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Free nucleotides of the lens in human senile cataract
Exptl Eye Re.s. (1969) 8, 4 0 1 - 4 0 5
Free
Nueleotides
of the Lens
in Human
Senile
Cataract*
~.JIOVANNI IX.I.ARAINI, FllANt'.'E,SCO CAII'I'A, I.~UCIANO JJRO.~JPEI~.I AND ANt; b:l,O ])F:~SCA'I'OI¢.I
Unicersity E//e Clinic, Parma, Italy (t?,'ceivcd 16 April 196"9, Lo~,do~.) The tot al ¢'ontertt of free ttu¢.lcotides is deerott~t.-d i~ compleLt: cortical cataract, us compared t~> il~<~o~,ph.tecortic~d opacitio~ ~tld Lo ttucle~xr ~tll(| ~:apsulttr c0,t~rt~cts; this d c c t ' e ~ is prob~tbly accompanied b'¢ tit*; di~ppt;araricc of UDP coe,zymes. No t~pprecitd}le ditl~rences ~l'e, Ol~ tilt" contr0.r2,', fotl/|¢] ill iota] triphospllat~ nurfleotide coalmrtt (as relbrre, d to dry weight) at~(l in the percentage distribution of adet~it~e nt,elcx~tid,:s. Tim t~omlibiliLyis ¢xJtmidered that t },e AT}" docrease Imv h-~as, detect~:¢l it~ complete <'orti<-al cttta~ract, is riot littked to ~t decrease.. in phospho~,latiot, btlt l~robabiy to a 1o~.~by leakage during the- terminal dc.'stt'ttct.it)tt of the
1. ]zJtroduction
"l'lw },roblei,, o f ener,Ay l,r¢,duct.ior~ is o b v i o u s l y of c o n s i d e r a b l e i m p o r t a / m e in t h e s~u(ly of t he n~ctabolic clm/lges t,h a t accotn]~any th e dcvelopt~tent of lens o p a c i t i e s , d u e to its intim~tte relationship t~, p r a c t i c a l l y all o f th e m o s t impor"cant m e t a b o l i c a c t i v i t i e s ,,f a cell. Previou.~ s~udies f r o m t h i s l a b o r a t o r y (M~r~dni, S a n t o r i a n d C a r l a ; 1967) have. s h o w n t h a t , in senih; c ~ t a r a c t , e n e r g y r e s e r v e s (as i n d i c a t e d b y AT:P level) u n d e r g o r e l a t i v e l y little c h a n g e u n t i l t h e l a t e s t a g e s of t h e disease, t h u s c o n f i r m i n g in m a n w h a t h a d b e e n d e m o n s t r a t e d by otb.er ~ u t h o r s in e x p e r i m e n t a l c a t a r a c t s ( K u c k , 1962; Kletifi, 1966; Sippel, 1966~. M o r e o v e r , a d i f f e r e n t b e h a v i o u r was r e c o r d e d d e p e n d i n g o n t h e clinical t y p e of t h e c a t a r a c t , t h e f,d] in t h e A T P level a p p e a r i n g o n l y w h e n t h e lens c o r t e x (i.e. t h e m o s t m e t a b o l i c a l l y a c t i v e p a r t o f t e n s fibres} wo~s completel3~- i a v o l v e d by t h e o p a c i t i e s . N u c l e a r , c a p s u l a r a n d i n c o m p l e t e ~cortical cataracZs h a d A T P leveI (as r e f e r r e d t o b o t h w e t w e i g h t a n d p r o t e i n s ) t h a t d i d n o t differ s i g n i f i c a n t l y f r o m t h o s e r e c o r d e d in n o r m a l lenses. On t h e o t h e r h a n d , t h a t t h e A T P d e c r e a s e in c o m p l e t e v o r t i c a l c a t a r a c t is real, a n d n o t s i m p l y l i n k e d to t h e i n c r e a s e in lens w a t e r , h a s a l r e a d y b e e n s h o w n b y M a l o n e a n d M a r a i n i (1968). I n t h e i n t e r p r e t a t i o n of t h e s e d ~ t a , in a d d i t i o n t o a p o s s i b l e d e c r e a s e of h i g h - e n e r g y b o n d p r o d u c t i o n , t h e p o s s i b i l i t y h a s t o be c o n s i d e r e d o f o t h e r m e c h a n i s m s o f A T P d e p l e t i o n , as for e x a m p l e t h e loss in t h e e x t r a l e n t i c u l a r s p a c e tl~rough t h e d a m a g e d cell m e m b r u n e s a n d le~s c a p s u l e d u r i n g d i s r u p t i o n of t h e fibres. I n r e c e n t y e a r s s o m e r e p o r t s h a v e a p p e a r e d s u g g e s t i n g t h a t t h i s c o u l d weLI be t h e case f o r o t h e r subs t a n c e s ( C h a r l t o n a n d ~ a n H e y n i n g e n , 1968), a n d for t h i s r e a s o n a s t u d y on t h e possible c h a n g e s o f t h e w h o l e n u c l e o b i d e s p e a t r u m o f t h e lens in t h e d i f f e r e n t t y p e s o f senile c a t a r a c t w a s d e e m e d of s o m e i ~ t e r e s t . * This investiga£ion was supported by a research grant 115.2323.0 4776 from the Coasiglio kNa~ion~le de|le Ricerche. 401
402
G. M A R A I N I ,
]". C A R T A ,
L. P R O S P E R I
A N D A. P E S C A T O R I
2. M a t e r i a l s and M e t h o d s Bovine a n d rabbi~ lenses were o b t a i n e d i m m e d i a t e l y after the d e a t h of the animals; senile c a t a r a c t s were o b t a i n e d after i n t r a c a p s u l a r extraction. The lenses were imnmdlately frozen at --196°C a n d stored a t this t e m p e r a t u r e until used. As in previous investigations, senile c a t a r a c t s were divided, d e p e n d i n g on the in r i v e bion~icroscopicaI aspect, into different groups a c c or di ng to the location and the extension of the opacities: I - - I n c o m p l e t e cortical c a t a r a c t s involving a b o u t one-half of the cortex. l I - - C o m p l e t e cortical cataracts. I I I ~ N u c [ e a r c a t a r a c t s (with clear cortex) and posterior capsular c a t a r a c t s (the r('maifider of the lens being practically free from opacities). A b o u t 8 g of lens (wet weight) were used for. eacl" experiment. The frozen lenses were weighed and homogenized in a fourfold volume of ice-cold 0-6 x perchloric ac.id (PCA). The h o m o g e n a t e was centrifiaged in a refrigerated centrifuge at 3000 rev /min for 15', the .~upern a t a n t was d e c a n t e d , anti the residue was again e x t r a c t e d with 0-2 .~" PCA and centrifuged as beforE. The s u p e r n a t a n t s were pooled, neutralized with 5 .~" K O I t and alh)wed to s t a n d o v e r n i g h t at 2°C. The potassium perch]orate was centrifuged out and the ext ract.~ were p u t on an anion excha'nge resin c o h m m of Dowex-1 (200-4(0 mesh, length 22 cm. d i a m e t e r 1-2 cm). After the nucleotide solution bad run th ro u g h the resin, tlle column was was]ted with several volumes of distilled w ater until the e l u a t c no longer absorbed at 260 mtt. G r a d i e n t elution with formic a c i d - a m m o n i u m f~)rmate was t h e n performed (Caldarera. Budini, Barbiroli and Rabbi. 1962). The m i x i n g flask contained at the start .)00 ml of w a t e r a n d the following solutions were successively fed into it from a reservoir: 0-5 .x~ formic acid (500 ml), ] .~t formic acid (500 ml), 2 M formic acid (500 ml). -t .~t formic aci,! (500 ml), 0.2 M a m m o n i u m f or m a t e in 4 ,~x formic acid (900 ml), 0.,t .~t : u m n o n i u m formate in 4 ~t formic acid (500 ml), 0-8 .~t a m m o n i u m formate in .I ~ formic acid (250 ml). Fra,:tions of 5 nd were collected. On each fraction the absorption at 260 and 275 mt~ was d e t e r m i n e d with a [ : n i t am .qP 500 s p e c t r o p h o t o m e t e r ; the different peaks were identified according to tlurlberr, Schmitz. 13rumm and P o t t e r (1(,)54). The absorption values .,at: 276 rote were of help in the interpret,ation of the profiles since tlie ~'27s: E,2Go ratio is characteristic of the differe,~t nucleotides The molar e x t i n c t i o n values of know n pure nucleotides were used for the calculation of t.he coiment.ration of the pe a k. Three e x p e r i m e n t s were carried out, on bovine lenses, two on rabbit lenses, one on iltcomplet~ cortical c a t a r a c t s , two on complete cortical c a t a r a c t s and one on :mclear and posterior c a t a r a c t s .
3. Results and D i s c u s s i o n T h e r e s u l t s a r e s u m m a r i z e d i n t h e T a b l e s a n d i n F i g s 1-5. F r o m t h e a n a l y s i s o f t h e s e d a t ~ a c e r t a i n n u m b e r o f c o n s i d e r a t i o n s arise. T h e first c o n c e r n s t h e v a l i d i t y of cons i d e r i n g as " n o r m a l " w i t h r e g a r d t o h u m a n c a t a r a c t o u s lenses t h e r e s u l t s o b t a i n e d in t h e lenses o f d i f f e r e n t a n i m a l s ( d u e to t h e ob~dous i m p o s s i b i l i t y o f o b t a i n i n g a n a d e q u a t e n u m b e r of n o r m a l h u m a n lenses); t h e r e s u l t s of p r e v i o u s i n v e s t i g a t i o l l s ( M a r a i n i e t al., 1967) s h o w i n g a n o r m a l A T P c o n t e n t p e r g p r o t e i n in c a t a r a c t o u s l e n s e s as c o m p a r e d to n o r m a l h u m a n lenses, a n d t h e p r e s e n t d a t a o n A T P c o n t e n t (/z.~t/g d r y weight,) in h u m a n c a t a r a c t o u s a n d n o r m a l a n i m a l lenses s h o w t h a t t h i s d e a r l y is n o t t h e case. M o r e o v e r , a p p r e c i a b l e d ifferen ces m a y be n o t i c e d b e t w e e n t h e free n u c l e o t i d e s p e e t r u m l o f r a b b i t a n d b o ~ i n e lenses; in r a b b i t lenses, f u r t h e r m o r e , a n u n i d e n t i f i e d p e ~ (no. 5) h a s b e e n r e c o r d e d w h i c h w a s n o t f o u n d a t all i n b o v i n e
]:I~]~;E
NUC],I,',O,I:I
D].,,_
OF
/,1,;N8
IN
27A a L E
I~UMAN
,S],]NIL~;
CATAI%A(
1
40:1
[
Achl-soh~ble .free nudeotides of h u m a n cxctaraetous and normal animal len.s'e.s" (values expressed as txmoles/lO0 (t dr]/wt) ] ncomplele cortical .N ut:~eot id(:s
Qata
('omph,l~. cortical
f i t ~ t,.'S
Nuclear atul capsular
C { t ~ l Far." t.%
(%)
caLara
(%)
CM l ' N A I)
27-o
A 31 t' (_;.%1I' l" .~I | ' I .M 1' AD}'
54 ~ 94; 7.3 -. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49--~ 3S-4 2G- I
L" I ) } ' A ( ;
1."I ) P( ;
(;])]' (q'}' (U].)I') ATI'
11)54 ~
16..!
13.8
. . . . .
-. . . . . . . . . . . . . 13~.~-2 151.7
. .
. . . . . . . 112-7
86" 1
] e I 11-542S
l(abbit% ] 12l l S ( ? s
( . ,.o~ ...
( o,,.o)
15- !
34-4
] 28' 1
35"3 t racers t races t rac~.:s 76.G
39.7 traces traces traces 99"4
12 .¢J
. 4.q.2
174.3
31-3
~(~-0
Boviue*
(%)
t rac(,~
( ;TI'
9.,5
63"4
108-0
62"7
1041 trac<:~ 210-9 55.b;
9-7 t r a "ees 272-9 42-1)
48.0
29.1
103-0
1" T I '
L,ucle<,~ ide.,
e t~
43-7
405.u
30~ .9
234-9
35:t-(;
579.0
756.5
*_; _' ). '-"~_ ")
_'~ ."", ,-- -~'
187-7
277.3
314 -7
344-0
Triphosphat~. l ' l l l ( ' ] t ~ ( ) l I( ~ . ~
* 3|~,~ valuea frolu three exp(,thoent~. "~ .Mean v a l u e s f r o m t w o cxl)~, tcnt-s. The following ahbreviation~ are tts,~: CMI', cytidine monophosphatc; ~NAD n i c o t i n a m i d e a d c t a i n e d i " t u c l e o t i d e ; AM1", a d e n o s i n e m o n o p h o s p h a t ¢ ' . : G M P g u a n o ~ i r m m o n o p h o s p h a t ¢ ; UStP, uridine monophosphate : ISIP, inoaine naonophosphat~ : ADP, adenosine diphosphate; UD/~AG, tu-idine diphosphate aeetylgluct~mine: U J D ] ' G , u r i d i n e d i p h o s p h a t e g | u t x ) s e ; G D ] ). g u a n o s i n e d i p h o s p h a t ~ . ; C"£P, c V t i d i n e t r i p h o s p h a c e ; U D I ' , uridiJ~e d i p h o s p h a t e ; A T P , a d e n o ~ i u c t r i p h o . ~ p h a t e ; G T P , g u a n o s i n e t r i p h o s p h a t e ; UTP urid~ne triphob~)hate.
Pe~'rz,n~age distribution of adenine nucleotides i~ hunmn, catara~ous and normal ani~m! le~se.,_"
Nucleol.lde
Incomplete cortical eataract~
Complete cortical cataracts
(%) ASI.P AT)P AT]?.
9"5 95.7 71-6
(%) 4-8 19.2 75-8
~'uclear and capsular cataracts
(%) 4-9 17-8 77-1
5.1 20-8 74-0
Bo~-ine lenses
Rabbit lenses
(%)
(%)
10.9 23-7 65-4
9-6 24.1 66-2
-t04
O. M A R A I N I , !.8 _ 1"6
~'. C A R T A ,
L. PROSPEI~I
AND
A. P E S C A T O R - I
2
IO
1"4
P
I-2I'0 -
otD 0.8 U4 0"6-
4
0'4 " 0.2-
3
Fr~t~ H20
5
/ ~ A
Form.am 0-SM
II 12
~6 788 9 j
700 800 600 Form.oc.4~+ Fo,'m.oc.4M + Am.fccmO4M A m . f o r m O 8M
100 200 300 400 500 Form.ac, Form.a6~'. F o r m . a t . Form no. 4M -4II< 2M 5M A m . f o ~ m 0.2 M
F]G. 1. C h r o m a t o g r a m f r o m r ~ h b i t lenses. (1) C M P ; (2) N A D ; (3) A M P ; (4) A D P ; (5) non.identL c o m p o u n d ; (6) U D P A G , (7) U D P Q : (8) G D P ; (9) C T I ' ( U D P ) ; (10) A T P ; (11) G T P ; (1 ° ) U T P .
IO
1"6 1"4 I2 oE FO r
,+2 4
t,j
6+7
0.6t -
0.4 ~ 0-2 _
Froclion
0
100 Form.oe. 0-5 M
HzO
_
_
200
Form.oc. |M • "]O.
300
Form,oc, 2M
±
400
Form.oc. 4 M
500
600
V o r t n o c . 4 u JrAm.form 0 2M
700
800
Fo~m,oc.4M4- FOrm.OC4M 4,~-'TLformO'4M ;~rn.formOSM
2. C h r o m a t o g r a m from b o v i n e lenses.
0.7
I0
0.6
=Lo.5 oE 0.,¢
4
0-2 0-1
~
~ _
I
Fractlen 0
H~o
.....
I
500 100 2O0 4CO300 Form,at, Forrr~oc. Form.at. FOCmLaC. Form.ac. 4 M + 0,5 M IM 2M 4M /~m.form. 0'2 M ~c.
.__
I
!
coo
.
.
.
-zoo
.
auo
Focm.oc.4U+ Form ~ 4 ~ + J~'n.,form 0 4 M Am.loom 0-8 M
3. C h r o m a t o g r a m f r o m n u c l e a r a n d c~psular senile c a t a r a c t s .
°-7I
I0
0"6
::tO.5
o.4 F
14-2
.
Froctk)n 0
H2o
100
200
Forn~oc.
Form.oc.
0-SM
(M
300 Fo,'rn.oc, 2M
400
Form.at. 4 M
500 Form.oc. 4 e + Am.for:re.O-2 M
.
~oo
.
.
!
zoo
eoo
FormaC.4M+ Forrr~oc. 4 M + A m . ' f o r m 0 4 M Am.forrn.O.SM
:Fzo. 4. Chromatogrnax f r o m i n o o m p l e t e corticM senile catars0to.
F I { , E F NU(.'I, E O T I D E S
OF LIgNS IN HUMAN
SENILE
CATAFCAf:T
405
0-7
0"6
Io
~.0.5 0O4 ID
02 OI
I-t-2
10o
Froction 0
F120
Form oc. 05M
200 300 400 500 Form.ac. Form.oc. Form.oc. Form.at 4M + IM
2 M
ZI'M
A m . f o r r n . 0 2 ta
600
700
800
FOrm.aC.4M ~- FOrm.o(;.4 M + A m f o r m 0 4 M ,am.form.OaM
Fro. 5. Chromrttogram frr,m COml)let.o cortical senile cataract.
],roses. I t is therefore considered Ix) be much more reliabh-, in the present disctLssion to limit the analysis, as far as l)ossiMe, t~ the variations e v e n t u a l l y found within the t h r e e grou[~s o f h u m a n
cataractous
lenses.
1"o1" these reasons it is imi,ossiMe to state if total free nucleotide c o n t e n t is normal in rmclcar and posterior (.,apsular cataracts, and in the earl), seages of cortical catara('t~" w h a t can, on the c o n t r a r y , be aflirtned with reasonable c e r t a i n t y is t h a t total ~'~)rtical ol)acification of the senile lens is accompanied by a m a r k e d fall of t o t a l tYee nu(.leotides with respect to the earlier phases of the disea~se. This decrease of free rluc:leot.i(les seem~ to be charact~.,rized b y the lack of apprcciabh: a m o u n t s of U'DP nu('h,othles in the (:hromatogram; these mmleotides of t h e metabolic type, wkich are well rcpres(:nted in normal bovine and rabt)it lenses, m a y on tbe c o n t r a r y be detected, ~b(m~h in limited amounts, in the other types of senile c a t a r a c t a n d in incomplete cortical opacities. Total c o n t e n t of t r i p h o s p h a t e nucleotides (referred to d r y weight) does not show ;great ,lifferenees between the three groups of c a t a r a c t o u s lenses; in a n y case, t,he lowest value is found in total cortical cataract, the highest in nuclear a n d capsular forms. The percentage d i s t r i b u t i o n of adelxine nueleotides (Table II) does liot show differ('nees in the three groups of c a t a r a c t o u s len~ses and. is p r o b a b l y n o r m a l (a c e r t a i n decrease of A M P is found with respect to bovine a n d r a b b i t lenses but, for tile reasons previously considered, this difference is difficult to i n t e r p r e t ; a t a n y rate, no relative increase of A M P m a y be recorded). This seems to rule our the possibility t h a t t h e decrease of high-energy bonds p e r lens, p r e v i o u s l y reported in complete cortical cataract (Maione a n d Maraini, 1968), is l i r ~ e d to a decrease of p h o s p h o r y l a t i o n . T h e present data, a n d t h e above reported eom~iderations, are n o t against, t h e possibility of a loss by leakage of these c o m p o u n d s in a d v a n c e d cortical c a t a r a c t d u r i n g t h e termfiml disruption of the lens fibres. REFERENCES Caldarera, C. 3L, Budizfi, R., Barbiroli, A. F. and JRabbi, A. (1962). CaTw~erRe.s.22~ 1026. Charlton, d. M. a n d v a n H e y ~ i n g e n , 1~. (1968). Exptl Eye Rex. 7, 47. I~[urlbert, R. B., ScJamitz, I=L, B r u m m , A. F. a n d P o t t e r , V. R . (1954). J . B/eL Chem. 200, 23. Klethi, d. (1966). Q u o t e d b y N o r d m a m a , J . (1965). Invest. Ophthalmol. 4, 385. :Kuck, J . F., J r . (1962). Invest. Ophthal.moL 1, 390. ]~Iaione, M_ a n d l ~ a i n i , G. (1968). Bull. Sac. Fran~. Ophtalmol. Paris, 81, 303. Maraini, G., Santori, M. a n d Carts, F. (1967). Exptl ,Eye Re~'. 6, 126. Sippol,.T.O. (1966). !nve~t. OphthalmoL 5, 576.
Free
Nueleotides
of the Lens
in Human
Senile
Cataract*
~.JIOVANNI IX.I.ARAINI, FllANt'.'E,SCO CAII'I'A, I.~UCIANO JJRO.~JPEI~.I AND ANt; b:l,O ])F:~SCA'I'OI¢.I
Unicersity E//e Clinic, Parma, Italy (t?,'ceivcd 16 April 196"9, Lo~,do~.) The tot al ¢'ontertt of free ttu¢.lcotides is deerott~t.-d i~ compleLt: cortical cataract, us compared t~> il~<~o~,ph.tecortic~d opacitio~ ~tld Lo ttucle~xr ~tll(| ~:apsulttr c0,t~rt~cts; this d c c t ' e ~ is prob~tbly accompanied b'¢ tit*; di~ppt;araricc of UDP coe,zymes. No t~pprecitd}le ditl~rences ~l'e, Ol~ tilt" contr0.r2,', fotl/|¢] ill iota] triphospllat~ nurfleotide coalmrtt (as relbrre, d to dry weight) at~(l in the percentage distribution of adet~it~e nt,elcx~tid,:s. Tim t~omlibiliLyis ¢xJtmidered that t },e AT}" docrease Imv h-~as, detect~:¢l it~ complete <'orti<-al cttta~ract, is riot littked to ~t decrease.. in phospho~,latiot, btlt l~robabiy to a 1o~.~by leakage during the- terminal dc.'stt'ttct.it)tt of the
1. ]zJtroduction
"l'lw },roblei,, o f ener,Ay l,r¢,duct.ior~ is o b v i o u s l y of c o n s i d e r a b l e i m p o r t a / m e in t h e s~u(ly of t he n~ctabolic clm/lges t,h a t accotn]~any th e dcvelopt~tent of lens o p a c i t i e s , d u e to its intim~tte relationship t~, p r a c t i c a l l y all o f th e m o s t impor"cant m e t a b o l i c a c t i v i t i e s ,,f a cell. Previou.~ s~udies f r o m t h i s l a b o r a t o r y (M~r~dni, S a n t o r i a n d C a r l a ; 1967) have. s h o w n t h a t , in senih; c ~ t a r a c t , e n e r g y r e s e r v e s (as i n d i c a t e d b y AT:P level) u n d e r g o r e l a t i v e l y little c h a n g e u n t i l t h e l a t e s t a g e s of t h e disease, t h u s c o n f i r m i n g in m a n w h a t h a d b e e n d e m o n s t r a t e d by otb.er ~ u t h o r s in e x p e r i m e n t a l c a t a r a c t s ( K u c k , 1962; Kletifi, 1966; Sippel, 1966~. M o r e o v e r , a d i f f e r e n t b e h a v i o u r was r e c o r d e d d e p e n d i n g o n t h e clinical t y p e of t h e c a t a r a c t , t h e f,d] in t h e A T P level a p p e a r i n g o n l y w h e n t h e lens c o r t e x (i.e. t h e m o s t m e t a b o l i c a l l y a c t i v e p a r t o f t e n s fibres} wo~s completel3~- i a v o l v e d by t h e o p a c i t i e s . N u c l e a r , c a p s u l a r a n d i n c o m p l e t e ~cortical cataracZs h a d A T P leveI (as r e f e r r e d t o b o t h w e t w e i g h t a n d p r o t e i n s ) t h a t d i d n o t differ s i g n i f i c a n t l y f r o m t h o s e r e c o r d e d in n o r m a l lenses. On t h e o t h e r h a n d , t h a t t h e A T P d e c r e a s e in c o m p l e t e v o r t i c a l c a t a r a c t is real, a n d n o t s i m p l y l i n k e d to t h e i n c r e a s e in lens w a t e r , h a s a l r e a d y b e e n s h o w n b y M a l o n e a n d M a r a i n i (1968). I n t h e i n t e r p r e t a t i o n of t h e s e d ~ t a , in a d d i t i o n t o a p o s s i b l e d e c r e a s e of h i g h - e n e r g y b o n d p r o d u c t i o n , t h e p o s s i b i l i t y h a s t o be c o n s i d e r e d o f o t h e r m e c h a n i s m s o f A T P d e p l e t i o n , as for e x a m p l e t h e loss in t h e e x t r a l e n t i c u l a r s p a c e tl~rough t h e d a m a g e d cell m e m b r u n e s a n d le~s c a p s u l e d u r i n g d i s r u p t i o n of t h e fibres. I n r e c e n t y e a r s s o m e r e p o r t s h a v e a p p e a r e d s u g g e s t i n g t h a t t h i s c o u l d weLI be t h e case f o r o t h e r subs t a n c e s ( C h a r l t o n a n d ~ a n H e y n i n g e n , 1968), a n d for t h i s r e a s o n a s t u d y on t h e possible c h a n g e s o f t h e w h o l e n u c l e o b i d e s p e a t r u m o f t h e lens in t h e d i f f e r e n t t y p e s o f senile c a t a r a c t w a s d e e m e d of s o m e i ~ t e r e s t . * This investiga£ion was supported by a research grant 115.2323.0 4776 from the Coasiglio kNa~ion~le de|le Ricerche. 401
402
G. M A R A I N I ,
]". C A R T A ,
L. P R O S P E R I
A N D A. P E S C A T O R I
2. M a t e r i a l s and M e t h o d s Bovine a n d rabbi~ lenses were o b t a i n e d i m m e d i a t e l y after the d e a t h of the animals; senile c a t a r a c t s were o b t a i n e d after i n t r a c a p s u l a r extraction. The lenses were imnmdlately frozen at --196°C a n d stored a t this t e m p e r a t u r e until used. As in previous investigations, senile c a t a r a c t s were divided, d e p e n d i n g on the in r i v e bion~icroscopicaI aspect, into different groups a c c or di ng to the location and the extension of the opacities: I - - I n c o m p l e t e cortical c a t a r a c t s involving a b o u t one-half of the cortex. l I - - C o m p l e t e cortical cataracts. I I I ~ N u c [ e a r c a t a r a c t s (with clear cortex) and posterior capsular c a t a r a c t s (the r('maifider of the lens being practically free from opacities). A b o u t 8 g of lens (wet weight) were used for. eacl" experiment. The frozen lenses were weighed and homogenized in a fourfold volume of ice-cold 0-6 x perchloric ac.id (PCA). The h o m o g e n a t e was centrifiaged in a refrigerated centrifuge at 3000 rev /min for 15', the .~upern a t a n t was d e c a n t e d , anti the residue was again e x t r a c t e d with 0-2 .~" PCA and centrifuged as beforE. The s u p e r n a t a n t s were pooled, neutralized with 5 .~" K O I t and alh)wed to s t a n d o v e r n i g h t at 2°C. The potassium perch]orate was centrifuged out and the ext ract.~ were p u t on an anion excha'nge resin c o h m m of Dowex-1 (200-4(0 mesh, length 22 cm. d i a m e t e r 1-2 cm). After the nucleotide solution bad run th ro u g h the resin, tlle column was was]ted with several volumes of distilled w ater until the e l u a t c no longer absorbed at 260 mtt. G r a d i e n t elution with formic a c i d - a m m o n i u m f~)rmate was t h e n performed (Caldarera. Budini, Barbiroli and Rabbi. 1962). The m i x i n g flask contained at the start .)00 ml of w a t e r a n d the following solutions were successively fed into it from a reservoir: 0-5 .x~ formic acid (500 ml), ] .~t formic acid (500 ml), 2 M formic acid (500 ml). -t .~t formic aci,! (500 ml), 0.2 M a m m o n i u m f or m a t e in 4 ,~x formic acid (900 ml), 0.,t .~t : u m n o n i u m formate in 4 ~t formic acid (500 ml), 0-8 .~t a m m o n i u m formate in .I ~ formic acid (250 ml). Fra,:tions of 5 nd were collected. On each fraction the absorption at 260 and 275 mt~ was d e t e r m i n e d with a [ : n i t am .qP 500 s p e c t r o p h o t o m e t e r ; the different peaks were identified according to tlurlberr, Schmitz. 13rumm and P o t t e r (1(,)54). The absorption values .,at: 276 rote were of help in the interpret,ation of the profiles since tlie ~'27s: E,2Go ratio is characteristic of the differe,~t nucleotides The molar e x t i n c t i o n values of know n pure nucleotides were used for the calculation of t.he coiment.ration of the pe a k. Three e x p e r i m e n t s were carried out, on bovine lenses, two on rabbit lenses, one on iltcomplet~ cortical c a t a r a c t s , two on complete cortical c a t a r a c t s and one on :mclear and posterior c a t a r a c t s .
3. Results and D i s c u s s i o n T h e r e s u l t s a r e s u m m a r i z e d i n t h e T a b l e s a n d i n F i g s 1-5. F r o m t h e a n a l y s i s o f t h e s e d a t ~ a c e r t a i n n u m b e r o f c o n s i d e r a t i o n s arise. T h e first c o n c e r n s t h e v a l i d i t y of cons i d e r i n g as " n o r m a l " w i t h r e g a r d t o h u m a n c a t a r a c t o u s lenses t h e r e s u l t s o b t a i n e d in t h e lenses o f d i f f e r e n t a n i m a l s ( d u e to t h e ob~dous i m p o s s i b i l i t y o f o b t a i n i n g a n a d e q u a t e n u m b e r of n o r m a l h u m a n lenses); t h e r e s u l t s of p r e v i o u s i n v e s t i g a t i o l l s ( M a r a i n i e t al., 1967) s h o w i n g a n o r m a l A T P c o n t e n t p e r g p r o t e i n in c a t a r a c t o u s l e n s e s as c o m p a r e d to n o r m a l h u m a n lenses, a n d t h e p r e s e n t d a t a o n A T P c o n t e n t (/z.~t/g d r y weight,) in h u m a n c a t a r a c t o u s a n d n o r m a l a n i m a l lenses s h o w t h a t t h i s d e a r l y is n o t t h e case. M o r e o v e r , a p p r e c i a b l e d ifferen ces m a y be n o t i c e d b e t w e e n t h e free n u c l e o t i d e s p e e t r u m l o f r a b b i t a n d b o ~ i n e lenses; in r a b b i t lenses, f u r t h e r m o r e , a n u n i d e n t i f i e d p e ~ (no. 5) h a s b e e n r e c o r d e d w h i c h w a s n o t f o u n d a t all i n b o v i n e
]:I~]~;E
NUC],I,',O,I:I
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OF
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IN
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I~UMAN
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1
40:1
[
Achl-soh~ble .free nudeotides of h u m a n cxctaraetous and normal animal len.s'e.s" (values expressed as txmoles/lO0 (t dr]/wt) ] ncomplele cortical .N ut:~eot id(:s
Qata
('omph,l~. cortical
f i t ~ t,.'S
Nuclear atul capsular
C { t ~ l Far." t.%
(%)
caLara
(%)
CM l ' N A I)
27-o
A 31 t' (_;.%1I' l" .~I | ' I .M 1' AD}'
54 ~ 94; 7.3 -. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49--~ 3S-4 2G- I
L" I ) } ' A ( ;
1."I ) P( ;
(;])]' (q'}' (U].)I') ATI'
11)54 ~
16..!
13.8
. . . . .
-. . . . . . . . . . . . . 13~.~-2 151.7
. .
. . . . . . . 112-7
86" 1
] e I 11-542S
l(abbit% ] 12l l S ( ? s
( . ,.o~ ...
( o,,.o)
15- !
34-4
] 28' 1
35"3 t racers t races t rac~.:s 76.G
39.7 traces traces traces 99"4
12 .¢J
. 4.q.2
174.3
31-3
~(~-0
Boviue*
(%)
t rac(,~
( ;TI'
9.,5
63"4
108-0
62"7
1041 trac<:~ 210-9 55.b;
9-7 t r a "ees 272-9 42-1)
48.0
29.1
103-0
1" T I '
L,ucle<,~ ide.,
e t~
43-7
405.u
30~ .9
234-9
35:t-(;
579.0
756.5
*_; _' ). '-"~_ ")
_'~ ."", ,-- -~'
187-7
277.3
314 -7
344-0
Triphosphat~. l ' l l l ( ' ] t ~ ( ) l I( ~ . ~
* 3|~,~ valuea frolu three exp(,thoent~. "~ .Mean v a l u e s f r o m t w o cxl)~, tcnt-s. The following ahbreviation~ are tts,~: CMI', cytidine monophosphatc; ~NAD n i c o t i n a m i d e a d c t a i n e d i " t u c l e o t i d e ; AM1", a d e n o s i n e m o n o p h o s p h a t ¢ ' . : G M P g u a n o ~ i r m m o n o p h o s p h a t ¢ ; UStP, uridine monophosphate : ISIP, inoaine naonophosphat~ : ADP, adenosine diphosphate; UD/~AG, tu-idine diphosphate aeetylgluct~mine: U J D ] ' G , u r i d i n e d i p h o s p h a t e g | u t x ) s e ; G D ] ). g u a n o s i n e d i p h o s p h a t ~ . ; C"£P, c V t i d i n e t r i p h o s p h a c e ; U D I ' , uridiJ~e d i p h o s p h a t e ; A T P , a d e n o ~ i u c t r i p h o . ~ p h a t e ; G T P , g u a n o s i n e t r i p h o s p h a t e ; UTP urid~ne triphob~)hate.
Pe~'rz,n~age distribution of adenine nucleotides i~ hunmn, catara~ous and normal ani~m! le~se.,_"
Nucleol.lde
Incomplete cortical eataract~
Complete cortical cataracts
(%) ASI.P AT)P AT]?.
9"5 95.7 71-6
(%) 4-8 19.2 75-8
~'uclear and capsular cataracts
(%) 4-9 17-8 77-1
5.1 20-8 74-0
Bo~-ine lenses
Rabbit lenses
(%)
(%)
10.9 23-7 65-4
9-6 24.1 66-2
-t04
O. M A R A I N I , !.8 _ 1"6
~'. C A R T A ,
L. PROSPEI~I
AND
A. P E S C A T O R - I
2
IO
1"4
P
I-2I'0 -
otD 0.8 U4 0"6-
4
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3
Fr~t~ H20
5
/ ~ A
Form.am 0-SM
II 12
~6 788 9 j
700 800 600 Form.oc.4~+ Fo,'m.oc.4M + Am.fccmO4M A m . f o r m O 8M
100 200 300 400 500 Form.ac, Form.a6~'. F o r m . a t . Form no. 4M -4II< 2M 5M A m . f o ~ m 0.2 M
F]G. 1. C h r o m a t o g r a m f r o m r ~ h b i t lenses. (1) C M P ; (2) N A D ; (3) A M P ; (4) A D P ; (5) non.identL c o m p o u n d ; (6) U D P A G , (7) U D P Q : (8) G D P ; (9) C T I ' ( U D P ) ; (10) A T P ; (11) G T P ; (1 ° ) U T P .
IO
1"6 1"4 I2 oE FO r
,+2 4
t,j
6+7
0.6t -
0.4 ~ 0-2 _
Froclion
0
100 Form.oe. 0-5 M
HzO
_
_
200
Form.oc. |M • "]O.
300
Form,oc, 2M
±
400
Form.oc. 4 M
500
600
V o r t n o c . 4 u JrAm.form 0 2M
700
800
Fo~m,oc.4M4- FOrm.OC4M 4,~-'TLformO'4M ;~rn.formOSM
2. C h r o m a t o g r a m from b o v i n e lenses.
0.7
I0
0.6
=Lo.5 oE 0.,¢
4
0-2 0-1
~
~ _
I
Fractlen 0
H~o
.....
I
500 100 2O0 4CO300 Form,at, Forrr~oc. Form.at. FOCmLaC. Form.ac. 4 M + 0,5 M IM 2M 4M /~m.form. 0'2 M ~c.
.__
I
!
coo
.
.
.
-zoo
.
auo
Focm.oc.4U+ Form ~ 4 ~ + J~'n.,form 0 4 M Am.loom 0-8 M
3. C h r o m a t o g r a m f r o m n u c l e a r a n d c~psular senile c a t a r a c t s .
°-7I
I0
0"6
::tO.5
o.4 F
14-2
.
Froctk)n 0
H2o
100
200
Forn~oc.
Form.oc.
0-SM
(M
300 Fo,'rn.oc, 2M
400
Form.at. 4 M
500 Form.oc. 4 e + Am.for:re.O-2 M
.
~oo
.
.
!
zoo
eoo
FormaC.4M+ Forrr~oc. 4 M + A m . ' f o r m 0 4 M Am.forrn.O.SM
:Fzo. 4. Chromatogrnax f r o m i n o o m p l e t e corticM senile catars0to.
F I { , E F NU(.'I, E O T I D E S
OF LIgNS IN HUMAN
SENILE
CATAFCAf:T
405
0-7
0"6
Io
~.0.5 0O4 ID
02 OI
I-t-2
10o
Froction 0
F120
Form oc. 05M
200 300 400 500 Form.ac. Form.oc. Form.oc. Form.at 4M + IM
2 M
ZI'M
A m . f o r r n . 0 2 ta
600
700
800
FOrm.aC.4M ~- FOrm.o(;.4 M + A m f o r m 0 4 M ,am.form.OaM
Fro. 5. Chromrttogram frr,m COml)let.o cortical senile cataract.
],roses. I t is therefore considered Ix) be much more reliabh-, in the present disctLssion to limit the analysis, as far as l)ossiMe, t~ the variations e v e n t u a l l y found within the t h r e e grou[~s o f h u m a n
cataractous
lenses.
1"o1" these reasons it is imi,ossiMe to state if total free nucleotide c o n t e n t is normal in rmclcar and posterior (.,apsular cataracts, and in the earl), seages of cortical catara('t~" w h a t can, on the c o n t r a r y , be aflirtned with reasonable c e r t a i n t y is t h a t total ~'~)rtical ol)acification of the senile lens is accompanied by a m a r k e d fall of t o t a l tYee nu(.leotides with respect to the earlier phases of the disea~se. This decrease of free rluc:leot.i(les seem~ to be charact~.,rized b y the lack of apprcciabh: a m o u n t s of U'DP nu('h,othles in the (:hromatogram; these mmleotides of t h e metabolic type, wkich are well rcpres(:nted in normal bovine and rabt)it lenses, m a y on tbe c o n t r a r y be detected, ~b(m~h in limited amounts, in the other types of senile c a t a r a c t a n d in incomplete cortical opacities. Total c o n t e n t of t r i p h o s p h a t e nucleotides (referred to d r y weight) does not show ;great ,lifferenees between the three groups of c a t a r a c t o u s lenses; in a n y case, t,he lowest value is found in total cortical cataract, the highest in nuclear a n d capsular forms. The percentage d i s t r i b u t i o n of adelxine nueleotides (Table II) does liot show differ('nees in the three groups of c a t a r a c t o u s len~ses and. is p r o b a b l y n o r m a l (a c e r t a i n decrease of A M P is found with respect to bovine a n d r a b b i t lenses but, for tile reasons previously considered, this difference is difficult to i n t e r p r e t ; a t a n y rate, no relative increase of A M P m a y be recorded). This seems to rule our the possibility t h a t t h e decrease of high-energy bonds p e r lens, p r e v i o u s l y reported in complete cortical cataract (Maione a n d Maraini, 1968), is l i r ~ e d to a decrease of p h o s p h o r y l a t i o n . T h e present data, a n d t h e above reported eom~iderations, are n o t against, t h e possibility of a loss by leakage of these c o m p o u n d s in a d v a n c e d cortical c a t a r a c t d u r i n g t h e termfiml disruption of the lens fibres. REFERENCES Caldarera, C. 3L, Budizfi, R., Barbiroli, A. F. and JRabbi, A. (1962). CaTw~erRe.s.22~ 1026. Charlton, d. M. a n d v a n H e y ~ i n g e n , 1~. (1968). Exptl Eye Rex. 7, 47. I~[urlbert, R. B., ScJamitz, I=L, B r u m m , A. F. a n d P o t t e r , V. R . (1954). J . B/eL Chem. 200, 23. Klethi, d. (1966). Q u o t e d b y N o r d m a m a , J . (1965). Invest. Ophthalmol. 4, 385. :Kuck, J . F., J r . (1962). Invest. Ophthal.moL 1, 390. ]~Iaione, M_ a n d l ~ a i n i , G. (1968). Bull. Sac. Fran~. Ophtalmol. Paris, 81, 303. Maraini, G., Santori, M. a n d Carts, F. (1967). Exptl ,Eye Re~'. 6, 126. Sippol,.T.O. (1966). !nve~t. OphthalmoL 5, 576.