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Ascorbic acid in the lens of the naphthalene-fed rabbit
E z p t l Eye [~'r..,,'. {1.97( 0 9, 3:~-.18
A s c 0 r b i c A c i d in the L e n s o f the N a p h t h a l e n e - f e d ]{~;'r}l
\-A.N
Rabbit
}II.:YX~Xt:l.:x
.\' ,(#i,:]d i.,,bur,,,,~<,r.q ,,/" O]:hth,,I.,,~¢)lo:l.q, lY~,i,:,:r.~'3# of ()_@,rd, O.,f,,,.,l,
1(,, !/la,,d
( tlecei,:ed 20 ./~, rw: 1969, Londo,~ ) Id" the lel'l~ o1" t he rabbit, fed n~q,hl.hale~m (1 g / k g I,ody wcight/d~Ly } ,tov.~ n{,~ t)i.[ ()ill|* .-.'~,ver~;ly ~:~tara.e~)u.,, th,: cr~lt<:t.rttrtt~icnl, o:[" a.scorhi(: "~t:irl withiz~ it t.r'nds |~, in,.'rt:a~ pcogre.~sivL..ly w i l h t i m e . . i n v i t r o the.rt: i.q a n ineres~s,.: in t h e as,.orb;~.: acid ~:~meentr~ttior~ o f a h-n:~ int:ubals~(I ia at Jnr:,linw, cow,raining aseorbi~; a.~:{d ;tt)¢t t . 2 - d i h v d r o 1.2-dihy,lro_~y n a l ) h t h a l e n c {rmphtha.lcne
1. lntroduct.ion
(.!at:~ract~ in t.be nai)htAw.lenc-I'ed rabbit, is causF~(l by t~h~: })re~m~cc ~t" ~mpl~thal.~-Jl~. derival;ives in t}lC viGl'~.ous H.Il(} {I.qIICOIiS ]mm¢mr~ (Vail }[eyr~i])g(.~rt att,[ Pivi,'. t9~;7). O n e o f ~}]es,~ m e t ~ , b o l i t e s is na.j~hrh;~len,~ r.lio], i . e . t . 2 - d i h y d r o 1.2-dihy~h'oxy;~aphth%. l e n t [.F/~. l ( a ) ] ; 1;}li.s is o x i ~ l J z e d b y a.~ c;r~zvmr~, t.:~tzecbot r e d . u c t ; a s e l , r e . ~ c r i t in t.he l e n s ~tn(t or, h e r l ; i s s u e s t.d" ~,}~e ¢~ye, a n d N A t ) P ' * to g~',e "
([,;.f2. I . 3 . 1 . 5 )
N A I ) P . K a~,{ t , 2 - d i h y d r o x y n a p h t h M c n ( ; [.Fig. l(b)] [eq,.lat, io.n (1-)]; this co:~}prm.nd is rein'lily :-~utoo x i d i z e d al)n,.ui,rat p]"[ to yJe.id 1,2-napl~g}t,'.~.quinorm [.l"ig. l(c)] ar~,! Iii.,() z I)-quatkm (2)]. O t h e r met, ahoJitcs of" I|~|.][)}lt;]'l'-.l](:l)l? I£11,'5{) "f:aOt) ]%'i[;1'1 ('!]Z~'III(;.% i]'l I~}|(, "VII t<) -(tivl' 1,2-,lihy,troxyn~"pht.h a.hme al3~I thcn,;e 1,2-ha phthaqui,)o|~,~. "
N a.pbthaIen..-; (Uol + N-YJ)P v-,Mihyd.:'oxvm~pht.hMcc, e 4- XA 1)PI I-~- t { " I )i h y d r o x y
nIapIltohalc n (~-t- (.3,,--.- i n t l , i l t h o . ( t l l ilion('. -~- I-I .,( ).,. HO
(e}
H
OH
(b)
( 1) (.'))
0
~e)
3-'~,':. 1. l~h)nmdnc ,>f n~lal,~lic prodm:ts of n~phthalen~, rne¢tsb~,llszn. {a) t.2-l)~hydr,:,l,2-dilJs'dr~xymxithtl~ah,ne (naphtha|tm~.~ dio[), (b) J.2.dihydro×ynrq)hthaJeltc; (c) 1 , 2 - n o p b t b a q u i n o n c .
In ~,Lc pre~ence of asc'orl~ic acid the R)Howing fm'~hct- react,ion l.a, kes place [equatiou
1-3)1 Naph/Am quinor,<_'- -i- a.scor|m te.--~d ihyd r o x s n m p h t h a l e n e + d e h y d r o a ~ , ' o r bar~.
(3)
* A bb~ e v i a t i o n ~ ]]se~:l : ,%8 I|,. g.I u t a t h ioxmc : (;SSt-I. ,Jzitlized glutat.h ioJ~e I X.-k I) P.+ o.i M X A t ) P H, ozkli>'ed ~n:d r e d u c e d e,:,cuzyme~ nicot.inami:h: adcniv.c ¢]iuuch.~t.ide pho.'.,.ph;:tt,c.
3S
A,-;+'()['.I'~I( • A t ' I t )
IN " i ' I I F :
ItAI>,V, TT
I.b;Ns.
[
,"¢.q
\V,. h a v o .-+}jov.'n tl,.'t t. the.'~e r e a c t i o n s p r o t ~ a b l y t a k e p l a c e in I:},e e y e o f t,h e naphthah.~J,.*5",l r+t},bil ( v a n l l e v t , ir,g<:n a n d ['iri,.,. I.q~iT), :~nd t h;tt t h e (.t+.~(:ro:lsc0 c n n c e r ~ . r a t . i c ) u - f ;~::+,+n-hio ~,+'i+l i~, *:lw a+iu,:+,us nt,~ w i t h ~,:q,hrt,,quinrm,:+ [+++tuaf.ion (3)1. \V,., w+.r,..-,ur/,ri:<<,,l: t.],,.,',,f:)rv, to f i n d t h u t i~ s l , i t e o f t h e 1~,.<~ r>f aseorhi+: neJ,t t'ro:n t}~...... t:is.-.~0o.~, i0, t h o rloI+-¢';tt~tt','t+:tcJLl.~ ]t.'l+s Of +.he mtl>hl;hah'.ne-fc.+[ rabbit., the. eo+teent.c:, l i,:,n, ,f' :,-t'< ,r'} ,it' .,:i, l xvat~, i,wr,.aso+l. T h {~, [J:t p,'r +h,st:ri h c s a .'..tu,t y t,j" el, is p h e l l o , lie,lot+.
2. ~ l a t e r i a l s m~d N l e t h o d s I>u~.h ~:n,i a ! t , i t , , r.~hl,ir., u:,u~{fv 3 e'; ~v.mth:~ obl, wore u.~::d. 3Iethod.s o f (t~:~,~g~.: w i t h ~.~:,fdtth;,l,*r.,,:-;,lJ,l .x.,~.i~ut~i<,,~ <,f rim ev'e lta\'e b e e n ,ie'..:rilw,l p r e v i o u s l y ( v a n Heyninge+~ .+~'.,l .~'ir},:. l'.~1;7). 31o:~t , , f , hr: l,..r,:~es ,ts,..,l i~, tbe e x p e r i m e n t s d e s c r i b e d in t h i s p a p e r had n,) visible ,'ba,xzes, +,;" +,M'." ~,6ni iI~.] ,-I,.n.u,*.5 (...u+:h as ~+ few w a t e r ¢.-.Jeft.:~at. t h e p e r i p h e r y o r n t~w s u b e a p s u l a r v.:.+ :~o]eW. :\ b:w ha+l w~,t,r ,:left* all roilt)(1 tile p e r i p h e r y . I.+qt.~; ~t~t,t ~tq~teot~.s h u u l o u r were ¢tl~alysed i m m e , | i a t e l v a f t e r t h e r a b b i t w a s killed. - r ;it,.,." wet+ + ,.t,,re, I ;,t --':).0 ' u n t i l r e q u i r e d . \ V e i g h e d l e n s e s w e r e g r o u n d w i t h ,3 n d !.0°.{~ ( w / v ) :r;~+h]~wOa<:etir; ae.i,t: a f t e r ch;.llin.<_,, for 1 hr, prot.ein w a s r e m o v e d front t h e e x t r a c t b y :-*-td;ril',l~uti<,n. "I'o tht~ :,,lue,~u~ h u m , m r an e q u a l votum,- of t h e trichloroaeet.ie, acid= ~olu+ I r,,:j Was zld..l+'*J ;tti¢l tim pe,+cipit;,te, if +,nv. w a s r e m o v e d b y c e n t r i f u g a t i o n . /,,c,,b~lio~ c,f r u b h i ! le~,.~ T h e m e d i u n t wa.~ K r e b s ~ i n g e r ] ' h o s p h a t e , p H 7.(t ( C o h e n , 1949), c o n t a i n i n g g l u c o s e ( a p .l.u. .' w / v' • ~.e. " 5.+5 m:,~'~, ~,nd a s ¢ o r b a t e (20 ~! o£0"o ~t/lO ml m e d i u m ; i.e. 1-0 m~t). l n c u b a ti,,zt.~, we£-o Jt~ air, .'.it LI4'+. J:x |ells i l l e u b a t i o n ~ube8 ( 3 1 e r r i a m a n d K i n s e v : 1950; v a n f i e y n m g e u . . i <..~f.;.'.3)."{'e~t ml. o f ~,v.dium, e o,~tah+ting 3-0 m.~r n a p h t h a l e n e dioi or 0 . 0 5 - 0 - t ~,l:,~ , u + p h t h a q u i a o n e w h e n a i ~ p r o p r i a t e , w e r e p u t in t h e i n c u b a t i o n t u b e s in t h e w a t e r - b a t . h . T h e rM~bi;, wa.~s kil]e+l ,, rJd e a c h lens s l i p p e d {nto it.~ p r e p a r e d t u b e . S a m p l e s o f e u e h i r m u b n tJ<~it t)~c,!itll,t (7~0 I.d) v.'eV~: w i t h d r ~ t w n a t tntecx, nls o f 1.5 o r 30 rain a n d a,~sayed fl~r a s c o r b i e -oh}. l.,~ t h e ,:once~,tr:~.iol~ h a d f a i l e n a p p r e c i a b l y a c a l c u l a t e d v o l u m e o f 0-5 .',~ a s c o r b a t e w a ~ ; u b l e d t~, b r i n g t h e level b a c k to ~ , p p r o x i m a t e I y 1-0 m ~ . A t the. e n d of t h e e x p e r i t n e . n t t h e ]et~aes. w e r e r e m o v e d , r i n s e d b r i e f l y with_ d i s t i l l e d w a t e r a n d a d d e d t o a w e i g h e d t u b e ,-:~,nt;,i,,in~ 3 f) ml Jl)'}.~, ( w / v ) trichloroa.cet.ic..mht. T h e t u b e w a s r e - w e i g h e d an,1 t h e w e i g h t +-~f t h e len-.~ ~+as obt, a i n e d b y d i f f e r e n c e . T h e ie~,~es w e r e d i s i n t e g . r a t e , t w i t h a g l a s s rod a n d t h e m i x t u r e c h i i l e d for about- I h r ; t h e p r o t e i u w a s zh.en r e m o v e d h y c . e n t r i f u g a t i o n and. lho e x t r a c t ; , s s a y e ( l t ~ r a s c o r b i c a v i d a n d (.,+'5}[. l)eh3"droa.sc:~.+c-orbic ~,eid. h~td j u s t t.,tmt+, a d d e d , w a s s n b s t i t u t e d . T h e r e a s o n h~r t h i s ntanoet:tvre is t h e l a b i l i t y t;f dehy,Lroasco.,'bie avid a t pl-[ 7-0. :[t+:s h a l > l i f e a t pTt: 7-24 - t a d 3 0 ' i~ +l})ottt. 5 l a i n ( Ba }1, 19.'37 ; see a l s o 1.a n g h n +,t a n d H e n l d , 1.<~56).
A s c o r b i e ac.id wa.,:-det:ermi he,.1 b y ~it,rat.ion w i t h +2,6 .dichloropheu.ol ind<.~pkenol a m t t o t M a,~_corbic ueid b y t h e m e t h o d o f t~oe {I954). Dehyetro~,+corbic: a c i d w a s tak.er~ to be t l m d i f f e r e n c e b e t w e e u t h e s e t w o mea.~urement,~. T h i o t g r o u p s w e r e d e . t e r m h ~ e d h y t h e r n e t k o d of E l | m a n (I9~9), T o t a l thiol w a s a ~ a y e d in a lens e x t r a c t , G S H in the s u p e r n a t a n t after r e m o v a l of protein x~fit.h trichloro,qcetic acid, a n d proteiT~ ttdo| w a s taker, as the ddfferet~ce
40
ItUTII
}mt, wvm~ th~..so t w o v a l u t ' : . L ( _ ' ~ ( . I it t e d t u t l , a ~ e ( l ~ . . "~,n t t y "~0.". t, 1BG,3). •
",
VAN
H I':VN]NGI~N
xv:ts a s , , , a ) ' e d spectv,>t>h, to~netrically.',
u,~:irw, g h t l . a l l t i ~ , t t u
'
.llaler&~L~" ? 1,2-]-)ihyqtm 1..2-dihy, t r o x y n . ' t l d v l h a h ' n c 'a'.s a ,~it't fr'-.: Ih,ofe~s,-" Sir ].,,var~ .t,mv~ . i ~ l 1,2-naphthaq.ui:uone f r o n l 3Ir.'~ ,I. I h ~ e s . [ ) e ] t ' , . ' d r ~ v l . q c , ) r b i c il,.:id (()'-~ .'a) "~v~ls n l n ~ l t ' ]~v ,,.,,:hlizi~.,_*, amcorbic a c i d w i t h b r o m i n e , as f,,}hm's. As,.:orbic ~cid (|71i mlZ) wa,~ diss, Av,.'d i~ 2 mi waL,-r in ~t~ i,~e-bat.h, in a f u m e c . u p b o a r d . B r o m i n e w a s ~,,Ided ( a b o u t . 0-06 mI) m:l ii ..,. f a i n t y e l h , w ,.-,d,,ur p e r s i s t e d ; t.his w;,s r e m o v e d b y a d d i n g a. s p e c k o f as(:orbic ~,cid a~ul N z w a s ther, b u b b l e d tdn'ough the .solution f o r s e v e r a l ~ a i n u t e s to r e m o v e all t.races o f I~r,m~iue. I t w , ~ f r e n h l y p r e p q r e d for e , ch e.xpet'iutent .Ascorhic a c i d . 0-5 .~. n,:ut.rallzmt l o nh¢,~lt p H I~-:-' ','¢;th N a O f t a n d kept, iJt ice, w a s f r e s h l y lw,gpar~d, l',n" e:~,.b exp,eri ~ eul..
Ile~tct io~, between deh~droascorbic acid . m l tAiol,~ t~ovine l e n s w a s u s e d for t h i s e x p e r i m e n t . F o u r l e n s e s (~ ~) w e r e g m n n , [ ~,.'i~ h t 6 ml ,,f 0-1 3~ p h o s p h a t e bufthr, p t i 7,,1, i n s o l u b l e , p r o t e i n w~s r e m o v e d b y ceutrifug',tt, ion anti t h e e x t r a c t , w a s t,h e n d i M ) , s e d f o r ,3 da.ys a.t, .I ° a.g~inst dist.iiled w a t e r . T h e w;~t.e:" w a s s t i r r e d c o n t , i m m u s l y a n d c.hanged t w i c e d a i l y . T h e c o n t e n t s o f t,he dia.iv,ds s a c w e r e f r o z e n for 2 day,~: t,h e u t h a w e d a n d d.iluto.d w i t h a.n e q u a l w ) l u m e o f 0,1 .~r p h o s p h a t e b u l ] ' e r p}:I 7"0. T o 10 ml o f tills 1 hi 6 len~ e x t r a c t , w a s a d d e d 4-,t m g of t381-1 to ~ix, t: a t:oucenLratio:~ ~ t 1-46 re.M. ,.Samples w e r e w i t ~ h d r a w n f r o m t h e lens v,xtract.s, wit,It o r w i t h t m t adde~l G,%11, f o r a s s a y o.f iuitia.1 p r o t e i n a.nd GS.H t h i o l ; t o 5 :hi o f e a c h , Ixg 8 7 °, w a s a , b l e d 0,04 ml o f 0"g~ 51 D H A a n d nnd
ascorbic
samples
were withdra.wrt
at intcrvMs
over
the next
hour
for assay
o f tt:i~fl
acid.
3, R e s u l t s 1,1, "C'il.*.O exj;le, r4~itc gtl.'3
..tscorbic acid. in ,zqueo~l.s ]~u.'mou," ~o~,.;.len..s. I a b l e ] c o m p a r e s
l.he c o i J t e n t . r a t.icm ,,t"
,E/][,ct q/" .n.aphthaleneJ;.~edi.,.y on the conce'm.rution o f a,;'e,~rbic acM it~ the. aqueous h-~.~n~.ou.r o f zhe rabbit _-%s¢orbio acid in a q u e o u s (/ztnole~/re.i)
J(a.bbit, ,'to. 43 i t2* 32 "2 33 29* 3;5* 36 30" ] 61"
14
No. of do~'s o f na.ph t t~:tiene
hulnom
% 2-Naphruh alenc- fed
C,~l-tt'v~l
di ll'e ten<',- - ,~3 -- ~J -4S - - 77 --72
-2
0.' 7
t .02
'2
0-16
t . I ;3
,'3
1-02
] .96
,'~ 5 9 9 10 I0 15 23 28
0-25 0-45 0-3I 0-.18 | -36 0,99 .t .47 0.23 1"99
1 -I 0 1-59 0-77 1.07
] -!N) 1-90
--60
--- 55 --- 28
:?.,I S
-- 4 S -- 3:;
0.94 1"[4
---"76 "~ 75
."ffaphthaleno (J g ] k g b o d y wt,) w a s a d m i n i s t e r e d d ~ i J y . E a c h c o m p ~ r i B o n is b e t w e e n ]itterm~te.*. * W ~ t e r ¢teft.q a r o u n d 7periphery o f l e n s ; i u t h e r e m a i n d e r t i l e lene.ez s h o w e d n o , o r m i n i m a l c h a n g e . "~ l'rotcin in a q u e o u s
burnout, scvero cataract.
..k,~(.'(~]ll~[(" ACID
1N
'I'lll,3
ILABI~IT
LI,:N,q,
I
41
~t.~t:ort)i(:. ~tt:id i~ th,; aqtteous h u m o u r of t h e mq.)]~tlta.lenc-fed rabl)it; with tha.~ :)f ,m u~Crca.t(41 l i ~ c r m a t c ' . The. n o r m a l va.ltlcs r a n g e fronl 0-77 be 2.18 ~ m o l c s / m l 0rye.rag0 1 "35 I.tl~O]O.~/~)ll). 3~Vit]t 0110 ~tXCCl)tion tht: eonce~,tratiou in t;lm ~ q u c o u s l m m o u r o f t.'he ~,,ld, t,hale~w-fed r a b b i t was low(.:r th,'.t~ tl).a.~ in t.hc tlllt;rcltted r:~.bbit., t l o w o v e r , ~,.fte.r t,h," :~oeoa~d du.~o, t h e d iff(,rerw.c betw,,~:~ the. ]evel.~ in t h e t,re~O.t:(l at~(t re)treater[ :~ninmls (,,.~
200
7" 160
og g~u
L
>I.
-¢)¢*
~e-~
t
L
~
~
.," ~ . . . . . ~ £ . . _
4
~
__ ~
I~
16
!
I
I
20
Z4
~8
F.:~.;. 2. ,~,sc,.~z'bic a c i d i,t th(~ aClt~t;ou~ t~tmto(zt" o J" tire. J:ai~J~thale,~(> fed vttbbit,. ~ ' h o j)erce~xtag~, d i l t b r e t t c e I~('t..~vc*.'n t h e c(,z)cet~tcat o:) o f a s c o r h i c a c i t | ia~ t h e ;tqttt:otl.'~ hlllilo~.lt" o f t~|)o n,~pht.hale.ste.fed r a b b i t , a.~d "that i,~ a tittc~'ma.¢(~ c¢.qlt r~.A. i8 I.dCH.~(-d ' a g ~ i n s t I,he. n u m b e J - o f daJl S d o s ~ s o f Z'ml~/~thah-t~t,.
t.c,~ded t o ('~ecrea.ge as the. amYJl.)cr of (loses incrca.~c.d (Fig. 2); t h e c o n c e n t l ' a t i o ~ el~ as(:orbic acid i~ t.ljc ~queotls h u m o m " of a r~tbbil; w h i c h h a d h a d 28 doses was h i g h e r t,|,an t ha~. in t h e control. T h e o~e v~luc w h i c h doc~ no$ fit, iu~o t,his p a t t e r n , - 7 6 % ha r~;[)bit no. 16 a,fter 23 (loses, was o b t a i n e d t'roJlz ~13 a q u e o u s ] m m o u r wh-k3h in v i v o .~howed ~i, srxot~g flare. Secretion of 9,scorbic acid by vi~e ciliary b ~ l y m a y the, r0fore )~ aI X" [~ 'tj(,!I':l'l i I ' ~ l . p a i r e d . '.F.s ~, ~.~: I J.
..~.,'co,'bic e~cid its. tl~e .ser~tm
of a..na,19hthalenc.fe'd,
N~). oE dcy,,:~_.s
A,-~eorbi(: zteid (/~tlotes/nal)
(t
0.053
3
O'Ofi~3
9
0.108
13
0-1.20
rabbit
,t R~l)bit, no. 4 5 - - n a p h t h a l e . n o
(1 g / k g ) ~:as a d m i n i s t ¢ : r e d d a i l y .
Since tl:ere is s o m e e v i d e n c e t h a b ~jay,hvhalene-i%eding brings a b o u t a~ i n c r e a s e in t h e concentra, t i o n o f conjuga,ted g ! u c u r o n i c acids in t h e b l o o d a n d in t h e a q u e o a s a n d v i t r e o u s h.mnours (T.akagi a n d Ok-0mura, ] 9 6 1 ; K i u c h i , 1959), t h e g r a d u a l re~Ltrn ~o
4~
I(UTH
VAN
ttEYN/NGEN
n o r m a l v a l u e s with increw~iug mm~bcrs of (loses of nal~h~lmlenc ('C~,.Me [ a n d Fig. 2) m a y bc because less unconjugat:ed m e t a b o l i c product,s of ![ILtph0h.l]..[i3[l[2 I'(~1.t.'[~.t']l(? ~d}'t'. Dctoxica~ion b y i ltcrease(.[ s y n t l , e s i s of gLucuronidcs is o f t e n a.ecompan [t'.d by increase,[ s y n t h e s i s of ascorbic acid ~nd. in a single experin~eng, t|~i,s was ,sltown co (l~-.(21.ll" i.t}. ,~aphthMe.ne feeding. Blood, w i t h d r a w n .i'r<>~n it m~pitt.hatene-fed r a b b i t a~ ;n~e~vul.-, was a s s a y e d fi)r t o t a l ascori,ie ~ci~t by l;he ,~,e0u)d o f Roe (195d:). T a b l e .[1 .~l.mw.~ tirol. over v, p e r i o d of L3 d a y s , tho (:7)]ttJetltril,15iott it~creased r a t h e r more t b a n twoiiHd. Th,, aJnou~)t of aseorbic acid secrt,,l.ed into the a q u e o u s httrnOltr would g[t,:t'eI~re ~i[s() i~r i n c r e a s e d ( K i n s e y , t947). Fom~m.t.ion of glucuvonides m0-y therefore act. in two w a y s ~o b r i n g a-bout a. g r a d u a l increase i~t t h e ascorhic acid of ~cqucous tttttnou.r of ~;he ,'~d~bit, dose(.[ d a i l y w i t h naplatha lene : (1) b y p a r t i a l r e m o v a l of goxic napl~thMcne ~ teta boli ~e~ ; a n d (2) b y i n c r e a s e 4 synthe.~i,5 o[" a,';cot'l.'ic :tcitl. Tam.t.: Li L
f'~'ec't of "nap/d/~ale~,.e-feedim/ o,~. /he ('onve.atratio~t qf u:,c.,,t'bic ,w?4 ,-r~ tim/era,. ~.f the .~',¢L5;!
.Rabt,iL rio+
I 1 "2* 32 '2 31 * l:l* 39* 2 9 ~" 2l 33 :$5*:36 30* l[ 4
No. u|" d,~u's of naphthalrplt.
"2 13 :g 5 5 8 ~ 9 i) 9 10 10 15 2N
Ae~,otbie a c i d in Ics~.~ (/~. u,h,s/,,).. i\'~tpt~tha ten,: "~, -fed ( ' o n t vo! diJl~'t'euce
i • 14 2- 7 4 ! <16 l .7ti 3-'2:2 2.40 2-97 2.42 2..92 1.6(1 ;I,7i.I 3.01 1-2!~ 4.55
1-~ 4 1- 2 7 i" t 9 1-25 1-1,5 t .26 1-54 1.6~ I 61 I- ! 6 1.19 l -[,() 1 -Y,f* I ,7{J
0 -.'-- .[ I ~J -,- 1 ,, -i-4 t -l-lbl~ -.i. ;~ l .~- 9 3 .1-44 -i- ~.l -i. 4 3 -!-211 -l- 1 5 0 ~ 9 - " t 5N
N ' a p h t h a l e i m (I g / k g b o d y "~vt) w~uu a d m i n i s t e r e d d a i l y . 1,3acl~ e o m i ? a r i s o u is b e t w e e n lit.termal.es. * %ratec e i e f t s a r o u n d p e v i p h e t . y o f l e n s ; t.J~e o t h e r l e n s e s s ho~red rto, or m i n i m a l cl~ai~,._,c~.
Table. Ill. compm:es t}m conee.utrgtion o~Faseorbio acid in the leas of ~he txaphtlaa]e~"lefed r a b b i t w i t h t h a t of its m~treated l i t t e r mate. W h e r e a s there is a. deficit of ascorbic acid in t h e a q u e o u s hmnou.r of t,hc n a p h t h a l e n e - f e d r a b b i t (Table I a.nd F i g . 2) in the lens ascorbic acid accumtflates. T h e a v e r a g e value f0r t h e ascorbic acid in t h e 14 n o r m a l tenses is 1-37/xmoles/g (range 1-14-1-76). Irt t h e 14 lenue~ of the n aph~lm,leilel e d r a b b i t s the a v e r a g e value is 2-37 (range 1-14:-4.55). T h e r e was a t e n d e n c y for tile c o n c e n t r a t i o n in t h e lens of t h e trea.ted r a b b i t , c o m p a r e d wiga t h e u n t r e a t e d , to i n c r e a s e w i t h t h e n u m b e r of doses. (The lens of r ~ b b i t no. 30, fed 1.5 ¢~ys, is a m a r k e d exception.) A clear-eu~ difference betweet~ t-he e y e of t h e n:~plathaiene-fed a n d the c o n t r o l r a b b it is s h o w n in Fig. 3. I n t h e n o r m a l eye t h e coneentrai;imt of aseorbie acid in t h e tens v a r i e s f r o m 1.14 to 1-76 (av. 1-335), while the c o n c e n t r a t i o n i n t h e a q u e o u s h m n o u r
:k ,'-;(.' (~l }-.' Jll(.J . " ~ ( ' l l )
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.raHges t'ro~ 0.77 to :2,[8 (av. 1-32ix.moles/m] 1. .|u ~,he lens of ~he. napl~thah.'~c-fcd ra|,bil) em)ccnbr~.t, ions of ~sm:~rbic a.cid whic'l~ fall w i t h i n the ]mrmal range 0.re as,so~,i,~h.:d w i t h ~ow lev~ls in t h e ~queot]s humour:~ (Fig. 3), Vaht~s for ascorbie ar.,itl in t h e a.que~m:; Inumour appron.cl~ing c~:mt.rol v~tu(:s are a.~sot~ia~t:ed wit.h elev~ted lens con lt;vls as~:orh i~: c,.~nt,J'at.io~s. ]tl t]~.: ~ocnml lt:ns bIi¢: ca gio wa.s 1"01 :t-0"12; in tim [~qtleous asco~:[)i~ nzilfl~t.h~h.~c-fcd r a b b i t !,he ratio wa.s 4,22~:0-7"5..ln ca.leuhtting ~she re.tic for t.ho iml)ht.hah;H~M'~d ra.bbit,, one vatuc w h i c h fa.]ls f~u' .fi'on~ the or, hers a.ntt a m o n g those i'r(ml e.he ,.,yes of m)r~na] r a b b i t s Jm.s been omi~t, ed. I~ c o r r e s p o n d s ~;o r~~bbi~ no. 30 in whic"h !.he. v~]ue for ]en..~ ;~scorbic acid w a s excep~}onally low (Pa ~1(.. l I l ) .
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F~.;. :.~..L/~'k~t i,~,s!~ip bvt.we,.~n t h e c ~ m e c - n t r a ~ i o n o f a s e o r b i e ,~cid i~ t h e l e n s at~d in t h e a q u , - ~ m s h u m o ~ a r , ,1' : h ~: cxc..-, ,_,f ~m p h 1.1~! e , e- ~k.d a~ t~ unt~'ea~ e d r a b b i t s . O , U'~t.ru~tted r,-tbbi~s. ~ , n ~'q?I~t,h a l e ~ e - f e d r a b b i t s . V a l u e s tk~r a l l eye.s i~ w h i c h a s ~ ; o r b i e a c i d w a s a~st~yed in b o t h le~m a ~ d a q u ~ t o u s h e i n o u s " a r c p l o t t e d h~ t.hi.~ l~g,m-.. 'J'h~: m t m b t : r o f d¢:~o~ o f na.phthalen~.~ r a H g c d f i ' o m 2 t~.~ 28.
T h u s in the e}:~: of tlw, n.~pht.hs..lczm-fcd r a b b i t t h e concentra.tio~ of ~.tscorbie acid in ~he .!en:~ wa-s more t h a n 4- t.imes thal; in t h e ~tqueou.-: b u r n o u t , whereas in the n o r m a l eve ~he avm:t~ge vMues for ielLS an(l ~(tue, ous h u m o u r were alnlOS~ t h e s a m e . Adm~:-~dst.ration of ~,,a.pht,t~nlene diol -i-n. the for.n~ of eye d.rojps. N a p } ~ h ~ l c n e dioI was ;~i m i n i s t e r e d in t h e form of e y e d r o p s co !,be righ~ e y e of t b u r r;.~,bbi~:s (var~ I-Iey~iingen •trtd Pirie, 1967). I n t.bree cases ghere were c~earactous changes, bu.~ t~he lens(;s were no!, a n a l y s e d . I~a t h e fourg/a ra.bbiv t h e r e were no lens changes, but, i~ !.he righg eye the tens was f o u n d to eont, a.in more ascorbic acid, ~1~d t h e ~.queous h u m o u r less a.seorbic a.eid, tha.n i~;e tmgrea~ed lefb eye. T h e values were, for t h e righ'0 a n d . l e f t lene, 2-1 a n d 1-5 m ~ a n d for bhe right; a n d l e f t a(.lueou~s humout's 1,2 a n d 1-65 rn~!. Oe.ner~iIy, o n l y r e d u c e d ascorbic acid in tlle lens wa, s measured. O n a n u m b e r of occasions, however, regal ascorbic acid w a s a s s a y e d (reduced plus d e h y d r o a s e o r b i c ~cid) a~td it- was s h o w n ~hab litNe d e h y d r o a s c o r b i e acid was pre,~en6 in t h e lens. T h i s is hog surprising, in view of ghe h i g h c o n c e n t r a t i o n of ghiol g,roup-% of g l u t a g b i o n e a n d prot, e i a in t h e lens. Glutathione. J.t; is well k~uowt~ !hag d e h y d r o a s e o r b i e acid is rat~idly xecluceXl b y gtut, a t h i o n e , wit,h t h e formagion of ascorbie acid a n d oxidized glu~a~hione [eqtmt, ion (4-)]. This rea.e~ion is n o m e n z y m i c . The~e is no evidence t h a t t;he e n z y m e w h i c h cat, a15:ses t h e react.ion in aome anima.l t~ssues (]CJ[ughes, I96-1.) is p reseng i n t h e lena (tlughe:~,
4-1
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p e r s o n a l c,.)uimm~i,:ation). I n t.he ~ b s e n c e o~" g l u t a t h i o n e , p r o t e i n thiul is .~i~ui!.'~rlv oxJtlized (see later. Fig. -i): d e h vdroa:~corba~e + 2 LI~I-L .--itsc'orl.~ tt, .4-G,";,.'SI.~.
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T a b l e .LV .-,how.-, thai;, in t h e n o n - e a t . ; m s c t o u s lens o f rite impht.lm.lcz~e-tbd r;,l~l.;it, flu. conee.ntratk,t~ o[" glttt.athione w a s n o t greati}" , { i m i t d s h e d . \ V i t h O t l e excepti,>n (r.~t)f~i.+ no. 13) rite c o n c e n t r a t i o n of' (.IS.H w a s d,'crea~v
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N~q~htl,alene {!. g l ' k g b ~ l v w t . ) w a ~ , ' . u h n i ~ i s t e r e d d : d l v . t~]ac|l c o n l p a r i s o n i~ b~,t'~vt, e n l i t t e r . ' ~ t a t < . s , * ~,V~ter elet'ts around per[pl,ery u f l e n s : t h e o t i l ( ~ r [t~tl~c.~ at~.;~'~l~,.[ ;H). ~;r I[tilti~tt:~l ch:~I/K~.'.<,
It~ r'iD'o t.J:]>uri',tent.,." I.no~tb~+tiw~ o.r the te~s ~ci~h'~:+phthale~w dt,rh'aticcs a~;d oscorbic ,.cid.. :2,) invest.iFat~,. f u r t h e r t h e i n c r e a s e in c o n c e n t r a t i o n of ascw_'bic a c i d i n t,he h;n~ of rim n.~phdtat,;u,.'fed rr~bbir, t.he p h e n o m e n o n w a s r e p r o d u c e d as far a..a. l;o~sible in v i t r o . T h e ~wo ]e,ases of a. r~-rbbil; w e r e e a c h i n o u h a t e ( t in ~L m.et[ium in whiei. %be concent;ra.t.ion of as(,orbie acid was m a i n t ~ d n e d a t t h e level of a.botz~ 1 n~.xi (see 5[ateri,ds a n d 5I~t.hod.-:) addi~iol~ o f a u a p h g h a i e n e t-[e,rivat.ive w~Js m a d e t;o ~he mediun>, in w h i c h one, o f t h e lense.~ w a : incuba.ted...-kfter some h o u r s ~-he e x p e r i m e n t was s t o p p e d a,rM l:b.e t w o 1.ense:.s ;~.~a.ved .for a s e o r b i e acid a n d glutar.hione. T a b l e V, lin.e A gi.v~-,.s t,he results of ~hese e x p e r i ment, s. "When t h e l e n s was i n c u b a t e d w i t h 3 m ~ n a . p l r t h a l e n e d i o l t h e a s c o r b i e acid i.a the .r.ffedimn was oxidized. T h e m e d i u m b e c a m e b r o w a in colour al~d. t~he s u b s t a n c e of t h e l e n s (but, aqoe t:he c a p s u l e ) also became, c o l o m ' e d . ~\r~.te~ cleft;s a p p e . a r e d at~ t h e equai~or, s i m i l a r t o t h o s e s e e n m I&e l e n s of tim n a p t & h a | e n e - f ~ :I rabbi~ i~ vivo. "rite ascorbJo a c i d con~eni; o f t,ize l e n s i n c a b a t e d in dioL i , c r e a s e d in 21[ f o u r e x p e r i m e n t s b y a n a v e r a g e o f 21°,.o, w h e r e a s t h a t o f g l u r a I s h i o u e d e c r e a s e f t go 5 0 % ( T a b l e V, line ~.). r, q~c, a n d d e h y d r o a s e o r b i e acid were a s s a y e d a w l I n o n e of t h e f o u r experiLnents tx,_~,.T b o s h w e r e fom~d t o b e M>sen~.
ASC01:~B[{'
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lr~ ,gCOU~ed p r o b a b l e tha~ t h e efl\'ct of n a p h t h a l e n e diol. ia b r i n g i a g td}out t h e a.c'~tt|nuhtthul o.f a~c.orbie acid in t,lLt' lctls, ~,','¢vstitle tO tile l b r m a t i o n a n d p e n e t r a t i o n ~,t" ~[r-,hvdroaacorbie a c i d [ e q u a t i o n s ( l ) - ( 3 ) ] . T o test. ttLi;4, h.'.lises w e r e i n c u b a t e d iu at :ne(tiLttn cont~a.inlt~g , l e h y d r o a s t ' o r h i c acid. Stlld eomparc<[ wit& lenses incubat;od in a.-corl,ic a<'i~i, a n d ;,tso w i t h tu~incut)ated lenses. 'I';}}A~. V. line H. sl~ows that., with. d e h v d r o a s c o r h i c a c i d in t.he m e d i u m , t.he conc¢,ntration ,.d" a s e o r b i c a c i d iu t.hc lens increased, h:o~n 1.13 to 1.96 nt_~], a n d t.here w a s ~,, c h ~ t ~ e i~ t.be level of (ISt-[. 'FabIe V. line C, g i v e s l~he a v e r a g e level o f a s e o r b i c a c i d ~,n..! ~;SH; in fix-t: Immc~ inettbat.ed in m i l l i m o l a r aseorl)ic a~,i
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C~.~-~]2:~-}[~J!lff ~|lp[tI'.'|ll~*l/t I'-LI kVC]) Ill{; ,~'CtllL't'U'~.l~il[.itlll t i t ilbf}ll¢~ I 11131 :. I;t-'.," .~,. ~("))~:1; ![.il~." Eo I S 0 t L I : l i n e 1:', 2 0 0 j ~ } ; tiP-e. G . I S 0 1 t I ; Iitu:~ G a ~ , t ~-I:. n t ~ n e ; t o t h e ~ : o n t r o t |]l~_,di!t I.¢.~-t. th[~,:z t-q, .,J u ~ m k l , . d [o_ a l l t.ik.,es, l i n e g . a s e , ~ r b [ c a , ' h l ( , m i t r e d f r o m |IICC]i(IIII; t'Otltt'~)| ~ell~ t l l l { l t I ~ u ] ) u . t ~ . "" I llI~.~ I.Ii t) N[)l JlTId L'r~l~.[l'OI i~lL'~iia.
11; wa.~ o.t. fic.~(: no~. po~sibh', to Mmw (:ha.~ t.he prese.nce o f ~UWh~ltaq~thlone in t h e ~l)c, iiuJ~.~ wou.lct .bring a b o u t ~w.cumulatk)n o f a s e o r b i c a c i d in t h e lens; a s c o r b i e acid in t.he n~e-Iittm xx:~.~r a p i d l y o x i d i z e d b u t 1;here was a d e c r e a s e i.:'; I;hc concent-rat.ion o f b o t h aac0rbie a c i d a n d GSi:t in t h e le~,s (TM)le. V: line E). It, seeine(L po:,*.-.'ible t h a t ao sub.~t:.mt.ial a m o u n t o f d e h v d r o a . s c o r b i c acid w a s p e a e trat.ing t,t;e !en~ w i t h o u t b~ing r e d u c e d Lo a s c o r b i a acid, b u t t h i s wa.s s h o w n not, lm be ~o. T h e c o n c e n t r a t i o n o f du~hvdroa,scorbic a c i d in thes6 l e n s e s was f o t m d t o ba r~bout: O-09/_mmlea/g. o~.fiy abottb 1,5% t~ha~'of a s c o r b i c acid.. H o w e v e r , w h e n t]/e e n z y m e , c.~zca.lase w a s a d d e d t,o t h e m e d i u m t~o r e m o v e H~O~ f o r m e d [ e q u a t i o n (2)], b o t h n a . p h t h a q u i n o n e amd d i h y d r o x ~ m a p h t h ; - I e n e w e r e founcl to i n c r e a s e t h e concc.al;ration of ascorbic acid il~ the leas (Table V, lines F and G). The c o n c e n t r a t i o n of gluta.l~hione wa..,: diminished. Cata.tase alone (Table V. line .'([) ]tad no effect. The mflueace o f
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V.-~.N
}t]g?,'NIN(;EN
~-ata|ase on t.|m c:oucentr~t, ion of a.~corbie acid ill tlto lens int ulJaLc~t ill napht, hale~u.' diol was n o t s t u d i e d . H.zO,z rc~u.lily m~t,ers g]le Ien,~ (l-'irie, 1965) an~.l o w i n g t:o Lhe pres~.:nce of glu~at.hio~.: p,.~roxidaso (1_,.(..:).3. t,1) (Pirie, 19(;.'.5 .lq~oln~l)r.r,,, 196.), oxidizes GS[-I t.o (.,,~.'-C, [equa.bion (5)J" (5) 2 ,~ .....~ + ]:I ,a{'.),z- ~ C l b b ( ~ + _ ") H ° O . The loss of GSI-I fron~ th(- le~s i n , : u b a t e d in nal~I~J,rquinor~e was not, gr¢:.aBly ~ti mini.~h~-, [ when eat, alaxe was a d d e d a n d ib is t,hert:fore not: clear ]~ow |!._,O.2 prev~:nt.~ t,he l-mn~:trat,ion of dehy~lroascorbi(~ ~t(:id in ~oo t,h c tet~s. ]fea(.'tio~e. I)ettc~en tte.hydroet.scorbic acid ¢o~d le~..~" tl,.io~,s, T h e (-oncenvr~.~ion of glut~thi¢ m e i:~ d e c r e a s e d in l, h e lens of t h e napht, h:~lene-fed rabbit, ('Pat~le I.V) and il~ t, he Ir',:~ i n c u b a t e d in a m e d i u m con~ainin~ a~;cc~a'bi¢ acit{ v,.-it,h n a p h i , h a l e n e ~neEa.bolite., ( T a b l e V). T h i s is p r e s u m a b l y d u e t,o !she oxid~t, iol~ ,:~ft,hiol gq'out.~ by c!¢hyd~%~~..~corl~ic acid [equation (5)], naphtsh:~.quinone, a n d H,,()~ ( i l l t}l~; absence, of eat, alaq,~). 26
\
._g
\\ I.O
-5 \x
zt_ .......
I:t¢;. -1. l~c,aelio~n b c t w : . c ~
sk.hydroascorbie
J_ 15
~. . . . . 30 Titr,~elmir,)
!.,
,~5
acid. ~md. p r o t e i ~
I
~0
U~iol in t b c
l*:','.sc.nt'e a n d
,~h.-m~t.~: ~,f
/2] qJ~a~ h icm ~.,.
A ,lialys¢:¢l l e n s e x t r a c t w a ~ J n c u b o . t e d a t p H 7"0 a m l 3 7 : w ~ t h d e h y d r o a ~ c . o r b i ~ a c i d (4 t~m~,lt~'/'~J) ~,'ith atlt] x~'Jil~<~x~ba d d e d (:St-I. S a m p l e s w e r e w i t b d r ~ w n at. int,~r~'Ms a n d a s s a y e d For t h i o i a , d a ~ c o r b l c acid. I .... I, Glutathi,.mc; & ....... ~ . i)rot~:in tl)iol : i t l c u b t ~ t i o n irt tht'.p r e s e n e , : o f (.;,%F! ; i~- . . . . . . . . . . "' pmtei, bhicA; • . . . . . . . . . . . . O . a s e o r b i c ~mitl; i n t ' u l ) ~ t t i o n itt tim ~ - b ~ e ~ c c o f ( I S i ' I .
To t i n d out, if' dehydi, o~scorhic a.cid also oxidizes prol~ein t.h.iol, a dia.lyscd lens cxt.ra~.-t, was d.ivi~te.d i n t o t w o pargs, go on~ of whieh w a s a.dded. GSt~., at) :~pproxinmgely its coneentrat.ion be,fore dialysis. Bot, h part,.s were i n c u b a t e d ~dt,tl d e h y d r o a s c o r b i c acid. F i g u r e 4 s}mws ~ h a t dehyc!roascorbic acid r e a c t s t>ref~rm~t, ially wigh ..¢,_~.~.. p r e s e n c e of G S H , p r o t e i n t,hiol is n o r oxidized, bu~ in its absence, p r o t e i n tAm)l i~ q u i t e r a p i d l y oxidized, a n d t,he ~,qe.o,-hic acid f o r m e d aloe:: 1 h r (1.06 p.moles) is a.bout. ha.If t,he amoanf, of l~hiol r e m o v e d (2-06/xmoles) as expecIsed, f r o m equagion (5). P r e s , n 3 a b l y in t h e s e n e a r l y n o r m a l le~ases of na!.~hghalene-fed r a b b i t s (TaMe 1[I) t t.~¢.~ i)reseuce, of ~ S l t prot.eel~s p r o t e i n ~hiol £rom oxidal~ion, i n t,}~e s e v e r e l y eal~aractous b r o w n l e n s (van ]:{e3qaingen a n d Pirie, 1967; I3,ees a n d Pirie, t.967), however, tltc t.hiol g r o u p of t~ro,x..m ~ ~" ma, y be oxidized h y dehs,. dLroa.scorbie acid.. a.s well a.-; c o m b i n i n g wit, h naphl~haquinone. T h e conetu.sio~ t,ha~ t;he p r o t e i n ghio! of ghr~' lerls ~sppears r.o r e d u c e dehyd_roa.seorbie acid h a s been r e a c h e d by o~hers (Glostcr, 1956" J~amghant a n d ]:f.eald, 1956), b u t u n t i l i' r e c e n t l y (Ltlma.n, 1959) t h e r e t~as not, been a convenient,, reliaMe met, h o d or a s s a y i n g p r o t e i n thio].
A,qC¢~i:I;[C ACID IN 'I'HE IIAI~BI~T I(,EN,q. [
47
4. D i s c a s s i o n
Thot'~ at,: , few r e p o r t s in bhe l i t e r a t u r e ot" t,he mea.~urement of ~he asc.-orbic ~cid q.~n~tenL, of t h e lens of t h e naI~hthMe.ne-|i,~d rabl.~it, l ) u k e - I g l d e r (195,/) summarize.~, " . . . ib i.~ i,~erasf, ing thal~ a loss of v i t a m i n ~2 is a l a t e p h e n o m e n o n in napht.halene c.-, ta r;ic:t.". Nordnl:tun (1954.) qa.ys t h a t x:itamin C ,_lecrease~ in the nciueoas fm~,'~ottr ,. f~:w hours a ft:er n a t ) h t h a l e n e feeding, b u t is unoha~Jgec~ in t h e lens ; it decre:tsexs slowly, fr,,,~ t-he" fir.~t .~i~n of o a t a r a o t , a n d f i n a g y t h e value." are vor~- levy. I h a v e found no ;',,},r,rt of :m increase in lens aseorlfic aoM ;ts r~ result of rmpht.haletle t~e(ling. It. sho-uM he. stre.ss~;r[, however., tha.t n o n e of the lenses of t h e l m p h t h a [ e n e - f e d ,-,t,bit.~ exa~,~ine([ it, tshis p a p e r .,vo..l-O. seriously eat.aractous. S o m e h a d w a t e r eleft~s :~romM n,r,at, of tim l,eril~h-.ry, some h a d a few w a t e r ele.fg.~, aubeap.~ular vacuoles: or ,,I }~:~"sfigh t eFm t~e.~, ;~nd ~he re.~; a p pea red qlzite normal..-Lscorbio acid is n o t at, a h i g h c-~mr:entratiop, in the severely ( : a t a r a c t o u s aitd therefore p r o b a b l y " l e a k ) - " lens. [n t:.J,,, acj~,.e.ol~:.
T h i s is t h e o n l y report, of a n increase in concenbra¢ion of ascorbic aoid in t h e Ien~ as ~.~.r e s u l t of a d m i n i s t r a t i o n t o I:he a m m M of a. foreign substance. So f a r i t h a s ordy ~,een brought, abcm~ in x-ire b y naphVhalene~ feediug or b y a p p l y i n g naphl)halerm diet. ,[irectlv to ghe eye in t h e form of eye drops. D e c r e a s e in ascorbic acid h a s been rep o r t e d to occur in m~,n.v ~.vpes of cat, erect, t~hough nob usuaIIy in a. r e g u l a r or c o n s i s t e n t 1 ~1 a . l l l l e l ' . [,
_dt~tatl¢~one..l~t t h e leus of gha napI~Bha.lm~e-£e[l rabbig t h e content, rat, ion of (~rSH w~s, wit.h one e x c e p t i o n , d e c r e a s e d b y no more ~han..20°,~'~ a n d o f t e n b y m u c h le~sa (2"ab!e IV). GSSG-is for~ned in ~he lens w h e n G S H is oxidizecl w i t h d e h y d r o a s e o r b i c eriC| a n d aapht.haffoJnone. IK.,.O~, f o r m e d w h e n 1,2-dihydroxy~xaplithMer~e is s a t o x i d i z e d [ecluation (2)] oxidizes G S t f e n z y m i c a l l y [equ,n, Cion (5)]. T h e c o n c e n t r a t i o n of g l u t a t h ] o n e in ghe nou-c~.t, araei~ous ie~s is kep.e !tigh in api~e o f all c.bese oxidat;i~.-e reactions. T h e e x p l a n a t i o n is prea~ma.bly t h e presence of t h e euzy.me gtut,a~,hiozm r e d u e t a s e I
1
"
-iS
R U T t t VAN H E V N I N G B N
[equation (6)]. whi(:h, in t,he normal ~,ens. is p r o b a b l y responsil)h; fin" a ]a.rgc part, of the reoxidation of N A D P H f o r m e d in the pentose p h o s p h a t e p a t h w a y : GSSG+ NADPH+
H - - ' , 2 (l:q.'.H+ N .~,DP-%
(6)
.In the lens of the napl:,t, hMenc-thd r a b b i t , GSS()I-a-n(I nai)ht, h~.quil~one l~r¢.sltp.ta})I)" t e n , p e t e for this coenzyme. In the e x p l a n t e d lens there is a considerable lo.*s of G Sf[, Naphgh~.~qui~or,,~ (T~bte V, lines A,E,F,G) a n d H,,O.R (Table V, tim.'s A and. F) are bot, t~ a.t t'~r higher conee.ntrat.io~_~s t h a n in r i v e , a n d pencgrating the lens tenet-her wi~;h ,tehydroascorbic a.cid will ra.pidlv oxidize G S H to GSSG. N a . p h t h a q u i n o n e will also oxidize N : ~ ) P I t a n d t.hus inhit.,ig the efl;ective reoxidation of GSSG by glu~at, hi on¢~ reduc.base [equation (6)11- .In t.ta: absence of t h e quinone, a n d of H,,O,,, del~ydroascorbie acid penebrat, es t.hc l(n~s :~n(l is reduced to aseorbic acid with no re.~ulta.~g loss of (JSH (Table V. line g ) . ACKNOWL]£DGSI.ENT$ 1 t,lmnk my co!Ice,guts i,t girls laboratory for help and m;~ay useful disuussio~t~, a,nd M ~ G. St acey a~Jd Mrs S. Wheeler for skilled tecimical a.ssist.ance. Tb.is investiga~ioa w,~s supported {in })a.rt.) by an E~trl Wilson Award ,.,f the N~tti,Jmd Comici] ~co C,)mb:a. BiiJ~dhess, Inc., New York City. If E F E R i E N C E S
Ball, E. G. (1937). J. Biol. Chem. 118, 219. I3ergmeyzr, H. IJ. (t963). in 3Iethod,~ of E~tzy~natic .4~uzlysLs p. 363. (Ed. by Bergmeyer, it. t',l, Acadelnlc Press, Leaden. .Booth, J. and ]3oyland, E. (t949). Bioche,m..1.44, 36[. Cohen, P. P. (1949). In -]tanometric Tec~niqae,~' a~d Ti.s~'uc ~lletaboli~m, p. ~19. (Ed. hy Umbreit, W. ~,V.: :Burris, R. H. mxd Stauffer, J. F.). Burgess :Pub]ishing Co., M.in.neapolis. TJuke-EIder, ~:V. S. (1954). In Text Book of ()phllutlmogogy VI, p. ~iS06. Htmry Kimpto~. I.ondolL E]hnan, G. L. (1959). Arch. Bioche~t. t3iophys. 82, 70. G]oster, J. {1956). ]Jrit. J. OphthatmoL 40, 536. van 14eyningen, [l,. (1965). ]tbod,,cm. J. 96, 419. van tteyni~gen, ~. and Pirie, A- (1967). Biochem. J. 102, 842. Holmberg, N. J. (1968). Exptl Eye P,es. 7, 570. Hughes, R. E, (1964). Nature 203, 1068. Ki~mey, V. E. (t947). Am. J. Ophthalmot. 30, 1202, Kiuchi, K. (t959}. 5'hikoku, Aaa Med. 14, I1O. .. :LmLgham, M. E, amt Heald, K. (I956), Biochem. J. 73~ 52. Mar~in, G. g. (1961). Anu. N. ):, Acad. Svg. 92, 141. Mcrriam, F. C. a.nd Kirmey, V. E. (1950), Arch. Opht]t~l~n~*. 4,3, 979. Nordmann, J. (1954). In Bioloffie du Cri.~ta31in. P. Massou et, ~.~e, P~ris. Patterson, J. W. and Mastin, D. W. (195!). Am. J. Physivl. 167, 119, Pirie.. A. (1965). Biochem. J. 96, 224. Bfiih~, N. (1956), .Acla .Physiol. SextTut. 36, 291. .Ree~, J. ~. am:t Pirie, A, (1967). Bioche.m.J. 102, 853. Roe, J. ]-I. (1954), in Method.* oj' BiochemiclH Analysis l, 127. (Ed. by (Jlick, D. F,.). ]nterscience Publisher~ Inc., New York. Takagi, Y. and Okumura, It. (i961). Acta Sc~.,. Ophtlmlnwl. ,]a~. 65; 11.62. a;Vachtl, C. az~d Kin.sey, V. E, (t958). Am. J. OpMh,:dmol. 46, (5), part IT, p. 288.
A s c 0 r b i c A c i d in the L e n s o f the N a p h t h a l e n e - f e d ]{~;'r}l
\-A.N
Rabbit
}II.:YX~Xt:l.:x
.\' ,(#i,:]d i.,,bur,,,,~<,r.q ,,/" O]:hth,,I.,,~¢)lo:l.q, lY~,i,:,:r.~'3# of ()_@,rd, O.,f,,,.,l,
1(,, !/la,,d
( tlecei,:ed 20 ./~, rw: 1969, Londo,~ ) Id" the lel'l~ o1" t he rabbit, fed n~q,hl.hale~m (1 g / k g I,ody wcight/d~Ly } ,tov.~ n{,~ t)i.[ ()ill|* .-.'~,ver~;ly ~:~tara.e~)u.,, th,: cr~lt<:t.rttrtt~icnl, o:[" a.scorhi(: "~t:irl withiz~ it t.r'nds |~, in,.'rt:a~ pcogre.~sivL..ly w i l h t i m e . . i n v i t r o the.rt: i.q a n ineres~s,.: in t h e as,.orb;~.: acid ~:~meentr~ttior~ o f a h-n:~ int:ubals~(I ia at Jnr:,linw, cow,raining aseorbi~; a.~:{d ;tt)¢t t . 2 - d i h v d r o 1.2-dihy,lro_~y n a l ) h t h a l e n c {rmphtha.lcne
1. lntroduct.ion
(.!at:~ract~ in t.be nai)htAw.lenc-I'ed rabbit, is causF~(l by t~h~: })re~m~cc ~t" ~mpl~thal.~-Jl~. derival;ives in t}lC viGl'~.ous H.Il(} {I.qIICOIiS ]mm¢mr~ (Vail }[eyr~i])g(.~rt att,[ Pivi,'. t9~;7). O n e o f ~}]es,~ m e t ~ , b o l i t e s is na.j~hrh;~len,~ r.lio], i . e . t . 2 - d i h y d r o 1.2-dihy~h'oxy;~aphth%. l e n t [.F/~. l ( a ) ] ; 1;}li.s is o x i ~ l J z e d b y a.~ c;r~zvmr~, t.:~tzecbot r e d . u c t ; a s e l , r e . ~ c r i t in t.he l e n s ~tn(t or, h e r l ; i s s u e s t.d" ~,}~e ¢~ye, a n d N A t ) P ' * to g~',e "
([,;.f2. I . 3 . 1 . 5 )
N A I ) P . K a~,{ t , 2 - d i h y d r o x y n a p h t h M c n ( ; [.Fig. l(b)] [eq,.lat, io.n (1-)]; this co:~}prm.nd is rein'lily :-~utoo x i d i z e d al)n,.ui,rat p]"[ to yJe.id 1,2-napl~g}t,'.~.quinorm [.l"ig. l(c)] ar~,! Iii.,() z I)-quatkm (2)]. O t h e r met, ahoJitcs of" I|~|.][)}lt;]'l'-.l](:l)l? I£11,'5{) "f:aOt) ]%'i[;1'1 ('!]Z~'III(;.% i]'l I~}|(, "VII t<) -(tivl' 1,2-,lihy,troxyn~"pht.h a.hme al3~I thcn,;e 1,2-ha phthaqui,)o|~,~. "
N a.pbthaIen..-; (Uol + N-YJ)P v-,Mihyd.:'oxvm~pht.hMcc, e 4- XA 1)PI I-~- t { " I )i h y d r o x y
nIapIltohalc n (~-t- (.3,,--.- i n t l , i l t h o . ( t l l ilion('. -~- I-I .,( ).,. HO
(e}
H
OH
(b)
( 1) (.'))
0
~e)
3-'~,':. 1. l~h)nmdnc ,>f n~lal,~lic prodm:ts of n~phthalen~, rne¢tsb~,llszn. {a) t.2-l)~hydr,:,l,2-dilJs'dr~xymxithtl~ah,ne (naphtha|tm~.~ dio[), (b) J.2.dihydro×ynrq)hthaJeltc; (c) 1 , 2 - n o p b t b a q u i n o n c .
In ~,Lc pre~ence of asc'orl~ic acid the R)Howing fm'~hct- react,ion l.a, kes place [equatiou
1-3)1 Naph/Am quinor,<_'- -i- a.scor|m te.--~d ihyd r o x s n m p h t h a l e n e + d e h y d r o a ~ , ' o r bar~.
(3)
* A bb~ e v i a t i o n ~ ]]se~:l : ,%8 I|,. g.I u t a t h ioxmc : (;SSt-I. ,Jzitlized glutat.h ioJ~e I X.-k I) P.+ o.i M X A t ) P H, ozkli>'ed ~n:d r e d u c e d e,:,cuzyme~ nicot.inami:h: adcniv.c ¢]iuuch.~t.ide pho.'.,.ph;:tt,c.
3S
A,-;+'()['.I'~I( • A t ' I t )
IN " i ' I I F :
ItAI>,V, TT
I.b;Ns.
[
,"¢.q
\V,. h a v o .-+}jov.'n tl,.'t t. the.'~e r e a c t i o n s p r o t ~ a b l y t a k e p l a c e in I:},e e y e o f t,h e naphthah.~J,.*5",l r+t},bil ( v a n l l e v t , ir,g<:n a n d ['iri,.,. I.q~iT), :~nd t h;tt t h e (.t+.~(:ro:lsc0 c n n c e r ~ . r a t . i c ) u - f ;~::+,+n-hio ~,+'i+l i~, *:lw a+iu,:+,us nt,
2. ~ l a t e r i a l s m~d N l e t h o d s I>u~.h ~:n,i a ! t , i t , , r.~hl,ir., u:,u~{fv 3 e'; ~v.mth:~ obl, wore u.~::d. 3Iethod.s o f (t~:~,~g~.: w i t h ~.~:,fdtth;,l,*r.,,:-;,lJ,l .x.,~.i~ut~i<,,~ <,f rim ev'e lta\'e b e e n ,ie'..:rilw,l p r e v i o u s l y ( v a n Heyninge+~ .+~'.,l .~'ir},:. l'.~1;7). 31o:~t , , f , hr: l,..r,:~es ,ts,..,l i~, tbe e x p e r i m e n t s d e s c r i b e d in t h i s p a p e r had n,) visible ,'ba,xzes, +,;" +,M'." ~,6ni iI~.] ,-I,.n.u,*.5 (...u+:h as ~+ few w a t e r ¢.-.Jeft.:~at. t h e p e r i p h e r y o r n t~w s u b e a p s u l a r v.:.+ :~o]eW. :\ b:w ha+l w~,t,r ,:left* all roilt)(1 tile p e r i p h e r y . I.+qt.~; ~t~t,t ~tq~teot~.s h u u l o u r were ¢tl~alysed i m m e , | i a t e l v a f t e r t h e r a b b i t w a s killed. - r ;it,.,." wet+ + ,.t,,re, I ;,t --':).0 ' u n t i l r e q u i r e d . \ V e i g h e d l e n s e s w e r e g r o u n d w i t h ,3 n d !.0°.{~ ( w / v ) :r;~+h]~wOa<:etir; ae.i,t: a f t e r ch;.llin.<_,, for 1 hr, prot.ein w a s r e m o v e d front t h e e x t r a c t b y :-*-td;ril',l~uti<,n. "I'o tht~ :,,lue,~u~ h u m , m r an e q u a l votum,- of t h e trichloroaeet.ie, acid= ~olu+ I r,,:j Was zld..l+'*J ;tti¢l tim pe,+cipit;,te, if +,nv. w a s r e m o v e d b y c e n t r i f u g a t i o n . /,,c,,b~lio~ c,f r u b h i ! le~,.~ T h e m e d i u n t wa.~ K r e b s ~ i n g e r ] ' h o s p h a t e , p H 7.(t ( C o h e n , 1949), c o n t a i n i n g g l u c o s e ( a p .l.u. .' w / v' • ~.e. " 5.+5 m:,~'~, ~,nd a s ¢ o r b a t e (20 ~! o£0"o ~t/lO ml m e d i u m ; i.e. 1-0 m~t). l n c u b a ti,,zt.~, we£-o Jt~ air, .'.it LI4'+. J:x |ells i l l e u b a t i o n ~ube8 ( 3 1 e r r i a m a n d K i n s e v : 1950; v a n f i e y n m g e u . . i <..~f.;.'.3)."{'e~t ml. o f ~,v.dium, e o,~tah+ting 3-0 m.~r n a p h t h a l e n e dioi or 0 . 0 5 - 0 - t ~,l:,~ , u + p h t h a q u i a o n e w h e n a i ~ p r o p r i a t e , w e r e p u t in t h e i n c u b a t i o n t u b e s in t h e w a t e r - b a t . h . T h e rM~bi;, wa.~s kil]e+l ,, rJd e a c h lens s l i p p e d {nto it.~ p r e p a r e d t u b e . S a m p l e s o f e u e h i r m u b n tJ<~it t)~c,!itll,t (7~0 I.d) v.'eV~: w i t h d r ~ t w n a t tntecx, nls o f 1.5 o r 30 rain a n d a,~sayed fl~r a s c o r b i e -oh}. l.,~ t h e ,:once~,tr:~.iol~ h a d f a i l e n a p p r e c i a b l y a c a l c u l a t e d v o l u m e o f 0-5 .',~ a s c o r b a t e w a ~ ; u b l e d t~, b r i n g t h e level b a c k to ~ , p p r o x i m a t e I y 1-0 m ~ . A t the. e n d of t h e e x p e r i t n e . n t t h e ]et~aes. w e r e r e m o v e d , r i n s e d b r i e f l y with_ d i s t i l l e d w a t e r a n d a d d e d t o a w e i g h e d t u b e ,-:~,nt;,i,,in~ 3 f) ml Jl)'}.~, ( w / v ) trichloroa.cet.ic..mht. T h e t u b e w a s r e - w e i g h e d an,1 t h e w e i g h t +-~f t h e len-.~ ~+as obt, a i n e d b y d i f f e r e n c e . T h e ie~,~es w e r e d i s i n t e g . r a t e , t w i t h a g l a s s rod a n d t h e m i x t u r e c h i i l e d for about- I h r ; t h e p r o t e i u w a s zh.en r e m o v e d h y c . e n t r i f u g a t i o n and. lho e x t r a c t ; , s s a y e ( l t ~ r a s c o r b i c a v i d a n d (.,+'5}[. l)eh3"droa.sc
A s c o r b i e ac.id wa.,:-det:ermi he,.1 b y ~it,rat.ion w i t h +2,6 .dichloropheu.ol ind<.~pkenol a m t t o t M a,~_corbic ueid b y t h e m e t h o d o f t~oe {I954). Dehyetro~,+corbic: a c i d w a s tak.er~ to be t l m d i f f e r e n c e b e t w e e u t h e s e t w o mea.~urement,~. T h i o t g r o u p s w e r e d e . t e r m h ~ e d h y t h e r n e t k o d of E l | m a n (I9~9), T o t a l thiol w a s a ~ a y e d in a lens e x t r a c t , G S H in the s u p e r n a t a n t after r e m o v a l of protein x~fit.h trichloro,qcetic acid, a n d proteiT~ ttdo| w a s taker, as the ddfferet~ce
40
ItUTII
}mt, wvm~ th~..so t w o v a l u t ' : . L ( _ ' ~ ( . I it t e d t u t l , a ~ e ( l ~ . . "~,n t t y "~0.". t, 1BG,3). •
",
VAN
H I':VN]NGI~N
xv:ts a s , , , a ) ' e d spectv,>t>h, to~netrically.',
u,~:irw, g h t l . a l l t i ~ , t t u
'
.llaler&~L~" ? 1,2-]-)ihyqtm 1..2-dihy, t r o x y n . ' t l d v l h a h ' n c 'a'.s a ,~it't fr'-.: Ih,ofe~s,-" Sir ].,,var~ .t,mv~ . i ~ l 1,2-naphthaq.ui:uone f r o n l 3Ir.'~ ,I. I h ~ e s . [ ) e ] t ' , . ' d r ~ v l . q c , ) r b i c il,.:id (()'-~ .'a) "~v~ls n l n ~ l t ' ]~v ,,.,,:hlizi~.,_*, amcorbic a c i d w i t h b r o m i n e , as f,,}hm's. As,.:orbic ~cid (|71i mlZ) wa,~ diss, Av,.'d i~ 2 mi waL,-r in ~t~ i,~e-bat.h, in a f u m e c . u p b o a r d . B r o m i n e w a s ~,,Ided ( a b o u t . 0-06 mI) m:l ii ..,. f a i n t y e l h , w ,.-,d,,ur p e r s i s t e d ; t.his w;,s r e m o v e d b y a d d i n g a. s p e c k o f as(:orbic ~,cid a~ul N z w a s ther, b u b b l e d tdn'ough the .solution f o r s e v e r a l ~ a i n u t e s to r e m o v e all t.races o f I~r,m~iue. I t w , ~ f r e n h l y p r e p q r e d for e , ch e.xpet'iutent .Ascorhic a c i d . 0-5 .~. n,:ut.rallzmt l o nh¢,~lt p H I~-:-' ','¢;th N a O f t a n d kept, iJt ice, w a s f r e s h l y lw,gpar~d, l',n" e:~,.b exp,eri ~ eul..
Ile~tct io~, between deh~droascorbic acid . m l tAiol,~ t~ovine l e n s w a s u s e d for t h i s e x p e r i m e n t . F o u r l e n s e s (~ ~) w e r e g m n n , [ ~,.'i~ h t 6 ml ,,f 0-1 3~ p h o s p h a t e bufthr, p t i 7,,1, i n s o l u b l e , p r o t e i n w~s r e m o v e d b y ceutrifug',tt, ion anti t h e e x t r a c t , w a s t,h e n d i M ) , s e d f o r ,3 da.ys a.t, .I ° a.g~inst dist.iiled w a t e r . T h e w;~t.e:" w a s s t i r r e d c o n t , i m m u s l y a n d c.hanged t w i c e d a i l y . T h e c o n t e n t s o f t,he dia.iv,ds s a c w e r e f r o z e n for 2 day,~: t,h e u t h a w e d a n d d.iluto.d w i t h a.n e q u a l w ) l u m e o f 0,1 .~r p h o s p h a t e b u l ] ' e r p}:I 7"0. T o 10 ml o f tills 1 hi 6 len~ e x t r a c t , w a s a d d e d 4-,t m g of t381-1 to ~ix, t: a t:oucenLratio:~ ~ t 1-46 re.M. ,.Samples w e r e w i t ~ h d r a w n f r o m t h e lens v,xtract.s, wit,It o r w i t h t m t adde~l G,%11, f o r a s s a y o.f iuitia.1 p r o t e i n a.nd GS.H t h i o l ; t o 5 :hi o f e a c h , Ixg 8 7 °, w a s a , b l e d 0,04 ml o f 0"g~ 51 D H A a n d nnd
ascorbic
samples
were withdra.wrt
at intcrvMs
over
the next
hour
for assay
o f tt:i~fl
acid.
3, R e s u l t s 1,1, "C'il.*.O exj;le, r4~itc gtl.'3
..tscorbic acid. in ,zqueo~l.s ]~u.'mou," ~o~,.;.len..s. I a b l e ] c o m p a r e s
l.he c o i J t e n t . r a t.icm ,,t"
,E/][,ct q/" .n.aphthaleneJ;.~edi.,.y on the conce'm.rution o f a,;'e,~rbic acM it~ the. aqueous h-~.~n~.ou.r o f zhe rabbit _-%s¢orbio acid in a q u e o u s (/ztnole~/re.i)
J(a.bbit, ,'to. 43 i t2* 32 "2 33 29* 3;5* 36 30" ] 61"
14
No. of do~'s o f na.ph t t~:tiene
hulnom
% 2-Naphruh alenc- fed
C,~l-tt'v~l
di ll'e ten<',- - ,~3 -- ~J -4S - - 77 --72
-2
0.' 7
t .02
'2
0-16
t . I ;3
,'3
1-02
] .96
,'~ 5 9 9 10 I0 15 23 28
0-25 0-45 0-3I 0-.18 | -36 0,99 .t .47 0.23 1"99
1 -I 0 1-59 0-77 1.07
] -!N) 1-90
--60
--- 55 --- 28
:?.,I S
-- 4 S -- 3:;
0.94 1"[4
---"76 "~ 75
."ffaphthaleno (J g ] k g b o d y wt,) w a s a d m i n i s t e r e d d ~ i J y . E a c h c o m p ~ r i B o n is b e t w e e n ]itterm~te.*. * W ~ t e r ¢teft.q a r o u n d 7periphery o f l e n s ; i u t h e r e m a i n d e r t i l e lene.ez s h o w e d n o , o r m i n i m a l c h a n g e . "~ l'rotcin in a q u e o u s
burnout, scvero cataract.
..k,~(.'(~]ll~[(" ACID
1N
'I'lll,3
ILABI~IT
LI,:N,q,
I
41
~t.~t:ort)i(:. ~tt:id i~ th,; aqtteous h u m o u r of t h e mq.)]~tlta.lenc-fed rabl)it; with tha.~ :)f ,m u~Crca.t(41 l i ~ c r m a t c ' . The. n o r m a l va.ltlcs r a n g e fronl 0-77 be 2.18 ~ m o l c s / m l 0rye.rag0 1 "35 I.tl~O]O.~/~)ll). 3~Vit]t 0110 ~tXCCl)tion tht: eonce~,tratiou in t;lm ~ q u c o u s l m m o u r o f t.'he ~,,ld, t,hale~w-fed r a b b i t was low(.:r th,'.t~ tl).a.~ in t.hc tlllt;rcltted r:~.bbit., t l o w o v e r , ~,.fte.r t,h," :~oeoa~d du.~o, t h e d iff(,rerw.c betw,,~:~ the. ]evel.~ in t h e t,re~O.t:(l at~(t re)treater[ :~ninmls (,,.~
200
7" 160
og g~u
L
>I.
-¢)¢*
~e-~
t
L
~
~
.," ~ . . . . . ~ £ . . _
4
~
__ ~
I~
16
!
I
I
20
Z4
~8
F.:~.;. 2. ,~,sc,.~z'bic a c i d i,t th(~ aClt~t;ou~ t~tmto(zt" o J" tire. J:ai~J~thale,~(> fed vttbbit,. ~ ' h o j)erce~xtag~, d i l t b r e t t c e I~('t..~vc*.'n t h e c(,z)cet~tcat o:) o f a s c o r h i c a c i t | ia~ t h e ;tqttt:otl.'~ hlllilo~.lt" o f t~|)o n,~pht.hale.ste.fed r a b b i t , a.~d "that i,~ a tittc~'ma.¢(~ c¢.qlt r~.A. i8 I.dCH.~(-d ' a g ~ i n s t I,he. n u m b e J - o f daJl S d o s ~ s o f Z'ml~/~thah-t~t,.
t.c,~ded t o ('~ecrea.ge as the. amYJl.)cr of (loses incrca.~c.d (Fig. 2); t h e c o n c e n t l ' a t i o ~ el~ as(:orbic acid i~ t.ljc ~queotls h u m o m " of a r~tbbil; w h i c h h a d h a d 28 doses was h i g h e r t,|,an t ha~. in t h e control. T h e o~e v~luc w h i c h doc~ no$ fit, iu~o t,his p a t t e r n , - 7 6 % ha r~;[)bit no. 16 a,fter 23 (loses, was o b t a i n e d t'roJlz ~13 a q u e o u s ] m m o u r wh-k3h in v i v o .~howed ~i, srxot~g flare. Secretion of 9,scorbic acid by vi~e ciliary b ~ l y m a y the, r0fore )~ aI X" [~ 'tj(,!I':l'l i I ' ~ l . p a i r e d . '.F.s ~, ~.~: I J.
..~.,'co,'bic e~cid its. tl~e .ser~tm
of a..na,19hthalenc.fe'd,
N~). oE dcy,,:~_.s
A,-~eorbi(: zteid (/~tlotes/nal)
(t
0.053
3
O'Ofi~3
9
0.108
13
0-1.20
rabbit
,t R~l)bit, no. 4 5 - - n a p h t h a l e . n o
(1 g / k g ) ~:as a d m i n i s t ¢ : r e d d a i l y .
Since tl:ere is s o m e e v i d e n c e t h a b ~jay,hvhalene-i%eding brings a b o u t a~ i n c r e a s e in t h e concentra, t i o n o f conjuga,ted g ! u c u r o n i c acids in t h e b l o o d a n d in t h e a q u e o a s a n d v i t r e o u s h.mnours (T.akagi a n d Ok-0mura, ] 9 6 1 ; K i u c h i , 1959), t h e g r a d u a l re~Ltrn ~o
4~
I(UTH
VAN
ttEYN/NGEN
n o r m a l v a l u e s with increw~iug mm~bcrs of (loses of nal~h~lmlenc ('C~,.Me [ a n d Fig. 2) m a y bc because less unconjugat:ed m e t a b o l i c product,s of ![ILtph0h.l]..[i3[l[2 I'(~1.t.'[~.t']l(? ~d}'t'. Dctoxica~ion b y i ltcrease(.[ s y n t l , e s i s of gLucuronidcs is o f t e n a.ecompan [t'.d by increase,[ s y n t h e s i s of ascorbic acid ~nd. in a single experin~eng, t|~i,s was ,sltown co (l~-.(21.ll" i.t}. ,~aphthMe.ne feeding. Blood, w i t h d r a w n .i'r<>~n it m~pitt.hatene-fed r a b b i t a~ ;n~e~vul.-, was a s s a y e d fi)r t o t a l ascori,ie ~ci~t by l;he ,~,e0u)d o f Roe (195d:). T a b l e .[1 .~l.mw.~ tirol. over v, p e r i o d of L3 d a y s , tho (:7)]ttJetltril,15iott it~creased r a t h e r more t b a n twoiiHd. Th,, aJnou~)t of aseorbic acid secrt,,l.ed into the a q u e o u s httrnOltr would g[t,:t'eI~re ~i[s() i~r i n c r e a s e d ( K i n s e y , t947). Fom~m.t.ion of glucuvonides m0-y therefore act. in two w a y s ~o b r i n g a-bout a. g r a d u a l increase i~t t h e ascorhic acid of ~cqucous tttttnou.r of ~;he ,'~d~bit, dose(.[ d a i l y w i t h naplatha lene : (1) b y p a r t i a l r e m o v a l of goxic napl~thMcne ~ teta boli ~e~ ; a n d (2) b y i n c r e a s e 4 synthe.~i,5 o[" a,';cot'l.'ic :tcitl. Tam.t.: Li L
f'~'ec't of "nap/d/~ale~,.e-feedim/ o,~. /he ('onve.atratio~t qf u:,c.,,t'bic ,w?4 ,-r~ tim/era,. ~.f the .~',¢L5;!
.Rabt,iL rio+
I 1 "2* 32 '2 31 * l:l* 39* 2 9 ~" 2l 33 :$5*:36 30* l[ 4
No. u|" d,~u's of naphthalrplt.
"2 13 :g 5 5 8 ~ 9 i) 9 10 10 15 2N
Ae~,otbie a c i d in Ics~.~ (/~. u,h,s/,,).. i\'~tpt~tha ten,: "~, -fed ( ' o n t vo! diJl~'t'euce
i • 14 2- 7 4 ! <16 l .7ti 3-'2:2 2.40 2-97 2.42 2..92 1.6(1 ;I,7i.I 3.01 1-2!~ 4.55
1-~ 4 1- 2 7 i" t 9 1-25 1-1,5 t .26 1-54 1.6~ I 61 I- ! 6 1.19 l -[,() 1 -Y,f* I ,7{J
0 -.'-- .[ I ~J -,- 1 ,, -i-4 t -l-lbl~ -.i. ;~ l .~- 9 3 .1-44 -i- ~.l -i. 4 3 -!-211 -l- 1 5 0 ~ 9 - " t 5N
N ' a p h t h a l e i m (I g / k g b o d y "~vt) w~uu a d m i n i s t e r e d d a i l y . 1,3acl~ e o m i ? a r i s o u is b e t w e e n lit.termal.es. * %ratec e i e f t s a r o u n d p e v i p h e t . y o f l e n s ; t.J~e o t h e r l e n s e s s ho~red rto, or m i n i m a l cl~ai~,._,c~.
Table. Ill. compm:es t}m conee.utrgtion o~Faseorbio acid in the leas of ~he txaphtlaa]e~"lefed r a b b i t w i t h t h a t of its m~treated l i t t e r mate. W h e r e a s there is a. deficit of ascorbic acid in t h e a q u e o u s hmnou.r of t,hc n a p h t h a l e n e - f e d r a b b i t (Table I a.nd F i g . 2) in the lens ascorbic acid accumtflates. T h e a v e r a g e value f0r t h e ascorbic acid in t h e 14 n o r m a l tenses is 1-37/xmoles/g (range 1-14-1-76). Irt t h e 14 lenue~ of the n aph~lm,leilel e d r a b b i t s the a v e r a g e value is 2-37 (range 1-14:-4.55). T h e r e was a t e n d e n c y for tile c o n c e n t r a t i o n in t h e lens of t h e trea.ted r a b b i t , c o m p a r e d wiga t h e u n t r e a t e d , to i n c r e a s e w i t h t h e n u m b e r of doses. (The lens of r ~ b b i t no. 30, fed 1.5 ¢~ys, is a m a r k e d exception.) A clear-eu~ difference betweet~ t-he e y e of t h e n:~plathaiene-fed a n d the c o n t r o l r a b b it is s h o w n in Fig. 3. I n t h e n o r m a l eye t h e coneentrai;imt of aseorbie acid in t h e tens v a r i e s f r o m 1.14 to 1-76 (av. 1-335), while the c o n c e n t r a t i o n i n t h e a q u e o u s h m n o u r
:k ,'-;(.' (~l }-.' Jll(.J . " ~ ( ' l l )
~N ' l ' I ) l ' ;
l l . A ] 3 b I"~ l '
I , E N~, . .",q
-13
I
.raHges t'ro~ 0.77 to :2,[8 (av. 1-32ix.moles/m] 1. .|u ~,he lens of ~he. napl~thah.'~c-fcd ra|,bil) em)ccnbr~.t, ions of ~sm:~rbic a.cid whic'l~ fall w i t h i n the ]mrmal range 0.re as,so~,i,~h.:d w i t h ~ow lev~ls in t h e ~queot]s humour:~ (Fig. 3), Vaht~s for ascorbie ar.,itl in t h e a.que~m:; Inumour appron.cl~ing c~:mt.rol v~tu(:s are a.~sot~ia~t:ed wit.h elev~ted lens con lt;vls as~:orh i~: c,.~nt,J'at.io~s. ]tl t]~.: ~ocnml lt:ns bIi¢: ca gio wa.s 1"01 :t-0"12; in tim [~qtleous asco~:[)i~ nzilfl~t.h~h.~c-fcd r a b b i t !,he ratio wa.s 4,22~:0-7"5..ln ca.leuhtting ~she re.tic for t.ho iml)ht.hah;H~M'~d ra.bbit,, one vatuc w h i c h fa.]ls f~u' .fi'on~ the or, hers a.ntt a m o n g those i'r(ml e.he ,.,yes of m)r~na] r a b b i t s Jm.s been omi~t, ed. I~ c o r r e s p o n d s ~;o r~~bbi~ no. 30 in whic"h !.he. v~]ue for ]en..~ ;~scorbic acid w a s excep~}onally low (Pa ~1(.. l I l ) .
0
--.1.. 2 -o L3
0
'3'
.1. !
~ R Lens o s c o r b i c
i 3
-
.J 4
_J
acid (raM }
F~.;. :.~..L/~'k~t i,~,s!~ip bvt.we,.~n t h e c ~ m e c - n t r a ~ i o n o f a s e o r b i e ,~cid i~ t h e l e n s at~d in t h e a q u , - ~ m s h u m o ~ a r , ,1' : h ~: cxc..-, ,_,f ~m p h 1.1~! e , e- ~k.d a~ t~ unt~'ea~ e d r a b b i t s . O , U'~t.ru~tted r,-tbbi~s. ~ , n ~'q?I~t,h a l e ~ e - f e d r a b b i t s . V a l u e s tk~r a l l eye.s i~ w h i c h a s ~ ; o r b i e a c i d w a s a~st~yed in b o t h le~m a ~ d a q u ~ t o u s h e i n o u s " a r c p l o t t e d h~ t.hi.~ l~g,m-.. 'J'h~: m t m b t : r o f d¢:~o~ o f na.phthalen~.~ r a H g c d f i ' o m 2 t~.~ 28.
T h u s in the e}:~: of tlw, n.~pht.hs..lczm-fcd r a b b i t t h e concentra.tio~ of ~.tscorbie acid in ~he .!en:~ wa-s more t h a n 4- t.imes thal; in t h e ~tqueou.-: b u r n o u t , whereas in the n o r m a l eve ~he avm:t~ge vMues for ielLS an(l ~(tue, ous h u m o u r were alnlOS~ t h e s a m e . Adm~:-~dst.ration of ~,,a.pht,t~nlene diol -i-n. the for.n~ of eye d.rojps. N a p } ~ h ~ l c n e dioI was ;~i m i n i s t e r e d in t h e form of e y e d r o p s co !,be righ~ e y e of t b u r r;.~,bbi~:s (var~ I-Iey~iingen •trtd Pirie, 1967). I n t.bree cases ghere were c~earactous changes, bu.~ t~he lens(;s were no!, a n a l y s e d . I~a t h e fourg/a ra.bbiv t h e r e were no lens changes, but, i~ !.he righg eye the tens was f o u n d to eont, a.in more ascorbic acid, ~1~d t h e ~.queous h u m o u r less a.seorbic a.eid, tha.n i~;e tmgrea~ed lefb eye. T h e values were, for t h e righ'0 a n d . l e f t lene, 2-1 a n d 1-5 m ~ a n d for bhe right; a n d l e f t a(.lueou~s humout's 1,2 a n d 1-65 rn~!. Oe.ner~iIy, o n l y r e d u c e d ascorbic acid in tlle lens wa, s measured. O n a n u m b e r of occasions, however, regal ascorbic acid w a s a s s a y e d (reduced plus d e h y d r o a s e o r b i c ~cid) a~td it- was s h o w n ~hab litNe d e h y d r o a s c o r b i e acid was pre,~en6 in t h e lens. T h i s is hog surprising, in view of ghe h i g h c o n c e n t r a t i o n of ghiol g,roup-% of g l u t a g b i o n e a n d prot, e i a in t h e lens. Glutathione. J.t; is well k~uowt~ !hag d e h y d r o a s e o r b i e acid is rat~idly xecluceXl b y gtut, a t h i o n e , wit,h t h e formagion of ascorbie acid a n d oxidized glu~a~hione [eqtmt, ion (4-)]. This rea.e~ion is n o m e n z y m i c . The~e is no evidence t h a t t;he e n z y m e w h i c h cat, a15:ses t h e react.ion in aome anima.l t~ssues (]CJ[ughes, I96-1.) is p reseng i n t h e lena (tlughe:~,
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p e r s o n a l c,.)uimm~i,:ation). I n t.he ~ b s e n c e o~" g l u t a t h i o n e , p r o t e i n thiul is .~i~ui!.'~rlv oxJtlized (see later. Fig. -i): d e h vdroa:~corba~e + 2 LI~I-L .--itsc'orl.~ tt, .4-G,";,.'SI.~.
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T a b l e .LV .-,how.-, thai;, in t h e n o n - e a t . ; m s c t o u s lens o f rite impht.lm.lcz~e-tbd r;,l~l.;it, flu. conee.ntratk,t~ o[" glttt.athione w a s n o t greati}" , { i m i t d s h e d . \ V i t h O t l e excepti,>n (r.~t)f~i.+ no. 13) rite c o n c e n t r a t i o n of' (.IS.H w a s d,'crea~v
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N~q~htl,alene {!. g l ' k g b ~ l v w t . ) w a ~ , ' . u h n i ~ i s t e r e d d : d l v . t~]ac|l c o n l p a r i s o n i~ b~,t'~vt, e n l i t t e r . ' ~ t a t < . s , * ~,V~ter elet'ts around per[pl,ery u f l e n s : t h e o t i l ( ~ r [t~tl~c.~ at~.;~'~l~,.[ ;H). ~;r I[tilti~tt:~l ch:~I/K~.'.<,
It~ r'iD'o t.J:]>uri',tent.,." I.no~tb~+tiw~ o.r the te~s ~ci~h'~:+phthale~w dt,rh'aticcs a~;d oscorbic ,.cid.. :2,) invest.iFat~,. f u r t h e r t h e i n c r e a s e in c o n c e n t r a t i o n of ascw_'bic a c i d i n t,he h;n~ of rim n.~phdtat,;u,.'fed rr~bbir, t.he p h e n o m e n o n w a s r e p r o d u c e d as far a..a. l;o~sible in v i t r o . T h e ~wo ]e,ases of a. r~-rbbil; w e r e e a c h i n o u h a t e ( t in ~L m.et[ium in whiei. %be concent;ra.t.ion of as(,orbie acid was m a i n t ~ d n e d a t t h e level of a.botz~ 1 n~.xi (see 5[ateri,ds a n d 5I~t.hod.-:) addi~iol~ o f a u a p h g h a i e n e t-[e,rivat.ive w~Js m a d e t;o ~he mediun>, in w h i c h one, o f t h e lense.~ w a : incuba.ted...-kfter some h o u r s ~-he e x p e r i m e n t was s t o p p e d a,rM l:b.e t w o 1.ense:.s ;~.~a.ved .for a s e o r b i e acid a n d glutar.hione. T a b l e V, lin.e A gi.v~-,.s t,he results of ~hese e x p e r i ment, s. "When t h e l e n s was i n c u b a t e d w i t h 3 m ~ n a . p l r t h a l e n e d i o l t h e a s c o r b i e acid i.a the .r.ffedimn was oxidized. T h e m e d i u m b e c a m e b r o w a in colour al~d. t~he s u b s t a n c e of t h e l e n s (but, aqoe t:he c a p s u l e ) also became, c o l o m ' e d . ~\r~.te~ cleft;s a p p e . a r e d at~ t h e equai~or, s i m i l a r t o t h o s e s e e n m I&e l e n s of tim n a p t & h a | e n e - f ~ :I rabbi~ i~ vivo. "rite ascorbJo a c i d con~eni; o f t,ize l e n s i n c a b a t e d in dioL i , c r e a s e d in 21[ f o u r e x p e r i m e n t s b y a n a v e r a g e o f 21°,.o, w h e r e a s t h a t o f g l u r a I s h i o u e d e c r e a s e f t go 5 0 % ( T a b l e V, line ~.). r, q~c, a n d d e h y d r o a s e o r b i e acid were a s s a y e d a w l I n o n e of t h e f o u r experiLnents tx,_~,.T b o s h w e r e fom~d t o b e M>sen~.
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C~.~-~]2:~-}[~J!lff ~|lp[tI'.'|ll~*l/t I'-LI kVC]) Ill{; ,~'CtllL't'U'~.l~il[.itlll t i t ilbf}ll¢~ I 11131 :. I;t-'.," .~,. ~("))~:1; ![.il~." Eo I S 0 t L I : l i n e 1:', 2 0 0 j ~ } ; tiP-e. G . I S 0 1 t I ; Iitu:~ G a ~ , t ~-I:. n t ~ n e ; t o t h e ~ : o n t r o t |]l~_,di!t I.¢.~-t. th[~,:z t-q, .,J u ~ m k l , . d [o_ a l l t.ik.,es, l i n e g . a s e , ~ r b [ c a , ' h l ( , m i t r e d f r o m |IICC]i(IIII; t'Otltt'~)| ~ell~ t l l l { l t I ~ u ] ) u . t ~ . "" I llI~.~ I.Ii t) N[)l JlTId L'r~l~.[l'OI i~lL'~iia.
11; wa.~ o.t. fic.~(: no~. po~sibh', to Mmw (:ha.~ t.he prese.nce o f ~UWh~ltaq~thlone in t h e ~l)c, iiuJ~.~ wou.lct .bring a b o u t ~w.cumulatk)n o f a s e o r b i c a c i d in t h e lens; a s c o r b i e acid in t.he n~e-Iittm xx:~.~r a p i d l y o x i d i z e d b u t 1;here was a d e c r e a s e i.:'; I;hc concent-rat.ion o f b o t h aac0rbie a c i d a n d GSi:t in t h e le~,s (TM)le. V: line E). It, seeine(L po:,*.-.'ible t h a t ao sub.~t:.mt.ial a m o u n t o f d e h v d r o a . s c o r b i c acid w a s p e a e trat.ing t,t;e !en~ w i t h o u t b~ing r e d u c e d Lo a s c o r b i a acid, b u t t h i s wa.s s h o w n not, lm be ~o. T h e c o n c e n t r a t i o n o f du~hvdroa,scorbic a c i d in thes6 l e n s e s was f o t m d t o ba r~bout: O-09/_mmlea/g. o~.fiy abottb 1,5% t~ha~'of a s c o r b i c acid.. H o w e v e r , w h e n t]/e e n z y m e , c.~zca.lase w a s a d d e d t,o t h e m e d i u m t~o r e m o v e H~O~ f o r m e d [ e q u a t i o n (2)], b o t h n a . p h t h a q u i n o n e amd d i h y d r o x ~ m a p h t h ; - I e n e w e r e founcl to i n c r e a s e t h e concc.al;ration of ascorbic acid il~ the leas (Table V, lines F and G). The c o n c e n t r a t i o n of gluta.l~hione wa..,: diminished. Cata.tase alone (Table V. line .'([) ]tad no effect. The mflueace o f
4G
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~-ata|ase on t.|m c:oucentr~t, ion of a.~corbie acid ill tlto lens int ulJaLc~t ill napht, hale~u.' diol was n o t s t u d i e d . H.zO,z rc~u.lily m~t,ers g]le Ien,~ (l-'irie, 1965) an~.l o w i n g t:o Lhe pres~.:nce of glu~at.hio~.: p,.~roxidaso (1_,.(..:).3. t,1) (Pirie, 19(;.'.5 .lq~oln~l)r.r,,, 196.), oxidizes GS[-I t.o (.,,~.'-C, [equa.bion (5)J" (5) 2 ,~ .....~ + ]:I ,a{'.),z- ~ C l b b ( ~ + _ ") H ° O . The loss of GSI-I fron~ th(- le~s i n , : u b a t e d in nal~I~J,rquinor~e was not, gr¢:.aBly ~ti mini.~h~-, [ when eat, alaxe was a d d e d a n d ib is t,hert:fore not: clear ]~ow |!._,O.2 prev~:nt.~ t,he l-mn~:trat,ion of dehy~lroascorbi(~ ~t(:id in ~oo t,h c tet~s. ]fea(.'tio~e. I)ettc~en tte.hydroet.scorbic acid ¢o~d le~..~" tl,.io~,s, T h e (-oncenvr~.~ion of glut~thi¢ m e i:~ d e c r e a s e d in l, h e lens of t h e napht, h:~lene-fed rabbit, ('Pat~le I.V) and il~ t, he Ir',:~ i n c u b a t e d in a m e d i u m con~ainin~ a~;cc~a'bi¢ acit{ v,.-it,h n a p h i , h a l e n e ~neEa.bolite., ( T a b l e V). T h i s is p r e s u m a b l y d u e t,o !she oxid~t, iol~ ,:~ft,hiol gq'out.~ by c!¢hyd~%~~..~corl~ic acid [equation (5)], naphtsh:~.quinone, a n d H,,()~ ( i l l t}l~; absence, of eat, alaq,~). 26
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A ,lialys¢:¢l l e n s e x t r a c t w a ~ J n c u b o . t e d a t p H 7"0 a m l 3 7 : w ~ t h d e h y d r o a ~ c . o r b i ~ a c i d (4 t~m~,lt~'/'~J) ~,'ith atlt] x~'Jil~<~x~ba d d e d (:St-I. S a m p l e s w e r e w i t b d r ~ w n at. int,~r~'Ms a n d a s s a y e d For t h i o i a , d a ~ c o r b l c acid. I .... I, Glutathi,.mc; & ....... ~ . i)rot~:in tl)iol : i t l c u b t ~ t i o n irt tht'.p r e s e n e , : o f (.;,%F! ; i~- . . . . . . . . . . "' pmtei, bhicA; • . . . . . . . . . . . . O . a s e o r b i c ~mitl; i n t ' u l ) ~ t t i o n itt tim ~ - b ~ e ~ c c o f ( I S i ' I .
To t i n d out, if' dehydi, o~scorhic a.cid also oxidizes prol~ein t.h.iol, a dia.lyscd lens cxt.ra~.-t, was d.ivi~te.d i n t o t w o pargs, go on~ of whieh w a s a.dded. GSt~., at) :~pproxinmgely its coneentrat.ion be,fore dialysis. Bot, h part,.s were i n c u b a t e d ~dt,tl d e h y d r o a s c o r b i c acid. F i g u r e 4 s}mws ~ h a t dehyc!roascorbic acid r e a c t s t>ref~rm~t, ially wigh ..¢,_~.~.. p r e s e n c e of G S H , p r o t e i n t,hiol is n o r oxidized, bu~ in its absence, p r o t e i n tAm)l i~ q u i t e r a p i d l y oxidized, a n d t,he ~,qe.o,-hic acid f o r m e d aloe:: 1 h r (1.06 p.moles) is a.bout. ha.If t,he amoanf, of l~hiol r e m o v e d (2-06/xmoles) as expecIsed, f r o m equagion (5). P r e s , n 3 a b l y in t h e s e n e a r l y n o r m a l le~ases of na!.~hghalene-fed r a b b i t s (TaMe 1[I) t t.~¢.~ i)reseuce, of ~ S l t prot.eel~s p r o t e i n ~hiol £rom oxidal~ion, i n t,}~e s e v e r e l y eal~aractous b r o w n l e n s (van ]:{e3qaingen a n d Pirie, 1967; I3,ees a n d Pirie, t.967), however, tltc t.hiol g r o u p of t~ro,x..m ~ ~" ma, y be oxidized h y dehs,. dLroa.scorbie acid.. a.s well a.-; c o m b i n i n g wit, h naphl~haquinone. T h e conetu.sio~ t,ha~ t;he p r o t e i n ghio! of ghr~' lerls ~sppears r.o r e d u c e dehyd_roa.seorbie acid h a s been r e a c h e d by o~hers (Glostcr, 1956" J~amghant a n d ]:f.eald, 1956), b u t u n t i l i' r e c e n t l y (Ltlma.n, 1959) t h e r e t~as not, been a convenient,, reliaMe met, h o d or a s s a y i n g p r o t e i n thio].
A,qC¢~i:I;[C ACID IN 'I'HE IIAI~BI~T I(,EN,q. [
47
4. D i s c a s s i o n
Thot'~ at,: , few r e p o r t s in bhe l i t e r a t u r e ot" t,he mea.~urement of ~he asc.-orbic ~cid q.~n~tenL, of t h e lens of t h e naI~hthMe.ne-|i,~d rabl.~it, l ) u k e - I g l d e r (195,/) summarize.~, " . . . ib i.~ i,~erasf, ing thal~ a loss of v i t a m i n ~2 is a l a t e p h e n o m e n o n in napht.halene c.-, ta r;ic:t.". Nordnl:tun (1954.) qa.ys t h a t x:itamin C ,_lecrease~ in the nciueoas fm~,'~ottr ,. f~:w hours a ft:er n a t ) h t h a l e n e feeding, b u t is unoha~Jgec~ in t h e lens ; it decre:tsexs slowly, fr,,,~ t-he" fir.~t .~i~n of o a t a r a o t , a n d f i n a g y t h e value." are vor~- levy. I h a v e found no ;',,},r,rt of :m increase in lens aseorlfic aoM ;ts r~ result of rmpht.haletle t~e(ling. It. sho-uM he. stre.ss~;r[, however., tha.t n o n e of the lenses of t h e l m p h t h a [ e n e - f e d ,-,t,bit.~ exa~,~ine([ it, tshis p a p e r .,vo..l-O. seriously eat.aractous. S o m e h a d w a t e r eleft~s :~romM n,r,at, of tim l,eril~h-.ry, some h a d a few w a t e r ele.fg.~, aubeap.~ular vacuoles: or ,,I }~:~"sfigh t eFm t~e.~, ;~nd ~he re.~; a p pea red qlzite normal..-Lscorbio acid is n o t at, a h i g h c-~mr:entratiop, in the severely ( : a t a r a c t o u s aitd therefore p r o b a b l y " l e a k ) - " lens. [n t:.J,,, acj~,.e.ol~:.
T h i s is t h e o n l y report, of a n increase in concenbra¢ion of ascorbic aoid in t h e Ien~ as ~.~.r e s u l t of a d m i n i s t r a t i o n t o I:he a m m M of a. foreign substance. So f a r i t h a s ordy ~,een brought, abcm~ in x-ire b y naphVhalene~ feediug or b y a p p l y i n g naphl)halerm diet. ,[irectlv to ghe eye in t h e form of eye drops. D e c r e a s e in ascorbic acid h a s been rep o r t e d to occur in m~,n.v ~.vpes of cat, erect, t~hough nob usuaIIy in a. r e g u l a r or c o n s i s t e n t 1 ~1 a . l l l l e l ' . [,
_dt~tatl¢~one..l~t t h e leus of gha napI~Bha.lm~e-£e[l rabbig t h e content, rat, ion of (~rSH w~s, wit.h one e x c e p t i o n , d e c r e a s e d b y no more ~han..20°,~'~ a n d o f t e n b y m u c h le~sa (2"ab!e IV). GSSG-is for~ned in ~he lens w h e n G S H is oxidizecl w i t h d e h y d r o a s e o r b i c eriC| a n d aapht.haffoJnone. IK.,.O~, f o r m e d w h e n 1,2-dihydroxy~xaplithMer~e is s a t o x i d i z e d [ecluation (2)] oxidizes G S t f e n z y m i c a l l y [equ,n, Cion (5)]. T h e c o n c e n t r a t i o n of g l u t a t h ] o n e in ghe nou-c~.t, araei~ous ie~s is kep.e !tigh in api~e o f all c.bese oxidat;i~.-e reactions. T h e e x p l a n a t i o n is prea~ma.bly t h e presence of t h e euzy.me gtut,a~,hiozm r e d u e t a s e I
1
"
-iS
R U T t t VAN H E V N I N G B N
[equation (6)]. whi(:h, in t,he normal ~,ens. is p r o b a b l y responsil)h; fin" a ]a.rgc part, of the reoxidation of N A D P H f o r m e d in the pentose p h o s p h a t e p a t h w a y : GSSG+ NADPH+
H - - ' , 2 (l:q.'.H+ N .~,DP-%
(6)
.In the lens of the napl:,t, hMenc-thd r a b b i t , GSS()I-a-n(I nai)ht, h~.quil~one l~r¢.sltp.ta})I)" t e n , p e t e for this coenzyme. In the e x p l a n t e d lens there is a considerable lo.*s of G Sf[, Naphgh~.~qui~or,,~ (T~bte V, lines A,E,F,G) a n d H,,O.R (Table V, tim.'s A and. F) are bot, t~ a.t t'~r higher conee.ntrat.io~_~s t h a n in r i v e , a n d pencgrating the lens tenet-her wi~;h ,tehydroascorbic a.cid will ra.pidlv oxidize G S H to GSSG. N a . p h t h a q u i n o n e will also oxidize N : ~ ) P I t a n d t.hus inhit.,ig the efl;ective reoxidation of GSSG by glu~at, hi on¢~ reduc.base [equation (6)11- .In t.ta: absence of t h e quinone, a n d of H,,O,,, del~ydroascorbie acid penebrat, es t.hc l(n~s :~n(l is reduced to aseorbic acid with no re.~ulta.~g loss of (JSH (Table V. line g ) . ACKNOWL]£DGSI.ENT$ 1 t,lmnk my co!Ice,guts i,t girls laboratory for help and m;~ay useful disuussio~t~, a,nd M ~ G. St acey a~Jd Mrs S. Wheeler for skilled tecimical a.ssist.ance. Tb.is investiga~ioa w,~s supported {in })a.rt.) by an E~trl Wilson Award ,.,f the N~tti,Jmd Comici] ~co C,)mb:a. BiiJ~dhess, Inc., New York City. If E F E R i E N C E S
Ball, E. G. (1937). J. Biol. Chem. 118, 219. I3ergmeyzr, H. IJ. (t963). in 3Iethod,~ of E~tzy~natic .4~uzlysLs p. 363. (Ed. by Bergmeyer, it. t',l, Acadelnlc Press, Leaden. .Booth, J. and ]3oyland, E. (t949). Bioche,m..1.44, 36[. Cohen, P. P. (1949). In -]tanometric Tec~niqae,~' a~d Ti.s~'uc ~lletaboli~m, p. ~19. (Ed. hy Umbreit, W. ~,V.: :Burris, R. H. mxd Stauffer, J. F.). Burgess :Pub]ishing Co., M.in.neapolis. TJuke-EIder, ~:V. S. (1954). In Text Book of ()phllutlmogogy VI, p. ~iS06. Htmry Kimpto~. I.ondolL E]hnan, G. L. (1959). Arch. Bioche~t. t3iophys. 82, 70. G]oster, J. {1956). ]Jrit. J. OphthatmoL 40, 536. van 14eyningen, [l,. (1965). ]tbod,,cm. J. 96, 419. van tteyni~gen, ~. and Pirie, A- (1967). Biochem. J. 102, 842. Holmberg, N. J. (1968). Exptl Eye P,es. 7, 570. Hughes, R. E, (1964). Nature 203, 1068. Ki~mey, V. E. (t947). Am. J. Ophthalmot. 30, 1202, Kiuchi, K. (t959}. 5'hikoku, Aaa Med. 14, I1O. .. :LmLgham, M. E, amt Heald, K. (I956), Biochem. J. 73~ 52. Mar~in, G. g. (1961). Anu. N. ):, Acad. Svg. 92, 141. Mcrriam, F. C. a.nd Kirmey, V. E. (1950), Arch. Opht]t~l~n~*. 4,3, 979. Nordmann, J. (1954). In Bioloffie du Cri.~ta31in. P. Massou et, ~.~e, P~ris. Patterson, J. W. and Mastin, D. W. (195!). Am. J. Physivl. 167, 119, Pirie.. A. (1965). Biochem. J. 96, 224. Bfiih~, N. (1956), .Acla .Physiol. SextTut. 36, 291. .Ree~, J. ~. am:t Pirie, A, (1967). Bioche.m.J. 102, 853. Roe, J. ]-I. (1954), in Method.* oj' BiochemiclH Analysis l, 127. (Ed. by (Jlick, D. F,.). ]nterscience Publisher~ Inc., New York. Takagi, Y. and Okumura, It. (i961). Acta Sc~.,. Ophtlmlnwl. ,]a~. 65; 11.62. a;Vachtl, C. az~d Kin.sey, V. E, (t958). Am. J. OpMh,:dmol. 46, (5), part IT, p. 288.