Am J Clin Nutr 1991 Di Mascio 194S 200S

8/13/2019 Am J Clin Nutr 1991 Di Mascio 194S 200S

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1 9 4 S Am J C /in N u ir 199 1 :53 :19 4S-200S . P rin ted in U S A . 199 1 A m erican Soc ie ty fo r C lin ica l N u tr ition

A n tio x id a n t d e fe n s e s y s te m s : th e ro le o f c a ro te n o id sto c op h ero ls a n d th io ls 3P ao lo D i M a sc io M ich a e l E M u rph y a n d H e lm u t S ie s

A B S T R A C T R eac tive oxyg en spec ies occur in tissues an dcan dam age D N A , pro te ins , ca rbohy dra tes, and lip id s. T hesepo ten tia lly de le te riou s reac tio ns a re con tro lled by a sy stem ofen zym atic and n onenzym atic an tio x idan ts w hich e lim ina tep roox idan ts and scav enge free rad ica ls. T he ab ility o f the lip id -so lub le caro ten o ids to quench s ing le t m olecu lar oxygen m ayex p la in som e an tican cer p rop er tie s o fthe caro teno ids , indepen-den t o f the ir p rov itam in A ac tiv ity . T ocop hero ls a re the m os tab undan t and effic ien t scav en gers o f hydro peroxy l rad ica ls inb io lo g ica l m em b ranes. W ate r-so lub le an tiox idan ts inc lude as-co rba te and ce llu lar th io ls. G lu tath ion e is an im portan t su bstra tefo r enzym atic an tiox idan t func tions and is capab le o f non en -zy m atic rad ica l scaveng ing . T h io ls assoc ia ted w ith m em branepro te ins m ay a lso be im portan t to the an tiox id an t sys tem s. In -te rac tions b etw een the th io ls , tocophero ls , and o th er co m poun dsen hance the effec tiveness o f ce llu la r an tiox idan t defense .Am JC lin N u ir 1991 :53 :1945-2005 .K E Y W O R D S A ntiox id an ts , ca ro ten o ids, to co phe ro ls ,th io ls, sing le t m o lecu la r oxyg en , oxy gen rad ica ls, scav engers,p ro te in su lihy dry ls , lip id perox ida tion

O v erv iew of o x ida tive stre ssA erob ic m etabo lism en ta ils the genera tion of ox ygen spec ies

capab le o f dam aging D N A , pro te ins , ca rboh ydra tes, an d lip ids.T hese spec ies inc lude the superox id e an ion rad ical, h ydrog enperox ide , the h ydrox y l rad ica l, and s ing le t m o lecu la r o xygen .In the pa tte rn ofan tiox id an t defense , som e b io log ica l co m poun dslike caro teno ids , v itam ins E and C , and th io ls p lay a pro m in en tro le . O x ida tiv e d am ag e in f lic ted b y reac tiv e oxygen spec ies isa lso refe rred to as ox ida tive stress, and re flec ts a sh ift in theproox id an t-an tio x idan t b alance in favo r o f th e fo rm er (1 ). D i-verse b io log ica l p rocesses such as in flam m atio n , ca rc inog enesis,ag ing , rad ia tio n dam age , and pho tob io lo g ical e ffec ts app ear toin vo lve reactive oxyg en spec ies. T h is fie ld o f resea rch has pro -v ided im pac t in recen t years in a n um ber of fie lds su ch as b io -chem ica l ph arm aco log y , tox ico log y , rad ia tion b iochem istry , asw ell as pa th ophy sio logy and m edic in e .

O ne-e lec tron red uc tio n of o xygen has been w ide ly stud iedsince early th is cen tu ry . M ichae lis (2 ) p o in ted ou t th a t one-e lec -tron steps of o xygen reduc tion , lead ing to in te rm ed ia te fo rm ationof free rad ica ls, m ay be of g enera l im portance in b io log icalchem istry . T h ese rad ical fo rm s of oxyg en are 02 and O an dthe ir p ro tona ted fo rm s , H O and H O . T he perhyd roxy l rad ica lH O 2 and the hy drox y l rad ica l H O w ere recog n ized by H aber

and W eiss (3 ) in the ir s tudy on th e iron salt-ca talyzed d ecom -position of H 2O 2 as be ing im p ortan t in chem ica l, pho to ch em ica l,and e lec trochem ical p ro cesses . B io log ica l resea rch on thesu perox ide an ion rad ica l O received an excep tiona l s tim ulusa fter th e d iscovery of su perox ide d ism utase by M cC ord an dFridov ich (4 ).

O ther reac tiv e sp ec ies o f ox ygen are of a nonrad ica l na tu re .H yd rogen p erox ide (H 2O 2) as the tw o-e lec tron reduc tio n s ta tebecam e im po rtan t in th e deve lopm ent o f enzy m ology af te r thed isco very of ca ta lase C o m poun d I b y C hance (5 ) and the f irs tdem on stra tion of H 2O 2 in a m am m alian ce ll, th e h ep atocy te(6 ) . F urthe r, the e lectro n ica lly exc ited carbon y ls a re w orthy ofno te . K au tsky and de B ru ijn (7 ) rea lized q u ite ea rly the po ten -tia lly im portan t ro le o f a m etastab le ac tive o xygen spec ies(s ing le t m o lecu la r o xygen), w hereas F oo te (8 ) and G o lln ick (9 )m ade im po rtan t fun dam ental con tr ib u tio ns to the pho to ch em -istry o fpho to oxyg en ation . T he p resen t ch ap te r focuses on som eof the nonen zy m atic an tio x idan ts. T he enzym atic d efense sys-tem s have been rev iew ed in the recen t lite ra tu re s ee R ef 1 ).

Caro teno ids

C aro teno id p igm en ts a re w ide ly d is tribu ted in na tu re , w herethey p lay an im po rtan t ro le in p ro tecting ce lls and organ ism s .C aro ten o ids such as lycopene and fl-ca ro tene (F ig 1) a re im -por tan t b io log ica l co m poun ds th at can inac tiva te e lectro n ica llyex cited m olecu les, a p rocess te rm ed quench ing . S ing le t m olecu la ro xygen (02) is an exam ple of such an exc ited com po und andis genera ted by pho tochem ica l reac tions o r enzy m atica lly o r bythe p rocess o f lip id p erox ida tion of b io m em b ranes, ie , b y pho -toexc ita tion or by chem iexc itation . C aro teno ids m ay a lso par-tic ipa te in free rad ica l reac tion s. For exam ple , fl-caro ten e an dre la ted caro teno id s decreased th e ra te o f fo rm ation of m ethy llino lea te h ydrop erox ides (10) .

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2 Su pported b y the N ationa l F ound a tion fo r C ancer R esearch , W ash-ing ton , D C and the D eu tsche F orsch ungsgem einschaft, B o nn , FR G .M E M w as su pported by g ran ts from th e D eu tscher A kadem isch er A u s-tausch d ien st and th e Jung -S tiftung f# {252 }rissen schaft und F orsch ung .

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p oso m es aga ins t rap id perox ida tion un til it w as con sum ed . T h isco n trasted w ith o ur recen t observ a tion tha t pero x ida tio n in itia tedw ith Fe /A D P /N A D PH began a lready afte r on ly 15% of the v i-tam in E w as lost from m icrosom al m em branes p repared fro mrat live r (37). T h is p rom pted fu r th er an alyses o f th e ro le o f th isan tiox idan t, p articu la rly w ith resp ec t to its re la tive im portancevs d iffe ren t types o f p roox id an ts (37a).

F igure 4 , A an d B , sh ow s th e tem p oral rela tion sh ip be tw eenthe loss o f a -tocop hero l and the on se t o f lip id p erox ida tio n inthe experim en ts w ith th e f ree m etal ions . T here w as no la g periodbefo re e ithe r the chem ilum inescence in crease o r the accum u -la tion of th iobarb itu ric ac id -reac tive sub stances (T B A R S) in -d uced by 5 00 m o l FeS O 4 /L (F ig 4 , A ) ind ica ting tha t lip idpero x ida tio n sta rted a lthou gh v itam in E co n ten t w as a t in itia lleve ls. In con trast, a c lear lag period preceded bo th chem ilu -m inescen ce and T B A R S accu m ulation ind uced by 1 00 m oIC uS O 4/L (F ig 4 , B ) w hereas a -tocop hero l w as los t as qu ick ly asw ith o ther p roox idan ts and w ith ou t any apparen t lag . T he un iquefea tu re o f th is p roox id an t is th at the re is still a period lef t b efo relip id perox ida tion sta rts even fo llow in g subs tan tia l v itam in Edep le tio n . T h is re la tionsh ip is sim ila r to tha t no ted dur in g theC uC l2 -induced p erox ida tion of hum an low -density lipo pro -tein s (34) .

O th er p roox idan ts , inc lud in g A D P-ch ela ted iron w ith reduc-tan ts (N A D P H an d asco rba te ), o r co m pound s th at genera te per-o xy l ra dica ls tert-buty hy dropero x ide an d A A PH ) w ere tes tedand the resu lts a re sum m arized in T ab le 2 . T he site o f fo rm ation ,

0 .

ibFI G 2 . Q uench ing of the N D P O 2 genera ted sing le t ox ygen m onom o l

em ission signa l at 1 270 n m by lycopene . S ing le t oxyg en w as genera tedch em ica lly b y us ing the therm od isso c ia tion ofthe endo perox ide of 3 ,3 -(l,4 -n aph thy lidene)d ip rop iona te (N D P O 2) (1 9). E xperim en ts w ere per-fo rm ed a t 37 #{1 76} Cn e th an o l:ch lo ro fo rm :w ater (50 :5 0 : 1 ) p laced in a ther-m os ta ted g lass cu ve tte . T he in f rared em iss io n of O w as m easured afte rad d itio n of 20 L o f 0 .4 M N D P O 2 so lu tio n . T he lig h t em issio n w asth en m easu red us ing a liq u id n itro gen-coo led g erm an iu m photo d iodede tecto r (20).

F IG 3 . S te rn -V olm er p lo t o f lycopene , 13-ca ro tene , and lu te in . F ro mexperim en ts such as in F ig 2 , th e quench ing con stan t (kq) w as ca lcu la tedaccord ing to S te rn -V olm er p lo t (S 0 /S = 1 + (kq + k r) k [ Q]) , w he re

U sing phospha tidy lcho line lip oso m es as a m o de l m em brane , S and S are th e chem ilu m in escence in tensitie s in the presence o f theN ik i and co-w orkers (35 , 36) fou nd tha t w hen lip id perox id ation quen ch er , resp ective ly , k1 is the chem ica l reac tio n ra te cons tan t, k , isw as in itia ted by the pero xy l rad ica l-genera tin g co m pound 2 ,2 - th e 02 decay cons tan t in the so lven t (10 s ), and [Q I is the quen ch erazob is (2 -am id inop ropane) (A A PH ), v itam in E p ro tec ted the li- concen tra tion ; because kq > > kr, kr w a s n e gl ec te d.


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A N T IO X ID A N T D E FE N S E S Y S T E M S 1 9 7 ST A B L E 1S in g le t o x y g en q u en ch in g b y caro ten o id s , to co p h ero ls , th io ls an d o th erb io lo gical c om p ou nd s*

C o m p o u n d 1 ( ( i t k $ T issu e co n cen tratio n1 6 Ms

Ca ro ten o id sL y co p en e 3 1 0 0 0 n d II 0 .5 - 1 .0 m o l / Ly -Ca ro ten e 2 5 00 0 n dA s tax an th in 2 4 00 0 ndCa n th a xa n th in 2 1 00 0 n da-C aro ten e 1 9 0 0 0 n d 0 .0 5 -0 .1 m o l / L(3 -C aro ten e 1 4 0 0 0 n d 0 .3 -0 .6 m o l / LB ix in 1 4 0 0 0 n dZ eax an th in 1 0 00 0 n d 4 3 n g /re tin # { 2 2 4 }L u tein 8 0 0 0 n d 8 0 n g /re tin a

z eax an th in an d lu te in05 m o l /L

C ry p to x an th in 6 0 0 0 n d 0 .3 m o l/LC ro c in 1 1 0 0 n d

B iliru b in 3 2 0 0 n d 5 -2 0 m o l/LB iliv e rd in 2 3 0 0 n dB iliru b in d itau rate 1 2 0 0 n dT o co p h ero ls1

a-T o co p h ero l 2 8 0 ; 4 5 # { 2 1 6 } * * 3 .6 2 3 m o l/Lf i-T o co p h ero l 2 7 0 0 .2 3 2 .3 m o l/L-y -T o co p h ero l 2 3 0 2 .8 2 .3 m o l / L6 -T o co p h ero l 1 6 0 1 .7 0T ro lo x 4 7 # { 2 1 6 } * * n d

T h io lsC y s tein e 8 .3 3 0 -1 0 0 m ol/LG lu tath io n e 2 .4 3 -2 0 m o l/LL ip o ate 1 3 8 0 .5 -1 0 m m o l/LM e th io n in e 1 3 1 -3 0 m o l / L E x cep t as n o ted , ex p erim en ts w ith caro ten o id s an d to co p h ero ls w ere

p erf o rm ed at 3 7 # { 17 6 } Cn e th an o l:ch lo ro f o rm :w ate r (5 0 :5 0 :1 ); th io ls in D 20 /5 0 m mo l/L p h o sp h ate b u f f e r (p D 7 .4 ). R etin o ic acid , e tre tin ate , iso tre -tin o in , an d d ih y d ro lip o ate h av e n o q u en ch in g ab ility . E ste rif icatio n o re th e r f o rm atio n at th e 6 -p o sitio n in th e ch ro m an e rin g o f a-to co p h ero lab o lish ed th e q u en ch in g ab ility (2 1 C y stin e , G S S G , an d th e o x id atio np ro d u c ts o f m e th io n in e su ch as m e th io n in e su lf o n e an d m e th io n in esu lf o x id e h av e n o ef f ect o n th e p h y s ical an d ch em ical q u en ch in g o f 0 2 .

t k q v alu es w ere o b tain ed f ro m S te rn -V olm er p lo ts (2 0 ). D ata f ro mre f e ren ces (1 9 , 2 1 , an d u n p u b lish ed o b serv atio n s).

t k 1 v alu es w ere o b tain ed b y m easu rin g th e th io l d ep le tio n as a f u n c tio no f tim e, u sin g th e 5 ,5 -d ith io b is(2 -n itro b en z o ic ac id ) (D T N B) reac tio n(2 2 ) .

Plasm a co n cen tratio n s are g iv en f o r caro ten o id s an d to co p h ero l,ty p ical ce llu lar co n cen tratio n s are g iv en f o r th io ls . D ata co m piled f ro mre f e ren ces ( 1 9 , 2 1 , 2 3 , 2 4 ).

II n d , n o t d e te rm in ed (u su ally lo w co m p ared to k q ) .1 T oco p h ero ls w ere m easu red e lectro ch em ically u s in g rev ersed -p h aseH PL C (2 5 ).

S o lv en t w as D 2 0 /C 2 H 5 O H (1 :1 ) f o r so lu b ility reaso n s.tt Se e re f e ren ce 5 2 ; p red o m in an tly ch em ical q u en ch in g ,

lip id -so lu b ility , an d o x id atio n p o ten tial o f th e p ero x id iz in g ag en tsall in f lu en ce th e ir reac tiv ity w ith v itam in E an d lip id s . B ased o nth ese d if f e ren ces, it is co n c lu d ed th at th e im p o rtan ce o f v itam inE in p ro tec tin g m em b ran es d ep en d s o n th e ty p e o f p ro o x id an tu sed . T h e re lativ e im p o rtan ce o f v itam in E is d im in ish ed w h en

p ero x id atio n is in itiated b y co m p lex es co n tain in g Fe2 an d en -h an ced w h en p ero x id atio n in v o lv e s p ero x y l rad icals, e sp ec iallyth o se g en erated in p ro p ag atio n reac tio n s (T ab le 2 ). T h ere f o re ,alth o u g h th e f in d in g th at v itam in E is n early th e ex c lu s iv e p er-o x y l-rad ical scav en g in g co m p o u n d d e tected in m em b ran es (4 1 )su p p o rts th e co n ten tio n th at it is a m ajo r an tio x id an t (4 2 ) , o u rresu lts su g g est th at o th e r im p o rtan t an tio x id an t f ac to rs also p ro -tec t m em b ran es b e f o re o r af te r th e o x id atio n o f v itam in E .

A sco rb ic acid m ay b e a p h y sio lo g ically re lev an t en h an cer o fth e ef f e ctiv en ess o f v itam in E . Fo r ex am p le, it m ay reg en eratev itam in E f ro m th e ch ro m an o x y l rad ical an d so p ro lo n g th e lagp h ase (3 6 , 3 9 , 4 3 ). H o w ev er, h y d ro g en tran sf er f ro m asco rb ateto v itam in E rad icals, alth o u g h d irec tly o b se rv ed b y E S R inch em ical m o d e l sy s tem s (4 4 ), h as n o t y e t b een d em o n s tratedu s in g sim ilar tech n iq u es in b io lo g ical m em b ran es b ecau se o f th elo w en d o g en o u s lev e l o f to co p h ero ls.

T h io lsT h io l g ro u p s ac t as in trace llu lar an tio x id an ts b y scav en g in g

f ree rad icals an d th ro u g h en z y m atic reac tio n s. G lu tath io n e is

FIG 4 . T im e co u rse o f ch em ilu m in escen ce , th io b arb itu ric acid - reac tiv esu b stan ces(T B A R S) accu m ulatio n , an d v itam in F lo ss in itiated b y FeS O 4(A ) o r C u S O 4 (B ). M icro so m al f rac tio n s w ere p rep ared as d escrib ed (3 8 ) ,d ilu ted to 0. 5 m g p ro te in /m L w ith 0 .1 m ol p o tass iu m p h o sp h ate b u f f e r/L (p H 7 .4 ), an d b u b b led w ith o x y g en at 3 7 # { 1 7 6 } Cs p rev io u sly d esc rib ed(3 9 ). L o w lev el ch em ilu m in escen ce w as m easu red w ith a s in g le-p h o to nco u n tin g -sy s tem as d esc rib ed e lsew here (3 8 , 3 9 ). M alo n d iald eh y d e w asestim ated b y th e f orm atio n o fT B A RS (4 0 ). M alo nd iald eh y de -eq u iv alen tsw ere calcu lated u s in g = 1 5 6 m M cm a-T o co p h ero l w as m easu rede lec tro ch em ically in lip id f ractio n s u s in g rev ersed -p h ase H PL C (2 5 ) .Ferro u s su lf ate (A ) w as d is so lv ed in n itro g en -g assed w ate r an d in jec tedim m ed iate ly to g iv e a 5 0 0 z m o l/L f in al co n cen tratio n . C o p p er su lf ate(B ) d isso lv ed in w ater w as in jec ted to g iv e 1 0 0 M m ol/L f in al co n cen tratio n .


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In itiatio n m e ch an is m

Pero x y l rad icals f ro m E x o g e n o u sT yp e o f p ro o x id an t* o f th e lag b ef o re C L p ro pag atio n re ac tio ns p ero xy l rad ic als F e2 co mp lexes

C u S O 4 3. 3 1 .5 C u 2 cataly z ed A A P H 31 2 C ataly z ed b y cy t

P-4 5 0 (o x )A A PH -d eriv ed rad ical

Fe /A D P/A sc 4 4 1 C ataly z ed b y cy tP-4 5 0 (o x )

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cy t P4 5 0(red)

FeS O 4 l0 0 F e2a Pro o x id an ts : FeS O 4 , 5 0 0 m o 1 f e rro u s su lf ate /L ; Fe/A D P/N A D PH , 1 0 M FeCl3 ch e lated b y 1 m m o l A D P/L w ith 0 ,2 m m o l N A D PH /L as

red u c tan t; t-B O O H , 2 3 m ol te rt-b u ty l h y dro p ero x id e L m in ; Fe /A D P/A sc , Fe /A D P as b e f o re w ith 0 .4 m M asco rb ate as red u c tan t; A A PH ,2 5 m m o l 2 ,2 az o b is(2 -am id in o p ro p an e )/L ; C u S O 4 , 1 0 0 z m o l co p p er su lf ate /L . Fro m re f e ren ce 3 7 a.

t N ot a sig n if ican t in itiato r in th e p ro o x id an t sy s tem . C h em ilu m in escen ce (C L ) b eg an w ith o u t an y m easu rab le lag tim e .

th e m o s t im p o rtan t ce llu lar th io l, ac tin g as a su b strate f o r sev eraltran sf e rases, p ero x id ases, an d o th er en z y m es th at p rev en t o rm itig ate th e d e le terio u s e f f ec ts o f o x y g en f ree rad icals (4 5 ). T h ep ro tec tio n o f b io lo g ical m em b ran es ag ain s t lip id p ero x id atio nis an in te re s tin g asp ec t o f its f u n c tio n , s in ce th is w ater-so lu b leth io l p rev en ts d am ag e in a lip id en v iro n m en t. T h is p ro tec tio nis en z y m atically m ed iated an d is d ep en d en t o n v itam in E ; it h asb een attrib u ted to o x id an t n eu traliz in g , lip id p ero x id ase an d /o r

FI G 5 E f f ec t o f th io ls o n th e tim e co u rse o f ch em ilu m in escen ce an dp ro te in su lf f iy d ry l lo ss in itiated b y Fe /A DP an d N A D PH . M icro so m alf rac tio n s w ere in cu b ated as d escrib ed in Fig u re 4 ex cep t th at FeCI3 (1 2m ol/L ) ch e lated w ith A DP(l m M) w as ad d ed an d p ero x id atio n in itiatedw ith N A DPH (0 .2 m M ). C o n cen tratio n s o f g lu tath io n e (G S H ) an d d ih y -d ro lip o ate (D H L ) w ere ch o sen to p ro v id e sim ilar lag p erio d s. M easu re -m en t o f p ro te in th io ls w as m o d if ied f ro m Jo ce ly n (5 0 ), u sin g = 1 4 .2m M cm f o r th e p ro d u c t o f 2 ,2 -d in itro -5 ,5 -d ith io d ib en z o ic ac id(D T N B ) red u ctio n .

to co p h ery l-rad ical reg en eratin g activ ities , D ep en d in g o n ex p er-im en tal co n d itio n s , d ih y d ro lip o ate (4 6 , 4 7 ) an d o th er th io ls(3 9 )can also p ro tec t m em b ran es n o n en z y m atically , p rev en tin g lip idp ero x id atio n an d sp arin g to co p h ero l. T h is su g g es ted th at th eth io ls asso c iated w ith m em b ran e p ro te in s m ay also h av e an an -tio x id an t ro le , an d th at th e p ro tec tio n o f p ro te in th io ls b y g lu -tath io n e sh o u ld b e co n sid ered alo n g w ith th e p rev en tio n o f p er-o x id atio n an d sp arin g o f to co p h ero ls (4 8 , 4 9 ).

G lu tath io n e (G S H ) o r d ih y d ro lip o ate w ere b o th ab le to slo wth e lo ss o f p ro te in th io ls f ro m m icro so m es d u rin g lip id p ero x i-d atio n (Fig 5 T h is e f f ec t p aralle led th e ab ility o f th ese th io ls tod e lay th e o n se t o f ch em ilu m in escen ce an d (Fig 5 ) an d s lo w th elo ss o f to co p h ero l (4 7 ). O th er th io ls h av e v ary in g ab ilitie s top ro tec t m em b ran es (re su lts n o t sh o w n ), in co n tras t to p rev io u srep o rts, w h ich in d icate th at g lu tath io n e is th e o n ly th io l th atcan p rev en t lip id p ero x id atio n . H o w ev er, it w as n o ted th at th eb u f f e r co m p o sitio n in th e ex p erim en ts g reatly af f ected th is re su lt,s in ce w h en T ris ch lo rid e w as u sed to rep lace th e u su al p o tass iu mp h o sp h ate b u f f e r, th e re lativ e sp ec if ic ity f o r G S H in creased .

I f G S H en z y m atically reg en erate s to co p h ero l f ro m its o n e -e lec tro n o x id atio n p ro d u c t, th en th e p rev en tio n o f lip id p ero x -id atio n an d p ro tectio n o f p ro te in th io ls w o u ld b e seco n d ary an -tio x id an t e f f ec ts. T h e d irec t d em o n stratio n o f a o n e -e lectro ntran s f e r f ro m G S H to to co p h ery l rad icals b y E S R is ren d eredd if f icu lt b y th e lo w to co p h ero l co n ten t o f m em b ran es. H o w ev er,an en z y m atic m ech an ism w as su p p o rted in d irec tly b y p ro p ertie ssu ch as a su b s trate sp ec if ic ity f o r G S H (4 0 , 5 1 ). T h e sp ec if ic ityf o r to co p h ero l iso m ers w as an aly z ed b y p rep arin g m icro so m esw ith eq u al am o u n ts o f a- an d 6 -to co p h ero l. T h e re lativ e lo ss o fth e tw o iso m ers w ere ro u g h ly p aralle l d u rin g lip id p ero x id atio n ,an d th e ad d itio n o f G S H o r d ih y d ro lip o ate d id n o t g reatly f av o rth e p ro tec tio n o f e ith er iso m er co m p ared w ith co n tro ls (T ab le3 ). T h u s , n o iso m er sp ec if ic ity ex is ts to su p p o rt th e h y p o th es iso f en z y m atic to co p h ero l reg en eratio n . B


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A N T IO X ID A N T D E F E N S E S Y ST E M S 1 9 9 ST A B L E 3E ffec t o f th io ls on the onse t o f ch em ilum inescen ce an d the re la tive ra te o f a - vs . tocop hero l lo ss dur in g m icrosom al lip id perox ida tion

A ddi t ionsL ag b efo re

C L t R ate o f a -T H loss R a te o f #{ 244} -T Hos sR atio of a -T H

t o # {2 44 }- THos smm nmo/ mg min nmo/. mg min

C ontro l 17 2 3. 8 0 .3 3 .4 0. 3 1 .12 0 .09G lu ta th ione (0 .5 m mol/L ) 28 4f 2 .9 0 .2 2 .2 0 .2 t 1 .3 7 0 .16D ihydro lipo a te (1 m m ol/L ) 30 5 1. 4 0.2 1.5 0 .3 0 .91 0 .09

D ata a re unp ub lished ob serva tions (Scho lich e t a l) . C o ncen tration s o f g lu ta th ione and d ihydro lip oa te w ere ch osen to prov id e s im ila r lag periods .t C L , c h em i l um i n es c en c e.f Sign ifican tly d ifferen t from co n tro l va lue P < 0 .05) . Sign ifican tly d iffe ren t fro m 0.5 m m o l/L g lu ta th ione va lue P < 0 .05) .

Po rtio ns o f th is w ork w ere don e in coopera tion w ith T ho m as P AD ev asagayam , P S tep han K aise r, A s trid S chescho nka , and H ein erScho lich . W e than k U rsu la R abe fo r exce llen t techn ica l ass is tance .

R efe rences1 . S ies H . B iochem istry of ox ida tive s tress. A n gew C hem In t E d E ngI

1 9 8 6 ; 25 : l 05 8 7 1 .2 . M ichae lis L . C urren ts in b iochem ical resea rch , N ew Y o rk : In te r-

sc ie nc e, 1 94 6:2 07 .3 . H aber F , W eiss J. T he cata ly tic d eco m pos ition o f hyd rogen p erox ide

by iro n salts. P roc R S oc L o nd A l93 4 ;l4 7 :332-35 l.4 , M cC ord J , F ridov ich I. S upero x ide d ism utase . A n enzym atic func tion

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3 1 . C adenas E , G insb erg M , R abe U , S ies H . E va lu ation of a -tocophero lan tio x idan t ac tiv ity in m icrosom al lip id perox ida tion as de tec ted bylo w -leve l chem ilu m in escence . B iochem J 1984 :2 23 :755-9 .

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200S D I M A S C IO E T A L34 . Q uehenberg er 0 , J#{2 52}rgens , Z ad ravec 5 , E s te rbauer H . O xida tion

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Am J Clin Nutr 1991 Di Mascio 194S 200S