products of l ipid peroxidat ion,which in t roduces the PFB molecule in to these com-pounds for high sensi t ivi ty analys is .3! 4-Hydroxy-alken als hav e bee n rep orted b y Esterbauer ~2 to b e verybiologiC:a l ly ac t ive molecules . 4-Hy droxyalken alswere conve r ted to PFB oxime de r iva t ives and y ie ldedessent ia l ly the sam e sensi t ivi ty as the PFB es ters w hende tec ted by G C-M S unde r NICI condi t ions . Furthe r-mo re , i t w as poss ible to syn thes ize a suitable internals tandard for 4-hydrox ynon enal tha t carr ies two deu -


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t e r ium labe l s . R esu l t s on the quan t i t a t ive ana ly s i s o f4-hydroxyalkenals in var ious t i ssues wi l l be publ ishedsepa ra te ly .

V i i i . . C O N C L U D I N G F ,E M A R K SThe GC -MS assays p r esen ted p rov ide power fu l a l -

t e rna t ives to c l a ss i ca l me thods fo r measu remen t o fl ip id peroxidat ion in v ivo . Th e new m ethods descr ibedare not on ly more specif ic , bu t a lso g ive informat ionon the type of oxidat ion react ions that have taken p lace .A m a jo r th ru s t o f the me thodo logy i s based on de tec t ionofpho : ;phol ip id peroxide s , w hich are formed f i r st whenrad ica l s and oxygen a t t ack membrane phospho l ip id s .Al t e rna t ive ly , t he me thodo logy can be u sed to measu reperoxid ized t r ig lycer ides that tend to bui ld up in ad i-pose s tores . 2s The se approach es are com plem entedwi th a GC -MS method to de te rmine ma jo r a ldehyd icsecondary oxidat ion products that i s descr ibed br ief ly .C om bined m easu remen t o f phospho l ip id pe rox ides and4 - h y d r o x y a l k e n a l s b y G C - M S m a y p r o v i d e i n f o r m a -t ion about turnover of ox id ized phosphol ip ids in v ivo .The h igh sens i t iv i ty o f GC -M S techn iques appear s top rov ide rou te s to s tudy phys io log ica l l eve l s o f l i p idpe rox ida t ion p roduc t s in sma l l b io log ica l samp les .A c k n o w l e d g e m e n t s - - T h i s work was supported by grants from TheNetherlands Organizat ion f or the Advancement of Pure Research(ZWO ) ( to FJGMvK ) and the National Inst i tutes of Health ( to EAD).

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editor. Methods in enz ym olog y, Vol. 1 05. Orlando, FL: Aca-demic Press ; 1984 : 299 -305 .5. Pryor, W. A.; Cast le , L . Chemical m ethods for the detect ionof l ipid hydroperoxides. In: Packer. L . A., edi tor . Methods inenzymology, Vol. 10 5. Orlando, FL: Academic Press; 1984:2 9 3 - 2 9 9 .6. Asa kaw a, T.; Matsushita , S. A colorimetr ic m ierodeterminationof peroxide values utilizing alum inum ch loride as the catalyst.I . ip ids l& 965-967 ; 1980 .7. H eath, R. L .; Tappe l , A. L. A new sensi t ive assay for themeasurement of hydroperoxides. Anal. Biochem. 76: 184-191 ;1976.8. Shenstone, E S. U ltraviolet and visible spectroscopy of l ipids.

In: Johnson, A. R.; Davenport , J , A., edi tors . Biochemistry andmethodology of l ipids New Y ork: W iley lnterscience; 1971:2 1 9 - 2 3 0 .9. van Kuijk, E J . G. M .; Tho ma s, D. W.; Stephens, R. J . ; Dratz ,E . A . G as chrom atograph y-ma ss spec tromet ry method fo r de -termination of phospholipid peroxides. I . Transester if icat ion toform methyl esters . J . F ree Bad . B io l . Med . I : 215-2 25 ; 1985 .I0 . G ib ian , M. J . ; Ga law ay , R . A . Chemica l a spec t s o f l ipoxygen-ase react ions, in: van Tamelen E. E., edi tor . Biorganic chem.istry, Vol. ! . New York: Academic Press; 1977: 117-136.

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hour, K .; N ooi, J . R, Part ic ipation of s i trglet oxygen in pho-tosens it ized ox idat ion o f 1 ,4 -d ieR dic sys tems andphotooxidation of soybean oi l. d . Am. Oi l Chem. Soc. 5 0 : 3 2 5 -330; 1976.25. van Kuijk, E J . G. M.; Thom as, D. W.; Stephens. R. J . ; Dratz .E. A. Lipid peroxidation products in subcutaneous adiposeassociated with vi tamin E deficiency m easured by gas chroma-tography-mass spec t romet ry , in : Sevan ian , A . , ed i to r . Bio-logical e f fects o f l ipid peroxidation. In press; i 987.26. Str ife , R. J . ; Murphy, R. C. Stable isotope labeled 5-1ipoxy-genase m etabolites o f arachidonic acid: Analysis by negativeion chemical mass spectrometry. Prostaglandin Leukotrienesa n d M e d . 13: I -8 ; 1984 .27 . Le t s , H . J . ; Mal le, E . ; Moser , R . ; N im f , J . ; Kos tne r, G . M . ;Esterbauer , H.; G leisbach , 14. Preparat ion of s~O-labeled s tand-ards of hydroxy-eicosatetraenoic ,~cids and thromboxanes forquan ti ta t ive measurement b y gas ch rom atograp hy-m ass spec -t romet ry . Biomed. M ass Spectrom. 13: 483 -48 8 ; 1986 .28 . Bocymans , J. M.; Brash, A. R .; Oates, J. A .; Hubb ard, W. C.Preparat ion o f monohydroxy-eicosatetranoie acids. Anal . B io -chem. 104: 259-2 67 ; 1980 .29. Str ife , R. J . ; Murphy, R. C. Preparat ion of pental luorobenzylesters of arachidonic acid l ipoxy gena se metaboli tes. Analysis


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Detection of Phospholipid Peroxides in Biological Samples