E:*TJ. Eye Res. (1987) 45, 191-195

L E T T E R TO THE E D I T O R S

W a t e r G r a d i e n t s A c r o s s B o v i n e L e n s e s

The age d e p e n d e n c e of the non- f reezeable w a t e r c o n t e n t s h o w e d d i f fe ren t t r e n d s in h u m a n (Lahm, Lee and Be t t e lhe im, 1985) t h a n in r a t lenses (Castoro and Be t t e lhe im. 1986). A l t h o u g h the to ta l w a t e r c o n t e n t in bo th species decreases wi th age, especia l ly in t he nucleus , t h e non- f reezeab le (bound) w a t e r c o n t e n t increases in the r a t lens and decreases in the h u m a n lens wi th age. This showed d i f fe ren t i n v o l v e m e n t of syneres is in the ag ing .of the two species.

In bo th of the above s tudies lenses were o b t a i n e d f rom ind iv idua ls of d i f fe rent -ages and the w a t e r c o n t e n t s of t he cor tex , i n t e r m e d i a t e l ayer and the nucleus , were ana ly sed as a func t ion of chronologica l age. Howeve r , t he re exists also an in te rna l age i n d i c a t o r in the lens, since t he lens grows t h r o u g h o u t life. Most bu t no t all o f the g r o w t h is a c c o u n t e d for by an increase in the co r t ex (R.afferty. 1985). T h u s a g r a d i e n t going fi'om the a n t e r i o r (or poster ior) sur face to the c e n t e r o f the nuc leus a long the visual axis provides and ' i n t e rna l age g r a d i e n t '. M e a s u r e m e n t of such an ' i n t e rna l age g r a d i e n t ' would e l imina te the va r i a t ion due to the ind iv idua l i ty and d i f fe ren t h i s to ry of the samples . H o w e v e r , one m u s t be careful in c o m p a r i n g the " in ternal a g e ' index wi th t he chronologica l age of the lens. N e i t h e r o f t h e m is a t r u e age index. Tim ' i n t e r n a l age ' index would t r u l y ref lect the ag ing process if t he re would be rio c o m m u n i c a t i o n s be tween the t iber 'cells , no fluxes of wate r , me tabo l i t e s , etc. across the m e m b r a n e . U n d e r those condi t ions the first cor t ical fiber cell would be ze ro-year - old and the nuc leus would be the chronological age; the d i f fe ren t w a t e r g r a d i e n t s be tween t h e m would reflect the aging process. B u t because of water" fluxes across the m e m b r a n e s ne i the r t he d i f fe ren t wa t e r molecules in the nuc leus have the chronologica l age of t he an imal , nor t he equ i l ib r ium condi t ions be tween the d i f fe ren t w a t e r molecules m a y be t he s ame as prevai led a t b i r th . T h e chronologica l age o f the lens s imi la r ly can be re fe r red on ly to the nucleus .

Bov ine eyes (2-years-old) were ob ta ined f rom s laugh te rhouses . The lenses were worked up wi th in 3 hr. The lenses were placed in a r e f r ige ra ted m i c r o t o m e a t - -10°C and sec t ioned f rom an t e r i o r (or poster ior) sur face to t he nucleus. Six- to twen ty - f i ve - mi l l ig ram samples were i m m e d i a t e l y and h e r m e t i c a l l y encapsu l a t ed in p re -we ighed coa ted a l u m i n i u m sample pans. These were used to m e a s u r e t he f reezeable (free) w a t e r c o n t e n t o f t he lens by di f ferent ia l s cann ing c a l o r i m e t r y (DSC).

F o r t he d i f ferent ia l scann ing c a l o r i m e t r y (DSC) a he rme t i ca l l y sealed e ~ p t y coa ted a l u m i n i u m pan se rved as a . re ference . T h e s ample and re fe rence pan was j ) laced in a DSC ( D u P o n t 990, D u P o n t : Wi lming ton , D E ) cell and cooled to --30°(3 b y an ex t e rna l d r y ice ba th . DSC curves were o b t a i n e d by hea t i ng the sample a t a p r o g r a m m e d ra te of 3°(3 rain -~ to 30°C. The i n s t r u m e n t was ca l ib ra t ed wi th a s apph i r e disc and the DSC cell ca l ib ra t ion c o n s t a n t was o b t a i n e d per iodical ly . The DSC curves r eco rded the d i f ferent ia l h e a t flow (q) as a f unc t i on of t ime. The q was r e c o r d e d s i m u l t a n e o u s l y w i t h two d i f fe ren t sensi t ivi t ies , for example , 0 -5 mV cm -~, high sens i t iv i ty , and 10 mV cm -1, low sens i t iv i ty . T h e a r e a u n d e r the cu rve gives t h e n u m b e r o f J of h e a t used to m e l t the m e a s u r e d mass o f wa te r . Since it was our i n t en t ion to t r a n s l a t e the a rea of an e n d o t h e r m (in J per g sample) to a ce r ta in a m o u n t of f reezeable w a t e r per g sample , we ran a ca l ib ra t ion cu rve wi th dist i l led w a t e r and

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with aqueous NaCI so lu t ions wi th d i f ferent c o n c e n t r a t i o n s hav ing d i f fe ren t me l t i ng (freezing) points (Be t t e lhe im, Chr is t ian and Lee, 1983).

Af t e r t he DSC m e a s u r e m e n t s , t he tota l w a t e r c o n t e n t o f t he lens was m e a s u r e d . T h e pans were p u n c t u r e d , t a k i n g care no t to d i s tu rb t he samples . The pans were n e x t p laced in a t h e r m o g r a v i m e t r i c ana lyse r ( D u P o n t 951) a n d the to ta l w a t e r c o n t e n t o f t he samples ob t a ined f rom the we igh t loss which o c c u r r e d du r ing hea t ing the palls to and m a i n t a i n i n g t h e m a t 105°C. T h e non- f reezeab le w a t e r c o n t e n t was o b t a i n e d as t he di f ference be tween t h e t o t a l a n d freezeable w a t e r c o n t e n t , and i t was expressed as a p e r c e n t a g e of the to t a l w a t e r con ten t .

W A T E R G R A 1 ) I E N T S A C R ( ) S S B O V I N E 1 , E N S E S 193

T h e d e p e n d e n c e o f t h e d i f f e r e n t w a t e r c o n t e n t s o n t h e p o s i t i o n a l o n g t i m v i s u a l a x i s w a s o b t a i n e d b y r e g r e s s i o n a n a l y s i s u s i n g t h e M i n i t a b I I p r o g r a m in a P r i m e c o m p u t e r ( P r i m e : B o s t o n , M A ) . T h e s t a t i s t i c a l s i g n i f i c a n c e o f e a c h r e g r e s s i o n a n a l y s i s w a s e s t a b l i s h e d b y t h e s t a n d a r d S t u d e n t ' s t t e s t .

'I'A m,~: 1

Re.qression. analysis of lhe different water contents as a function, of distance ( i n r a m ) front the surface (anterior or posterior) of bovine lenses along the visual axis

la, t~s No. In tercept a Slope h S.D. r ~

Total water % of lens weight

1 79"4 -- 10'9 2"37 0"810* 2 75"2 - - 8"55 1 "37 0"867" 3 77"6 -- 3"86 0"23 0"963" 4 74" ! -- 6'09 I '23 0"891 * 5 79'5 -- 5" ! 0 0'49 0 '948" it 73"9 --5" ! 1 0"65 0"925*

F'reezable water % of lens weight

I 64-8 -- 12.7 3.27 0.752¢ 2 59.3 -- 10.9 !- 16 (}.936" 3 64.5 -- 5.26 0.35 0.954* 4 61-8 -- 7.86 1"71 ()'876~ 5 68"5 -- 6"23 0"47 0"966" 6 60"8 -- 5.57 ~ 0"46 0.967"

Non-freezable water % of lens weight

1 14"6 1"78 (H16 0"407§ 2 15"9 2-38 1"08 0"447~ 3 13" I 1 "41 0"36 0"579" 4 12"3 ! "77 ! "85 0"234 5 ! 1 "0 1 "07 (t"26 0"741 6 13"2 0'46 0"45 0" 177

Non-fi 'eezable water % of total water

1 ! 6"6 8"06 2"58 0"6ti I "}" 2 19"3 8"64 1"35 0"872* 3 15"6 3"72 0"59 11"784" 4 ! 5"8 5" 19 2'44 0"601 ~: 5 12"5 3"58 0"38 0"937" 6 16"2 3"10 0"70 0"796¢

Statist ically significant at, the 99-9(*), 99(t), .q5($), 90(§) % confidence levels.

T h e t o t a l w a t e r c o n t e n t g r a d i e n t o f a t y p i c a l b o v i n e l e n s is g i v e n in ]~ig. 1, t h e f r e e z e a b l e w a t e r c o n t e n t g r a d i e n t in F i g . 2 a n d t h e n o n - f r e e z a b l e w a t e r c o n t e n t in

F i g . 3. T h e c o r r e s p o n d i n g r e g r e s s i o n c o e f f i c i e n t s , s t a n d a r d d e v i a t i o n s a n d c o r r e l a t i o n c o e f f i c i e n t s a r e p r e s e n t e d in T a b l e 1 f o r e a c h o f t h e b o v i n e l e n s e s i n v e s t i g a t e d .

T h e t o t a l w a t e r c o n t e n t ( a s p e r c e n t a g e o f t h e l e n s w e i g h t ) h a s a n e g a t i v e g r a d i e n t

( i .e . d e c r e a s e s g o i n g f i ' o m c o r t e x t o n u c l e u s ) a s h a s b e e n s h o w n a l s o b y P h i l i p s o n

( 1 9 6 9 ) in r a t l e n s e s . T h i s i m p l i e s t h a t d u r i n g m a t u r a t i o n o f l e n s f i b e r s , w a t e r is l o s t

t o a c c o m m o d a t e a d e n s e r p a c k i n g o f l e n s p r o t e i n s . S i m i l a r l y t h e f r e e z e a b l e (fi-ee) w a t e r c o n t e n t o f t h e l e n s f r o m c o r t e x t o n u c l e u s a l s o h a s a n e g a t i v e g r a d i e n t . T h i s in t u r n

w o u l d i n d i c a t e t h a t w h a t e v e r w a t e r w a s l o s t f r o m t h e l ens f i b e r s d u r i n g ' a g i n g ' w a s

f l ' e e z e a b l e ( f r e e ) w a t e r . T h e n o n - f r e e z e a b l e ( b o u n d ) w a t e r p e r c e n t a g e o f b o v i n e l ens , o n

194 J . A . I ' A S ' i ' ( ) i { ( ) A N D I; .A. I l I ' : T T E I , H E I M

t i le otJ ier Jl i ind, Jlii,~ ll ilo.~iliv~. • 7 i - l id io i t t . i.t+. ili(,l-l,il.~t,s l~fl i l ig f l 'oin i.Ol' l t ,x l~ liUl,Jl,llS. "rhi,~ l n l s i t i v e g r t l d i en t is evi~n lnoro l i ron l )un( .ed wJlt, i i the non-tYeozettlJJe w a t e r i~ ~,alctuhll:i;d |is il llt, l '( .(,nl o f t im wat t , r ( .o l i to l i t (T i i t l l t , i ) .

( ) l ie l l l l i y h l i e r l l r e t the i l o s i l i v e ~i ' t ldi t , l i l (iJ" ilOll-[Fl'l,t+ziiJih, wi i le l " i.( int(, l l t, il,~i li i:Oli,¢ei'sion i i~ rl 'eozeliblo (J'l'O(t) wi t t t#r to noi i- | ' r t , i~zl,ill l le ( h o u n d w a t e r ) due to t i le ll i l l.J¢lil~ l i t l l ro t i , in liioh,l.uh,.~, l ' ]s imoi l t l ly i r the i i l le l¢ ing i,~ | in l igg i 'e t~ l t ion l l l 'ol ,e~s i h i ' o u g h e o v i i h , n t l l o n d i n g o r h ln -d i l l o le etu. h l te r i l c ' t ions , w a t e r n io lecu les t h a t had son ic t i ' i i n s J a t i l i n i i J - r o t a t i o n a i J'roiJdonl tJl'eViOtiSl,V w i l l IlL, en t ra l )pOd, lose these r i . l ,edolns to lnoi'(J r i g id t ied .~yinnil , l l ' i l . l l l o i ' ~ t i i i i z t i t i on and I ) l ;(~onle l i o n - ~ ' r e e z e a l j i o wtttOl',

Tl i l i .~ i loL o n l y t i le i i l~gt i t ive t o l i l i wiit(~l' ~ i ' i t l l i t , i iL b u t iil.~(i the l ) (mit iv l ; l lOli- f reezeal l le w i i te l ' g r i i d i l ; i i t Pi'oln c o r t e x to nuoJeils (<'ontl'iinlto,~ to the t o t a l r e f r a c t i v e h i d e x g r a d i e n t t h a t is llet;t~.sstiry to the re | ' rae i i ve l lower o f lll~, lens ( B e t i e l h e h n . l!),%5), .411 illt.l'ell,~,(; in the non- f i 'eezealde w a t e r Ool l te l l t |'i'Olit (~()l'tex to nuc leus, in essence, m a k e s the to ta l re f rac t ive index g r a d i e n t smal le r than it wouhi be i | ' on ly the ehailge in t o t a l w a t e r i~ontont, wouh l lie the e o n t r i l n i t i n g the tor .

T h i s s t u d y r l : -eml i l las izes t i l t ; nel.'l.,ssit.y ill" ( i i s l i n ( , t i on l iet.wet,n e l l l 'onologi (mi agillg o f the lens |is a who le l ind the ag ing due: to lens g r o w t h ~ le l i lonst ra te( i in the cor tex to n twleus gradients .

A ( ; K N ( ) W I , E I ) ( ; M E N T

This work was SUPllorted b y an N I H g r a n t No. EY 02571.

l)ep.rlmenl of ('hvmi.uh'!I, ,l o i l N A. (JANTOII() A N I) . .ldelphi t"niversity, I~'liI,;I)I,HIItJK A. J{I,71'TI,H,III,:IM (#,mh >. (,'it!y. N )" 11530, IT.N.A.

(Accepted 16 Jlt irch 195'7)

P, I.: I," E R E N(~ I ']S

l {e t te lhe inL F. A. (1985). l)t lysieal basis o f lens t ranstmrelmy. In The Oc .h . . l.e..~. ,%'h'm'l.re. Fum' l ion aml l 'al lmlofl!L (Ed..~!aisel, H.). Pp. 2(i5-3(1(1. 31. l ) , ;kker l tw : New Y . r k .

i {e t te lhe im, I,'. A. (197 I). E. r l . ; r ime. ta l l~hysical (.'hemi,slr!t. P. 149. Saumlers : l~hiladell lhia. i { t , t te lheim, F. A.. Chr is t ian. S. and l,ee, I,. K. (19g3). l) i t l 'erential st 'a iming , .a lor imetr ie

lll{,i_lSUl'(-llll'lltS ()ll h l i ln i l l t Jl'llS. (..'ll#'r. l~lff: 1~1".'~. 2. ~(),'{--N. (Tas toro . . l .A . alld l {et te lheim, !". A. (i!)8G). l ) i s t r i hu t i on , f t h ( ; to ta l and llon-t'reezable water

in ra t lenses. Exp. Eye Re.~'. 43, 185-91. l ,a i l ln . I)., Lee, I,. K . and l~etteiheim, F. A. (1985). Age dependenve of fi'eezabh; and non-

f'reezalfle water c(intent of normal human lenses. I,ce.st. Ophlhalmol. l'is. Nci. 26. 1162-5 .

Monk. G.S . (1963). Liffht. Prim:|ides aml ExlJerimr'nt.~ (2rid edn). Pp. 456-7. Dover l 'uldishing The: New York

i~hi l ipson. B. (1969). D is t r i bu t ion ot* l)rotein w i t h i n the normal ra t lens. lnce.~l. Ophthalmol. 8, 258-70.

Rafferty, N .S . (1985). Lens Morphoh~gy. In The Ocular Len.~, ,S'tructure, Fanclio~, a~td Palholoffy. (Ed. Maisel, H.). Pp. 1-60. M. l)ekker Inc: New York.

A p p e n d i x

T h e l )rocess t h a t increase s t i l e n o n - f r e e z e a b l e w a t e r c o n t e n t as o n e p r o c e e d s f rom c o r t e x t o n u c l e u s can be ca l led an i n v e r s e s y n e r e s i s . I t i n f l u e n c e s t i le ref l -act ive i n d e x

Please send reprint requests to l)r F. A. Bettelheim

WATER GRADIENTS ACROSS BOVINE LENSES 195

of the lens along the visual axis. A qual i ta t ive appreciat ion of this can be seen from ihe following considerations:

(1) The refractive index of water decreases when one goes from freezeable (free) water to non-freezeable (bound) water. The refractive index of free water a t 37°C is 1"331 while tha t of bound water, assuming ice-like structure, is 1"309 (ordinary) and 1"3104 (extraordinary) with sodium light (Monk, 1963).

(2) As a first approximation, we may assume tha t the molar refractivities in the lens are addit ive. If so, the following equation may apply (Bettelheim, 1971):

n ~ - - l M l ~_v n ~ - - l M 1 na 2 - 1 M 3 nsoln--1 P~°~nXln~ +2 Pl n~+2 P2 ~'Xana2+2 Pa

= ( 1 ) n~ol, + 2 X~ M 1 + X 2 M 2 + XaM~

where u is the refractive index and the subscripts 1,2,3 and soln refer to the fi'ee water, bound water, lens proteins and to the lens, respectively. M is the molecular weight, X is the mole fi'action and p is the density. The densi ty of the lens can be

al)l)roximated by : ! = w__21 + w_~ + w..~ (2)

P~o~n Pl P.z P3 where w is the weight fraction.

The right hand side of equat ion (1) increases as the refi'active index of the lens increases. The first two terms of the nomina tor in equat ion (1) shows the contr ibut ion of inverse syneresis to the change of the refractive index along the visual axis. In general, the inverse syneretic process lessens the increase in refractive index moving from cortex to nucleus, tha t would occur if one considers only changes in the protein (,oncentrations along the visual axis. The magni tude of such influence upon the refractive index gradient due to inverse syneresis depends on the relative contr ibut ions of the three terms of the nominator in equat ion (1). A sample calculation using the parameters of lens No.3 in Table I and the following densit ies: 0'99336, 0"99986 and 1.30 for free water, bound water and lens protein, respectively, was performed. The other parameters used were 1.331, 1.309 and 1"50 for the refractive indices of free water, bound water and lens proteins, respectively.

The average molecular weiglit of the lens l)roteins was approximated by using the value of 200000. The calculation yielded the following results:

(1) ~¥ith inverse syneresis [equation (I)] tim refi'active index increases by 0"005 units tbr every mm distance on the cortex to nucleus axis.

(2) Wi thou t inverse syneresis (when the first two terms in the brackets of the nominator are combined and one uses only n I and Pl) the same calculation would yield an increase of 0"00764 units in the refi.active index per mm distance.

This calculation should be taken only as an indication of the direction of the change contr ibuted by the inverse syneresis to the refractive index gra(iient. The basic assumption, i.e. the addi t iv i ty of molar refractivities may not apply to the lens and the density, the refractive index and the molecular weight of the lens proteins used in this calculation may be in error. These, however, do not change the general conclusions tha t inverse syneresis lessens the refractive index gradient produced by a change in protein concentrations, alone. In the above example, inverse syneresis exerted a 33% change in the refi'active index gradient .