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In vitro uptake of ascorbic acid by the guinea pig eye lens
Exptl Eye Res. (1970) 9, 175-I80
In vitro Uptake of Ascorbic Acid by the Guinea Pig Eye Lens
University of Wales Institute. of Science a,u'l Technoloff!/, Cardiff, ;Vales (Received 25 A.3)ril 1969, l.,o~Mon) h t v i t r o e x p e r i m e n t s iud_iea.t~ ~.ha¢, in t h e ri~t ant i g u i n e a pig, bot.h ,ascox.bic ar;id e n d deh v d r o a s c o r b i c acid e n t e r t h e e y e loire. T h i s i8 in contras% wit.h othxer biologieM s y s t e m s • ~There d c h y d r o a s t / o r b i e a c i d a l o n e w o u l d a p p e a r t o b e 1she f o r m in wlfich t h e vitn,min c r o s s ~ m e m b r a n d barriers. F u r t - h c r m o r e , t h e upt a& e o f ])-araboaseorbio a c i d (£ao&seorhic acid) b y ¢he lens is n o t s i g n i f i c a n t l y diftbren~ f r o m t h a t o f a~corbie acid---a fi~xdittg in c o n t r a s t w i t h ~he c o r r e s p o ~ d i n g ait.uaLion in t h e e r y t h r o c y t e w h e r e d e h y d r o g s o ~ s c o r b i e a c i d etxt~rs b u t slowly u n d e r e o n d i t i o u s t h a t p e r m i t ~ r a p i d ~pta.lte o f debych-oascorbio acid (1-£ughes a n d .~laton, ].91iS). S t o r a g e o f lenses in i s o t o n i c m e d i u m f o r p e r i o d s o f u p t o 7 d ~ y s reaulbs i n subsga,rttial increases in t h e m~bsequont r a t e o f u p t ~ k ~ o f a scorbio aci d, ~ p h e n o m e n o n ~ha~ ea~x he f u r t h e r m o d i f i e d b y ct~unging t h e n a t u r e , o f t.ho s t o r a g e m e d i u m . I ~ n s e s f r o m o l d g u i n e a pigs, h o w e v e r , I taw.. a m u c h l ow e r r a t e o f u p t a k e o f a s c o r b i c a c i d t h a n t h o s e fi'om y o u n g a.r~imMs.
1, I n t r o d u c t i o n
There is consideraMe evidence ~h~t v i t a m i n C (I.-xyloascorbic acid) preferen.tially cross~s biological m e m b r a n e s iu t h e oxidized tbrm, i.e. as dehydroascorbic acid (R~it~, ] 958). Tlds h a s recently been confirmed using t:b.e isolated erythroeyCe as a model systent {'or p e r m e a b i l i t y studies; dehydroaseorbie acid entered t h e er3rt,h r o e y t e m a n y t.imes more r a p i d l y t.ha~, ascorbic acid (Hughes a.nd Match, 1968)..Furbhermore, t h e eryt/lrocyte displays ~n interesting selectivity in this respect. U n d e r conditions t h a t permit- a rapid ~ p t a k e of dehydroascorbie acid, t h e r e is virtuMIy no u p t a k e of t h e i~omeric dchvdroisouseorbic acid (dehydro-:)-araboascorbic acid). The behaviour of the eye lens ha-s in m a n y respects been c o m p a r e d w i t h t h a t of t h e e.rythrocyte (Kinoshita, 1964). tll particular, b o t h systems h~ve been used for t h e in vitro investiga*~ion of t h e passage of molecules aclx)ss a biologicM m e m h r a t m a n 4 the work described in tiffs r e p o r t was originally d e s i r e d t,o compare t;he two systems in ~erms of ascorbie acid u p t a k e . Ascorbie acid lends itself snell to p e r m e a b i l i t y studies of t,h is t y p e since it is a water-soluble molecule thug can b e conven/ently and r a p i d l y determined a~ low coneen~r-ztiolls..Furthermore, t h e relatively high concentration of ~scorbic acid in t h e lehs is suggestive of a n i n v o l v e m e n t in lenticulax m e t a b o l i s m ; factors affecting the passage of asoorbie acid hxt~o t h e lens could therefor~ p r e s t m m b l y influence t h e overall m e t a b o l i s m of t h e lens (Iteabh, 1962). I n tlm e x p e r i m e n t s described, below prelimi~aary storage of t h e lens in a.n isotonic mediu.m was practised in order to remove, as f a r as possible, a n y endogenous ~ e o r b i e acid. Duxing t h e initial e x p e r i m e n t s it became appareut, t h a t b o t h tlxe period,, of storage and. the n a t u r e of t h e storage m e d i u m could influence ~he subsequent, u p t a k e of ascorbie acid. AdditionM e x p e r i m e n t s were therefore included t o deter,nin~ whegher t l f s " s t o r a g e : ' effect h a d a n y c o u n t e r p a r t in t e r m s of "natux~l a g e i n g " : t h i s w a s done by m e a s u r i n g the uI, t a k e of ascorbic acid b y lenses f r o m y o u n g a n d old guinea pig~. 175
17{]
R. E. f t U G H E S AND R. J. H U I ¢ L E Y 2. M e t h o d s
Reagents W h e r e v e r possible, Auatar grade reagents were used. L-Ascorbic acid (~-xyloascorbic acid) a n d x)-iso-ascorbic aei4 (D-ar,~boascorbic acid) were purchased front K o c h - L i g h t Laboratories Ltd. Dehydroa~corbio ~cid was p r e p a r e d i m m e d i a t e l y heft,re use by oxidation of ~-ascorbio acid with b r o m i n e (Hughes, 1956).
E,~]e lenses Male animals were used i.~ al~ experfme~ts. T h e y were killed b y atu~mlng and decapit:~-. rich. The eyes were removed, the le~se,~ quickly dissected ou~, rinsed ia isotoni~ saline a n d stored ~.t 5°0 ia t h e appropriate n l e d i u m (in most cases, isotonic sa,tine) for ~he required time. After 2da.L~m storage the buJk of tim ler~icaiar ascorbio acid h a d diffused outinvo t h e storage m e d i u m . :By depletiz~g the lens of ascorbio acid in this man:ter ~ubs~at~tiai u p t a k e s could later be o b t a i n e d d u r i n g shor~t, erm incubations. Lenses u~ed directly a|'ter r e m o v a l from. v itamin'0-suffioien~ animals con~aiue¢l s u b s t a n t i a l a m o u n t s of ascorbic acid a n d a n y s u b s e q u e n t u p t a k e d a r i n g i n c u b a t i o n was correspondingly reduced. "'Storage d e p l e t i o n " of asc.orbio acid also s t a n d a r d i z e d the procedure to the e x t e n t theft it enabled ascovbie acid u p t a k e s ~o be m e a s u r e d on ]enses with a p p r o x i m a t e l y the same residual bas~l levels of the v i t a m i n .
Incubation The a~corbie acid-depleted lenses were allowed to r e m a i n a t room t e m p e r ~ t u r e l~or 20 rain a.nd were t h e n tr~nsfe~Ted to ~he incub,utien m e d i u m (isot.onic phosphat-e buffer). The fit'at tens f r o m each pair wa~ used as a c:on~rol; i~ was i n c u b a t e d in 10 nil o[' the p h o s p h a t e buffer a t 37°C. The second lens w~s i n c u b a t e d u n d e r the same conditio~,~ with 10 ml of t h e m e d i u m i n which 5 m g of the appropz'iate test c o m p o u n d (,',.scorbic acki. denydroa,~corbm acid or isoascorbie acid) h a d been dissolved. I n c u b a t i o n wa~ carried silt in a 50 ml conical flask secured in a s h a k i n g i n c u b a t o r opera~,h~g a t 86 osc~llations/mi:~. At physiologicaA conditions of p H and t e m p e r a t u r e dehydroo,scorbia acid undergoes irreversible conversion into dioxogutordo acid. IJ~crea~e ia pI~ above 6.8 results :~n ~ r a p i d increase in t h e rate of ].oss of dehydroascorbic acid. To minimize such losses incuba~,iort was carried out in a. m e d i u m buffered at 6-8; a~ p H 6'8 50°/o of the dehyclroascorbic acid ~-X'asstit[ presenb iu the incub~rte after 16 ruin. The s~;~bility of ascorbic acid too decreases with increase in p H b u t losses can be p r e v e n t e d effectively b y ;ncorporating 2 mg of homocysteine in t h e i n c u b a t i o n m e d i u m (t{ughes, 1956). )Vtlen She incuba*ion m e d i u m contldtmd dehydroascorbio acid (Tables 1 a n d 3) homoscrine- was s u b s t i t u t e d for homocys~eine; this was done in an a.t~empt t o compensa,~e for a n y possible steric interference in~roduced into the system b y the homocys~eine (homocy~t,eine itself could n o t be used a,s it would eonve~t %he dehydroascorbic acid into ascorbio acid; ht)moserine is structurally similar to h o m o c y s t e i n e b u t i t has n o reducing ae~tion on t h e dehydr0ascorbic acid).
De~rJ).-i,mtion of ascorbic acid A t the end of t h e i n c u b a t i o n period each lens was r e m o v e d from the medium, washed quickly in three changes of isotonic saline, weighed.on a direct-.ceading analytical ba.lance and e x t r a c t e d with 1"8 m l of 50/o trichloroaeetic acid c o n t a i ~ n g 1 % norit. The e x t r a c t w~s centrifuged a n d 1 nfl of t h e clear s u p e r n a t a n t /~sed for t h e d e t e r m i n a t i o n of t o t a l aseorbJc acid using t h e 2 : 4 d i n i t r o p h e n y l h y d r a z i n e znetltod as clesoribed b y Riiih:4 (1958). S t a n d a r d curves were prepa.red using k n o w n a m o u n t s of oxidized ascorbic acid; ~he s t a n d a r d curves for ascorbie acid a n d i~oascorbic acid were identical.
I N VfTItO U P T A K E OF ASCORBIC ACID
177
3. R e s u l t s
F o u r set.~ of expe.rimcnts were done. T h e e x p e r i m e n t a l c o n d i t i o n s a n d t h e result.~ o b t a i n e d arc given in T a b l e s I - I V . Ta
m,~: i
Upotke of i~-ascorbic acid a~d &;hydroascorbic acid by isotated eye lens T i m e of incubation (m)
}'.e.t
15
A s e o r b i c a c i d ttptatke ( m g l [ 0 0 g l,iaaue)
Dehydroaseorbie acid uptake ( r a g / 1 0 0 g i,i~sue}
3"83 (4) F,=0.40
3-f)3 (4) :J=0"40
2-95 (6) -~0.4t
2"59 (7) ~0,20
( ; n i n e s pi~ 11)
.t.37 (7) ±0.25
15
5.66 (6) q-0.42
/
3-89 (8) -2:0"14 4-75 (7) ~-~.0'30
l n c u b a t i o n w a s ~t 37'=C it, 10 mI ~ootonic phosi)hat,e buffer (pie[ 6-8) contMl~.ing 5 m g a s c o r b i c a o i d (or d e h y d r o u s e o r h i e ~cid) a n d 2 m g h o m o e y s t e i n e (or ltomoaerine). Previotaa t o ~he o x p e r h n e n t t,lm lenses h a d b e e n s t o r e d in i~otonic s a l i n e a t 3~C for 24. h r (ra.ts) a n d 72 h r ( g u i n e a pigs). I~osult--3 ~,re g i v e n a s m e a n v a ] u e s w i t h s / , ~ n d a r d errors, T h e /~gurcs in p a r e n t h e s e s d e n o t e t h e n u m b e r o f d e t e r m i n a t i o ~ on w h i c h thv, m<,an v a l n e is bam.~l.
'~L~.BLI.; I I
Uptake of ascorbic acid ( L-rcyIoa.~.'eorblc a "~) a.ud isoascorbic acqxl (D-araboaxccrrbic acid) by ffuinea. Tig lenses
Aseorb~e acid.
Uptake (in r a g / 1 0 0 g lena tissue)
4"69 (~) -4=0-34 .
isoascorbic acid
4-'10 (8) -~-b0.42
Im~ubatio~ ~ a s for I0 m i ~ at 37~C in I0 m l bsotonic bt:ffer ( p H 6.8) ~t~iTxing 5 m g a.seorbio acid or ~oascorbic aei~ attd 2 m g homocyszeh.,e. Previous to tlae experiment the l e ~ had beolt ~ o ~rod in iso~onic saILuo at 3~C for 48 hr. Result~ are givert as meatt value~ with standard error~4 the figures in parentheses denoto the n u m b e r of determisaatiorL~ on which the m e a n is baa~I. N o significa n0 difference w a g o b s e r v e d be~wc~a n~e~rts.
178
It.. E .
I.IUGflE:3
A1N]) 'l"a a ~
11. J .
]IURLI~,k~
Ill
Effec~ of p H o~. .uptake: of ascorbic acid and delq/droascorbic avid by isolated gz,..in~, jr;,y eye lens
t)}[
A s c o r b i c at:id upt.'tke (mg/lO0 g ti~uc)
A 5.2
P, fi-o
C t;.n
4-20 (7)
4-:~I iS) :~ 0-1,t
-1-93 (~i) :~ 0-42
4-~:1 (3) :'-.0- I S
:1-5-1 (;5~ _~ 0-24
:.EO--15
1 ) e h y d r o a ~ c t ) r b i c a c i d * tll)l~k~ (mg/lt'lO g tissue)
4-60 14) •:3-0-3(~
T h e i n c u b a t i o n w a s t})r l 0 vain a t 370C' i~ 10 ml o f t h e u . p p r o p r i a t e lruffer c , ~ n t a i n i r g / i m g a.~corbie a c i d (or d e h y d r o a s c o r b i e a c i d ) v,n d 2 m g l~omocyuLeirm (or homoserit~e). Previt~us t o the- e x p e r i t m , t t t t h e le.nses h a d l~een s t o r e d i n i s o t o a i 0 s M i n o for 4 5 hx at. 3"C. l l e s u i t ~ a r e gi~-~:n ~u m e a n v a i m . s w i t h sta:~4a:-d er~v)rs; t h e f i g a r e a ~n p a r e n t h e s c ~ d e n o t ~ the. n u r n b , w o f d e t e r m i n a t l o n : , oft w h i c h tJ~e m e a n is b , , , - l . * S i g n i f i c g n e e level for d i f f e r e n c e o f m e a n s b e t w e e n g r o u p s A a n d C =-~ <:5';.~> I c';,.
TAtl I.I; i V
Ix.fleet o f sloraqe time a.J~d ,to.raffe nterliu.m on t/,c .sub.seque~,t t~jdake ~t/' ¢1.s-,:c,rbA; a¢'id b~,! lelt.ses f r o m !/o".~,',J and old flu ifu'. l)[ys
G~'oup
A 1~ C D E J~"
A g o ,o£ g u i n e a pigs (months)
:t :t 2>2,t 3 .'> 24 :~
btorage xnediu:n
i s o t o o i c atlhJe, 3 ' t" lsvto~fie aalh~e, 3~C lsot,.mic s a l i n e , 3 : C t~.rcbs-lRi~Jger b u f f e r 3:(.'. K r e b s - I l i n g e r b u I I b r 3 ~(; 2isotonic s:t|ine, 3~0
As.-:t,rbie aci,I upuLke (mg!ll)t)~ tissue
.qt~,t-a,~. trout, (tlay.~)
:2-58 (6) :5. 0--t7
! 3 :t 1; :¢ 7
5.4& (6)~-~' 0"61
3-[14 l 1 "89 8- 73 9"7I
(?):--0-SS (6)q- 0-44 (6) :b. 0- 33 (4)~_ l "34
A f t e r s~or~ge t h e l e n s w a s i n c u b a t e d , f o r l 0 r a i n at 37°C ".~ith l 0 m ! i s o t o n i c p h o s p h a t e b u f l b r ( p t [ 6-8) e o n t . a i n i n g 5 m g ~ s e o r b i e a c i d a n d 2 m g h o m o c y s t . e i n e . 2R.emfl~s ~ r e g i v e n a~ m e a n v a l u e s w i t h sl.~mdanl e r r o r s ; t h e figttre~ i n p a r e n t h e s e s de~tete t h e n u m b e r o f d e t e r m i n a t i o n s o n w l - i e h t h e 2heart is b a s e d . J ' < I ~ / o for diffcrer, ce of m e a n s o f r e | t o w i n g g r o u p ~ : A at,d I5; B ~nd Jr'; I ) a n d E .
4. D i s c u s s i o n
Available evidence indicates ~ha~ vitamin C preferentially crosses biological membrane,~ in the oxidized tbrm---,~ finding tb-ut has beex~ af.tributed to the gre~t.er solubili W of dehydro~scorbic acid in the lipid component(s) of mere branes (Riiihii, 1956; Martin, 1961; van .L{eyningen, 1970). Thus in the guine~ pig e~3~hroeyte there is vi~'tually no ~ptake of a~scorbie acid under conditions gha~ permit r~pid uptake of dehydroascorbie a n d (Hughes and Maton, 1968). Tile in vitro experiments summarized above however, clearly suggest t h a t both in the rat and the guinea pig the
IN VITRO
UPTAKE
OF
ASCORBIC
ACID
179
eye lens is freely permeal~le to both ascorbic acid and dehydroascorbic acid, and fm't,hermore, that t.'ae u p t a k e of ascr)rbic acid is somewhat, more r a p i d than t h a t of dehydroascorbic acid (Table I). I t m a y also be noted t h a t the p e r m e a b i l i t y of the guinea pig lehs to both "ascorbic acid and dehydroascorbic acid a p p e a r e d to l)e gI~ater t.han, that~ of the r a t lena ; so, too, the guinea pig er3rt}trocyte is m u c h more permeable to dehydroascorbic acid titan is t h e r a t eryghrocyte (Hughes a n d M a t e s , 1968). However, vhe stere-o-specificity displayed b y the e r y t h r o e y t e m e m b r a n e t o w a r d s dehydroascorbic a q d a n d dehyd~oisoascorbic acid, was a b s e n t in the, case of the lens (Table Ill. N o difference could be d e r a o n s t r a t c d bet~:een the u p t a k e of ascorbio acid at,(t isoascorbic acid {compounds which differ in tim positions of the H a n d O H groups ou ca.rbon 5); in the case of the ery~hrocyte, dehydroascorbic acid crosses the memt)ranc rapidly but dchydro/soaseorbic acid does not (Hughes a n d M a k m , t968). t'attersml (1965) has concluded t h a t t h e process of glucose tratmport in the lens is ,~imilar to the process for the transport, of glucose in the e r y t h r o 0 y t e ; the results summarized in Tables I a n d I I however, indicate t h a t in the case of ascorbic acid titere are obvious differences in ~he m e m b r a u e permeability properties. q'he uptake of dehydroascorbic acid b y the lens would however a p p e a r to t)e l)H-dependent (Table II1), a phenomenon which has a parallel ia t h e case of ~he e r y t h r o c y t c and which is Drobably most satisfactorily explained iu terms of changes in the degree of ionization of certain spatiaI~y-cri_ticM grottpings within the m e m b r a n e l~()re~. A n y discussion of m e m b r a n e properties of the lens must, as P a t t e r s o n ha.~ ~'emil~ded us, necessarily remain highly tlmoreticaI as one is deMing nob with a, single, ~hscrete, definable bou'hdary, b u t with three different poten~iM p e r m e a b i l i t y barriers .....the capsule: the epithelium a n d the fibre m e m b r a ~ e s (Patterson, 1965). The results recorded in this paper could reflect passage across the capsule aloi~e and accumulation at" ~scorbic acid and dchydroascorbic acid in t h e extracelt{flar space rattler t h a n ~,omplete passage int.o the lens fibres. This hob'ever is unlikely as both the k n o w n sta.bility of ascorbic acid in the lens, and the reduction of del~ydroascorbic acid to use orbit acid b y the lens protein thiol suggest th~t~ normally diffusion of ascorbic arM and dehydroascorbic ac~i4 into the eye lens is complete. Storage of the-tenses in isotonic m e d i u m significantly increased their p e r m e a b i l i t y to a.scorbic acid (Table IV, groul)s A, B ~ d F). SVorage-induced. change.g in the biological properties of systems are of eouxse not u n k n o w m Of greater interdst in the ease of" i:,]le eye lens is t h a t the n a t u r e of the storage m e d i u m exerted a m a r k e d effect upon t.he subsequen$ p e r m e a b i l i t y of the lens to aseorbic acid. Thus lenses stored in Idrebs-Idinger buffer for 3 d a y s displayed an aseorbic acid-uptake twice thug of lenses stored in isotonic sMine for the same period (Ta.ble IV, groups 13 a n d D). The ascorbic acid u p t a k e b y lenses stored in saline for 7 d a y s was less t h a n t h a t of those stored in K rebs-t2.inger for only 3 d a y s (groups D and F). I t / . s conceivable t h a t t h e effect of storage m e d i m u is a t least in p a r t a reflection of changes induced.initially in the e x t r a cellular s~pa.ce. Such an e x p l a n a t i o n would, however, require ar~ accumuIation of ascorbic acid in the cxtraceliular space, a l ~ p p e n i n g , which, as indicated above, is unlikely to occm'. ]~urthermore, fairly eonsii:lerable changes ia the volume of the extracellu3ar fluid would be necessary ix) account for t h e subst~ntiM increax,~ in u p t a k e of ascorbic acid b y "steered" l e n s e s ~ a l t h o u g h in t h e absence of extracellular space measurements, such a .possibility cannot, be completely discounted. F u r t h e r groups were included to deCerrniae whether this "in vit.ro" ageing effe~V had a eounVerpart in t,erms of n a t u r a l ageing. Lenses from two groups of o l d guinea pigs (:>2g m e n tits) were stored, in saline or Krebs-:Ringer a n d t h e s u b s e q u e n t u p t a k e
180
R, E. HUGHES AND R.. J. H U R L E Y
of ascorbic acid measured° Here, to(,, storage in Krebs-R.inger resulted in grcate.r uptakes of ascorbic acid than storage in ~Mine but in both c~ses ~hc ttpt, akcs were considerably below those obtained with lenses from young anima.ls (TaMe I I [, group.'; C and E ) , ~ a finding th~,.t has a possible, eomlterpar~ in the absorpt, ion of as('orbit: acid fl'om the gastrointestinal tract of the guinea pig where a neg~tt,ive ('orrelation with age has been indicated (Hughes a n d Jones .... unpublish(,d dat, a). T i m , the in vitro ageing would appear to be a quite separate pheno~mnon from the p(,rlno, al)ility changes t,h a t a c c o m p a n y n a t u r a l ageing. I t is of interest to recall t h a t in cataract, ous eyes (in hmnans, an a.ge-eorrel;~ted condition) lowered ascorbie acid levels h a v e been reported (He'tt.h, 1962). Falls in lcnticulm' ascorbic acid are associa.t.cd too with cataracts induced by microw~Lvc t~rcatmer~t and b y X - r a y s (Carpenter, 1962; Pirie and van Hcy~ingcn, 1953). X-ra.ys h~vc of course been used b y certhiu investigators to simubm~ or a.ccelcratc cert, a.in somatic changes held to be characteristic of the, ageing process, a,nd it is lX)S.~ibie t.hat tlte changed aseorbic acid levels in eat:m~ct are 0, redection of a.n a.ccent:uar.~:,,1 ~.geassociated change in the permcabili~;y of the tens membranes. Pat.teflon an(I B u n t i n g (t964) have indeed d e m o n s t r a t e d t h a t iu the case of experinmntally-induced cat.armLet i31 y o u n g anima.ls there is a reduced p e r m e a b i l i t y to cert;fin small molecule,-.. T]uu'a ia ~./o evidence, however, bhat changed p e r m e a b i l i t y to ascorbic acid is a ca.us;d 5toter in the incidence of eat.araet. As H e a t h has pointed out, the d i m i n i s h e d levels of a~cort)ic. acid observed in ea~aractous lenses are consequential changes rat.her t h a n causal one~ (Heath, 1962), H e a t h relat, ed t h e m to ehtmges in the rnet.abolic processes in t.h~', eataractous lens b u t t h e y could e q u a l l y well be a consequence ot' ~, ctumged ~ e m brau,~ p e r m e a b i l i t y similar in n a t u r e to the age-correhd)ed ones described in this rei)¢)rt:. The effect of changes in lens p e r l n e a b i l i t y (whether age-corrclm;cd or cataractous in origin) upon lenticular v i t a m i n C level r e m a i n s to be de~ermined. F u r t h e r m o r e t,hc anomalous b e b a v i o u r of the lens m e m b r a n e s when compared wit.h more clearlydefinable st,ruct;ares such as the eryt, hroeyte m e m b r a n e , imposes severe limitation.~ upon its use as a model s y s t e m for pemneabiiit.y sgud.ies. ACKN O W L E D G M E N T This work was supported by a Medical Research Council Grant. REFEItENCES Carpenter, E. L. (1962), Quoted by Winter, ~b".C. in ~rch. Ophthal.mol. (Chicago) 75, 713. Heath, H, (1962), Exptl Eye/i~es. I~ 362. van Heyningea, R. (1979). Exjotl Eye/~es. 9, 38. /-Iughes, R. E. (1956)..~i~hem. J. 6!t~ 203. Hughes, R. E. and Matxm, S. C. (1968).~Brgt. J. Haematol. 14, 247. Kinoshita, J. H. (1964). Arch. 02ohthaZmol. (Chicago) 72~ 554. Martin, G. tl. (I961). Ar~u. ALJz. Acad. ,Sci. 92, 141. Ptitterst)n, 5. W. (1965). SymTosium on the Le~t.s, p. 253. tgd. by J. E. liar fla. St.. Lo~ds. Patterson, J. W. an4 :Bunting, K. W. (1964). Prec.. goc. Exptl Biol. Med. 1315, ] 156. Pirie, A. and vau He)'ningen, R.. (1953). Bioche-m. J. 54~ 682. Riiihii, N. (I958). Ae2a physioL Sca~,M. 45~ suppl. 155 p. 24.
In vitro Uptake of Ascorbic Acid by the Guinea Pig Eye Lens
University of Wales Institute. of Science a,u'l Technoloff!/, Cardiff, ;Vales (Received 25 A.3)ril 1969, l.,o~Mon) h t v i t r o e x p e r i m e n t s iud_iea.t~ ~.ha¢, in t h e ri~t ant i g u i n e a pig, bot.h ,ascox.bic ar;id e n d deh v d r o a s c o r b i c acid e n t e r t h e e y e loire. T h i s i8 in contras% wit.h othxer biologieM s y s t e m s • ~There d c h y d r o a s t / o r b i e a c i d a l o n e w o u l d a p p e a r t o b e 1she f o r m in wlfich t h e vitn,min c r o s s ~ m e m b r a n d barriers. F u r t - h c r m o r e , t h e upt a& e o f ])-araboaseorbio a c i d (£ao&seorhic acid) b y ¢he lens is n o t s i g n i f i c a n t l y diftbren~ f r o m t h a t o f a~corbie acid---a fi~xdittg in c o n t r a s t w i t h ~he c o r r e s p o ~ d i n g ait.uaLion in t h e e r y t h r o c y t e w h e r e d e h y d r o g s o ~ s c o r b i e a c i d etxt~rs b u t slowly u n d e r e o n d i t i o u s t h a t p e r m i t ~ r a p i d ~pta.lte o f debych-oascorbio acid (1-£ughes a n d .~laton, ].91iS). S t o r a g e o f lenses in i s o t o n i c m e d i u m f o r p e r i o d s o f u p t o 7 d ~ y s reaulbs i n subsga,rttial increases in t h e m~bsequont r a t e o f u p t ~ k ~ o f a scorbio aci d, ~ p h e n o m e n o n ~ha~ ea~x he f u r t h e r m o d i f i e d b y ct~unging t h e n a t u r e , o f t.ho s t o r a g e m e d i u m . I ~ n s e s f r o m o l d g u i n e a pigs, h o w e v e r , I taw.. a m u c h l ow e r r a t e o f u p t a k e o f a s c o r b i c a c i d t h a n t h o s e fi'om y o u n g a.r~imMs.
1, I n t r o d u c t i o n
There is consideraMe evidence ~h~t v i t a m i n C (I.-xyloascorbic acid) preferen.tially cross~s biological m e m b r a n e s iu t h e oxidized tbrm, i.e. as dehydroascorbic acid (R~it~, ] 958). Tlds h a s recently been confirmed using t:b.e isolated erythroeyCe as a model systent {'or p e r m e a b i l i t y studies; dehydroaseorbie acid entered t h e er3rt,h r o e y t e m a n y t.imes more r a p i d l y t.ha~, ascorbic acid (Hughes a.nd Match, 1968)..Furbhermore, t h e eryt/lrocyte displays ~n interesting selectivity in this respect. U n d e r conditions t h a t permit- a rapid ~ p t a k e of dehydroascorbie acid, t h e r e is virtuMIy no u p t a k e of t h e i~omeric dchvdroisouseorbic acid (dehydro-:)-araboascorbic acid). The behaviour of the eye lens ha-s in m a n y respects been c o m p a r e d w i t h t h a t of t h e e.rythrocyte (Kinoshita, 1964). tll particular, b o t h systems h~ve been used for t h e in vitro investiga*~ion of t h e passage of molecules aclx)ss a biologicM m e m h r a t m a n 4 the work described in tiffs r e p o r t was originally d e s i r e d t,o compare t;he two systems in ~erms of ascorbie acid u p t a k e . Ascorbie acid lends itself snell to p e r m e a b i l i t y studies of t,h is t y p e since it is a water-soluble molecule thug can b e conven/ently and r a p i d l y determined a~ low coneen~r-ztiolls..Furthermore, t h e relatively high concentration of ~scorbic acid in t h e lehs is suggestive of a n i n v o l v e m e n t in lenticulax m e t a b o l i s m ; factors affecting the passage of asoorbie acid hxt~o t h e lens could therefor~ p r e s t m m b l y influence t h e overall m e t a b o l i s m of t h e lens (Iteabh, 1962). I n tlm e x p e r i m e n t s described, below prelimi~aary storage of t h e lens in a.n isotonic mediu.m was practised in order to remove, as f a r as possible, a n y endogenous ~ e o r b i e acid. Duxing t h e initial e x p e r i m e n t s it became appareut, t h a t b o t h tlxe period,, of storage and. the n a t u r e of t h e storage m e d i u m could influence ~he subsequent, u p t a k e of ascorbie acid. AdditionM e x p e r i m e n t s were therefore included t o deter,nin~ whegher t l f s " s t o r a g e : ' effect h a d a n y c o u n t e r p a r t in t e r m s of "natux~l a g e i n g " : t h i s w a s done by m e a s u r i n g the uI, t a k e of ascorbic acid b y lenses f r o m y o u n g a n d old guinea pig~. 175
17{]
R. E. f t U G H E S AND R. J. H U I ¢ L E Y 2. M e t h o d s
Reagents W h e r e v e r possible, Auatar grade reagents were used. L-Ascorbic acid (~-xyloascorbic acid) a n d x)-iso-ascorbic aei4 (D-ar,~boascorbic acid) were purchased front K o c h - L i g h t Laboratories Ltd. Dehydroa~corbio ~cid was p r e p a r e d i m m e d i a t e l y heft,re use by oxidation of ~-ascorbio acid with b r o m i n e (Hughes, 1956).
E,~]e lenses Male animals were used i.~ al~ experfme~ts. T h e y were killed b y atu~mlng and decapit:~-. rich. The eyes were removed, the le~se,~ quickly dissected ou~, rinsed ia isotoni~ saline a n d stored ~.t 5°0 ia t h e appropriate n l e d i u m (in most cases, isotonic sa,tine) for ~he required time. After 2da.L~m storage the buJk of tim ler~icaiar ascorbio acid h a d diffused outinvo t h e storage m e d i u m . :By depletiz~g the lens of ascorbio acid in this man:ter ~ubs~at~tiai u p t a k e s could later be o b t a i n e d d u r i n g shor~t, erm incubations. Lenses u~ed directly a|'ter r e m o v a l from. v itamin'0-suffioien~ animals con~aiue¢l s u b s t a n t i a l a m o u n t s of ascorbic acid a n d a n y s u b s e q u e n t u p t a k e d a r i n g i n c u b a t i o n was correspondingly reduced. "'Storage d e p l e t i o n " of asc.orbio acid also s t a n d a r d i z e d the procedure to the e x t e n t theft it enabled ascovbie acid u p t a k e s ~o be m e a s u r e d on ]enses with a p p r o x i m a t e l y the same residual bas~l levels of the v i t a m i n .
Incubation The a~corbie acid-depleted lenses were allowed to r e m a i n a t room t e m p e r ~ t u r e l~or 20 rain a.nd were t h e n tr~nsfe~Ted to ~he incub,utien m e d i u m (isot.onic phosphat-e buffer). The fit'at tens f r o m each pair wa~ used as a c:on~rol; i~ was i n c u b a t e d in 10 nil o[' the p h o s p h a t e buffer a t 37°C. The second lens w~s i n c u b a t e d u n d e r the same conditio~,~ with 10 ml of t h e m e d i u m i n which 5 m g of the appropz'iate test c o m p o u n d (,',.scorbic acki. denydroa,~corbm acid or isoascorbie acid) h a d been dissolved. I n c u b a t i o n wa~ carried silt in a 50 ml conical flask secured in a s h a k i n g i n c u b a t o r opera~,h~g a t 86 osc~llations/mi:~. At physiologicaA conditions of p H and t e m p e r a t u r e dehydroo,scorbia acid undergoes irreversible conversion into dioxogutordo acid. IJ~crea~e ia pI~ above 6.8 results :~n ~ r a p i d increase in t h e rate of ].oss of dehydroascorbic acid. To minimize such losses incuba~,iort was carried out in a. m e d i u m buffered at 6-8; a~ p H 6'8 50°/o of the dehyclroascorbic acid ~-X'asstit[ presenb iu the incub~rte after 16 ruin. The s~;~bility of ascorbic acid too decreases with increase in p H b u t losses can be p r e v e n t e d effectively b y ;ncorporating 2 mg of homocysteine in t h e i n c u b a t i o n m e d i u m (t{ughes, 1956). )Vtlen She incuba*ion m e d i u m contldtmd dehydroascorbio acid (Tables 1 a n d 3) homoscrine- was s u b s t i t u t e d for homocys~eine; this was done in an a.t~empt t o compensa,~e for a n y possible steric interference in~roduced into the system b y the homocys~eine (homocy~t,eine itself could n o t be used a,s it would eonve~t %he dehydroascorbic acid into ascorbio acid; ht)moserine is structurally similar to h o m o c y s t e i n e b u t i t has n o reducing ae~tion on t h e dehydr0ascorbic acid).
De~rJ).-i,mtion of ascorbic acid A t the end of t h e i n c u b a t i o n period each lens was r e m o v e d from the medium, washed quickly in three changes of isotonic saline, weighed.on a direct-.ceading analytical ba.lance and e x t r a c t e d with 1"8 m l of 50/o trichloroaeetic acid c o n t a i ~ n g 1 % norit. The e x t r a c t w~s centrifuged a n d 1 nfl of t h e clear s u p e r n a t a n t /~sed for t h e d e t e r m i n a t i o n of t o t a l aseorbJc acid using t h e 2 : 4 d i n i t r o p h e n y l h y d r a z i n e znetltod as clesoribed b y Riiih:4 (1958). S t a n d a r d curves were prepa.red using k n o w n a m o u n t s of oxidized ascorbic acid; ~he s t a n d a r d curves for ascorbie acid a n d i~oascorbic acid were identical.
I N VfTItO U P T A K E OF ASCORBIC ACID
177
3. R e s u l t s
F o u r set.~ of expe.rimcnts were done. T h e e x p e r i m e n t a l c o n d i t i o n s a n d t h e result.~ o b t a i n e d arc given in T a b l e s I - I V . Ta
m,~: i
Upotke of i~-ascorbic acid a~d &;hydroascorbic acid by isotated eye lens T i m e of incubation (m)
}'.e.t
15
A s e o r b i c a c i d ttptatke ( m g l [ 0 0 g l,iaaue)
Dehydroaseorbie acid uptake ( r a g / 1 0 0 g i,i~sue}
3"83 (4) F,=0.40
3-f)3 (4) :J=0"40
2-95 (6) -~0.4t
2"59 (7) ~0,20
( ; n i n e s pi~ 11)
.t.37 (7) ±0.25
15
5.66 (6) q-0.42
/
3-89 (8) -2:0"14 4-75 (7) ~-~.0'30
l n c u b a t i o n w a s ~t 37'=C it, 10 mI ~ootonic phosi)hat,e buffer (pie[ 6-8) contMl~.ing 5 m g a s c o r b i c a o i d (or d e h y d r o u s e o r h i e ~cid) a n d 2 m g h o m o e y s t e i n e (or ltomoaerine). Previotaa t o ~he o x p e r h n e n t t,lm lenses h a d b e e n s t o r e d in i~otonic s a l i n e a t 3~C for 24. h r (ra.ts) a n d 72 h r ( g u i n e a pigs). I~osult--3 ~,re g i v e n a s m e a n v a ] u e s w i t h s / , ~ n d a r d errors, T h e /~gurcs in p a r e n t h e s e s d e n o t e t h e n u m b e r o f d e t e r m i n a t i o ~ on w h i c h thv, m<,an v a l n e is bam.~l.
'~L~.BLI.; I I
Uptake of ascorbic acid ( L-rcyIoa.~.'eorblc a "~) a.ud isoascorbic acqxl (D-araboaxccrrbic acid) by ffuinea. Tig lenses
Aseorb~e acid.
Uptake (in r a g / 1 0 0 g lena tissue)
4"69 (~) -4=0-34 .
isoascorbic acid
4-'10 (8) -~-b0.42
Im~ubatio~ ~ a s for I0 m i ~ at 37~C in I0 m l bsotonic bt:ffer ( p H 6.8) ~t~iTxing 5 m g a.seorbio acid or ~oascorbic aei~ attd 2 m g homocyszeh.,e. Previous to tlae experiment the l e ~ had beolt ~ o ~rod in iso~onic saILuo at 3~C for 48 hr. Result~ are givert as meatt value~ with standard error~4 the figures in parentheses denoto the n u m b e r of determisaatiorL~ on which the m e a n is baa~I. N o significa n0 difference w a g o b s e r v e d be~wc~a n~e~rts.
178
It.. E .
I.IUGflE:3
A1N]) 'l"a a ~
11. J .
]IURLI~,k~
Ill
Effec~ of p H o~. .uptake: of ascorbic acid and delq/droascorbic avid by isolated gz,..in~, jr;,y eye lens
t)}[
A s c o r b i c at:id upt.'tke (mg/lO0 g ti~uc)
A 5.2
P, fi-o
C t;.n
4-20 (7)
4-:~I iS) :~ 0-1,t
-1-93 (~i) :~ 0-42
4-~:1 (3) :'-.0- I S
:1-5-1 (;5~ _~ 0-24
:.EO--15
1 ) e h y d r o a ~ c t ) r b i c a c i d * tll)l~k~ (mg/lt'lO g tissue)
4-60 14) •:3-0-3(~
T h e i n c u b a t i o n w a s t})r l 0 vain a t 370C' i~ 10 ml o f t h e u . p p r o p r i a t e lruffer c , ~ n t a i n i r g / i m g a.~corbie a c i d (or d e h y d r o a s c o r b i e a c i d ) v,n d 2 m g l~omocyuLeirm (or homoserit~e). Previt~us t o the- e x p e r i t m , t t t t h e le.nses h a d l~een s t o r e d i n i s o t o a i 0 s M i n o for 4 5 hx at. 3"C. l l e s u i t ~ a r e gi~-~:n ~u m e a n v a i m . s w i t h sta:~4a:-d er~v)rs; t h e f i g a r e a ~n p a r e n t h e s c ~ d e n o t ~ the. n u r n b , w o f d e t e r m i n a t l o n : , oft w h i c h tJ~e m e a n is b , , , - l . * S i g n i f i c g n e e level for d i f f e r e n c e o f m e a n s b e t w e e n g r o u p s A a n d C =-~ <:5';.~> I c';,.
TAtl I.I; i V
Ix.fleet o f sloraqe time a.J~d ,to.raffe nterliu.m on t/,c .sub.seque~,t t~jdake ~t/' ¢1.s-,:c,rbA; a¢'id b~,! lelt.ses f r o m !/o".~,',J and old flu ifu'. l)[ys
G~'oup
A 1~ C D E J~"
A g o ,o£ g u i n e a pigs (months)
:t :t 2>2,t 3 .'> 24 :~
btorage xnediu:n
i s o t o o i c atlhJe, 3 ' t" lsvto~fie aalh~e, 3~C lsot,.mic s a l i n e , 3 : C t~.rcbs-lRi~Jger b u f f e r 3:(.'. K r e b s - I l i n g e r b u I I b r 3 ~(; 2isotonic s:t|ine, 3~0
As.-:t,rbie aci,I upuLke (mg!ll)t)~ tissue
.qt~,t-a,~. trout, (tlay.~)
:2-58 (6) :5. 0--t7
! 3 :t 1; :¢ 7
5.4& (6)~-~' 0"61
3-[14 l 1 "89 8- 73 9"7I
(?):--0-SS (6)q- 0-44 (6) :b. 0- 33 (4)~_ l "34
A f t e r s~or~ge t h e l e n s w a s i n c u b a t e d , f o r l 0 r a i n at 37°C ".~ith l 0 m ! i s o t o n i c p h o s p h a t e b u f l b r ( p t [ 6-8) e o n t . a i n i n g 5 m g ~ s e o r b i e a c i d a n d 2 m g h o m o c y s t . e i n e . 2R.emfl~s ~ r e g i v e n a~ m e a n v a l u e s w i t h sl.~mdanl e r r o r s ; t h e figttre~ i n p a r e n t h e s e s de~tete t h e n u m b e r o f d e t e r m i n a t i o n s o n w l - i e h t h e 2heart is b a s e d . J ' < I ~ / o for diffcrer, ce of m e a n s o f r e | t o w i n g g r o u p ~ : A at,d I5; B ~nd Jr'; I ) a n d E .
4. D i s c u s s i o n
Available evidence indicates ~ha~ vitamin C preferentially crosses biological membrane,~ in the oxidized tbrm---,~ finding tb-ut has beex~ af.tributed to the gre~t.er solubili W of dehydro~scorbic acid in the lipid component(s) of mere branes (Riiihii, 1956; Martin, 1961; van .L{eyningen, 1970). Thus in the guine~ pig e~3~hroeyte there is vi~'tually no ~ptake of a~scorbie acid under conditions gha~ permit r~pid uptake of dehydroascorbie a n d (Hughes and Maton, 1968). Tile in vitro experiments summarized above however, clearly suggest t h a t both in the rat and the guinea pig the
IN VITRO
UPTAKE
OF
ASCORBIC
ACID
179
eye lens is freely permeal~le to both ascorbic acid and dehydroascorbic acid, and fm't,hermore, that t.'ae u p t a k e of ascr)rbic acid is somewhat, more r a p i d than t h a t of dehydroascorbic acid (Table I). I t m a y also be noted t h a t the p e r m e a b i l i t y of the guinea pig lehs to both "ascorbic acid and dehydroascorbic acid a p p e a r e d to l)e gI~ater t.han, that~ of the r a t lena ; so, too, the guinea pig er3rt}trocyte is m u c h more permeable to dehydroascorbic acid titan is t h e r a t eryghrocyte (Hughes a n d M a t e s , 1968). However, vhe stere-o-specificity displayed b y the e r y t h r o e y t e m e m b r a n e t o w a r d s dehydroascorbic a q d a n d dehyd~oisoascorbic acid, was a b s e n t in the, case of the lens (Table Ill. N o difference could be d e r a o n s t r a t c d bet~:een the u p t a k e of ascorbio acid at,(t isoascorbic acid {compounds which differ in tim positions of the H a n d O H groups ou ca.rbon 5); in the case of the ery~hrocyte, dehydroascorbic acid crosses the memt)ranc rapidly but dchydro/soaseorbic acid does not (Hughes a n d M a k m , t968). t'attersml (1965) has concluded t h a t t h e process of glucose tratmport in the lens is ,~imilar to the process for the transport, of glucose in the e r y t h r o 0 y t e ; the results summarized in Tables I a n d I I however, indicate t h a t in the case of ascorbic acid titere are obvious differences in ~he m e m b r a u e permeability properties. q'he uptake of dehydroascorbic acid b y the lens would however a p p e a r to t)e l)H-dependent (Table II1), a phenomenon which has a parallel ia t h e case of ~he e r y t h r o c y t c and which is Drobably most satisfactorily explained iu terms of changes in the degree of ionization of certain spatiaI~y-cri_ticM grottpings within the m e m b r a n e l~()re~. A n y discussion of m e m b r a n e properties of the lens must, as P a t t e r s o n ha.~ ~'emil~ded us, necessarily remain highly tlmoreticaI as one is deMing nob with a, single, ~hscrete, definable bou'hdary, b u t with three different poten~iM p e r m e a b i l i t y barriers .....the capsule: the epithelium a n d the fibre m e m b r a ~ e s (Patterson, 1965). The results recorded in this paper could reflect passage across the capsule aloi~e and accumulation at" ~scorbic acid and dchydroascorbic acid in t h e extracelt{flar space rattler t h a n ~,omplete passage int.o the lens fibres. This hob'ever is unlikely as both the k n o w n sta.bility of ascorbic acid in the lens, and the reduction of del~ydroascorbic acid to use orbit acid b y the lens protein thiol suggest th~t~ normally diffusion of ascorbic arM and dehydroascorbic ac~i4 into the eye lens is complete. Storage of the-tenses in isotonic m e d i u m significantly increased their p e r m e a b i l i t y to a.scorbic acid (Table IV, groul)s A, B ~ d F). SVorage-induced. change.g in the biological properties of systems are of eouxse not u n k n o w m Of greater interdst in the ease of" i:,]le eye lens is t h a t the n a t u r e of the storage m e d i u m exerted a m a r k e d effect upon t.he subsequen$ p e r m e a b i l i t y of the lens to aseorbic acid. Thus lenses stored in Idrebs-Idinger buffer for 3 d a y s displayed an aseorbic acid-uptake twice thug of lenses stored in isotonic sMine for the same period (Ta.ble IV, groups 13 a n d D). The ascorbic acid u p t a k e b y lenses stored in saline for 7 d a y s was less t h a n t h a t of those stored in K rebs-t2.inger for only 3 d a y s (groups D and F). I t / . s conceivable t h a t t h e effect of storage m e d i m u is a t least in p a r t a reflection of changes induced.initially in the e x t r a cellular s~pa.ce. Such an e x p l a n a t i o n would, however, require ar~ accumuIation of ascorbic acid in the cxtraceliular space, a l ~ p p e n i n g , which, as indicated above, is unlikely to occm'. ]~urthermore, fairly eonsii:lerable changes ia the volume of the extracellu3ar fluid would be necessary ix) account for t h e subst~ntiM increax,~ in u p t a k e of ascorbic acid b y "steered" l e n s e s ~ a l t h o u g h in t h e absence of extracellular space measurements, such a .possibility cannot, be completely discounted. F u r t h e r groups were included to deCerrniae whether this "in vit.ro" ageing effe~V had a eounVerpart in t,erms of n a t u r a l ageing. Lenses from two groups of o l d guinea pigs (:>2g m e n tits) were stored, in saline or Krebs-:Ringer a n d t h e s u b s e q u e n t u p t a k e
180
R, E. HUGHES AND R.. J. H U R L E Y
of ascorbic acid measured° Here, to(,, storage in Krebs-R.inger resulted in grcate.r uptakes of ascorbic acid than storage in ~Mine but in both c~ses ~hc ttpt, akcs were considerably below those obtained with lenses from young anima.ls (TaMe I I [, group.'; C and E ) , ~ a finding th~,.t has a possible, eomlterpar~ in the absorpt, ion of as('orbit: acid fl'om the gastrointestinal tract of the guinea pig where a neg~tt,ive ('orrelation with age has been indicated (Hughes a n d Jones .... unpublish(,d dat, a). T i m , the in vitro ageing would appear to be a quite separate pheno~mnon from the p(,rlno, al)ility changes t,h a t a c c o m p a n y n a t u r a l ageing. I t is of interest to recall t h a t in cataract, ous eyes (in hmnans, an a.ge-eorrel;~ted condition) lowered ascorbie acid levels h a v e been reported (He'tt.h, 1962). Falls in lcnticulm' ascorbic acid are associa.t.cd too with cataracts induced by microw~Lvc t~rcatmer~t and b y X - r a y s (Carpenter, 1962; Pirie and van Hcy~ingcn, 1953). X-ra.ys h~vc of course been used b y certhiu investigators to simubm~ or a.ccelcratc cert, a.in somatic changes held to be characteristic of the, ageing process, a,nd it is lX)S.~ibie t.hat tlte changed aseorbic acid levels in eat:m~ct are 0, redection of a.n a.ccent:uar.~:,,1 ~.geassociated change in the permcabili~;y of the tens membranes. Pat.teflon an(I B u n t i n g (t964) have indeed d e m o n s t r a t e d t h a t iu the case of experinmntally-induced cat.armLet i31 y o u n g anima.ls there is a reduced p e r m e a b i l i t y to cert;fin small molecule,-.. T]uu'a ia ~./o evidence, however, bhat changed p e r m e a b i l i t y to ascorbic acid is a ca.us;d 5toter in the incidence of eat.araet. As H e a t h has pointed out, the d i m i n i s h e d levels of a~cort)ic. acid observed in ea~aractous lenses are consequential changes rat.her t h a n causal one~ (Heath, 1962), H e a t h relat, ed t h e m to ehtmges in the rnet.abolic processes in t.h~', eataractous lens b u t t h e y could e q u a l l y well be a consequence ot' ~, ctumged ~ e m brau,~ p e r m e a b i l i t y similar in n a t u r e to the age-correhd)ed ones described in this rei)¢)rt:. The effect of changes in lens p e r l n e a b i l i t y (whether age-corrclm;cd or cataractous in origin) upon lenticular v i t a m i n C level r e m a i n s to be de~ermined. F u r t h e r m o r e t,hc anomalous b e b a v i o u r of the lens m e m b r a n e s when compared wit.h more clearlydefinable st,ruct;ares such as the eryt, hroeyte m e m b r a n e , imposes severe limitation.~ upon its use as a model s y s t e m for pemneabiiit.y sgud.ies. ACKN O W L E D G M E N T This work was supported by a Medical Research Council Grant. REFEItENCES Carpenter, E. L. (1962), Quoted by Winter, ~b".C. in ~rch. Ophthal.mol. (Chicago) 75, 713. Heath, H, (1962), Exptl Eye/i~es. I~ 362. van Heyningea, R. (1979). Exjotl Eye/~es. 9, 38. /-Iughes, R. E. (1956)..~i~hem. J. 6!t~ 203. Hughes, R. E. and Matxm, S. C. (1968).~Brgt. J. Haematol. 14, 247. Kinoshita, J. H. (1964). Arch. 02ohthaZmol. (Chicago) 72~ 554. Martin, G. tl. (I961). Ar~u. ALJz. Acad. ,Sci. 92, 141. Ptitterst)n, 5. W. (1965). SymTosium on the Le~t.s, p. 253. tgd. by J. E. liar fla. St.. Lo~ds. Patterson, J. W. an4 :Bunting, K. W. (1964). Prec.. goc. Exptl Biol. Med. 1315, ] 156. Pirie, A. and vau He)'ningen, R.. (1953). Bioche-m. J. 54~ 682. Riiihii, N. (I958). Ae2a physioL Sca~,M. 45~ suppl. 155 p. 24.