Lipid peroxidation as a possible cause of cataract

Mechanisms of Ageing and Development, 44 (1988) 69--89 Elsevier Scientific Publishers Ireland Ltd.

69

LIPID PEROXIDATION AS A POSSIBLE CAUSE OF CATARACT

MARK A. BABIZHAYEV*, ANATOLY I. DEYEV and LEONID F. LINBERG Moscow Heirahoitz Eye Diseases Research Institute, 103064 Sadovaya-Chernogryaz.skaya, 14/19

Moscow (U.S.S.R.) (Received September 9th, 1987)

SUMMARY

The role of free-radical-induced lipid oxidation in the development of human lens opacity was studied. Physico-chemical parameters of the lens fiber membranes at different stages of cataract have been investigated. The deterioration of lens fiber plasma membranes structure preceding formation of large aggregates in lenticular matter, leading to lens opacity, was observed by electron microscopy. Initial stages of cataract were characterized by the accumulation of primary (diene conjugates, cetodienes) lipid peroxidation (LPO) products, while in the later stages there was a prevalence of end LPO fluorescent products. Reliable increase in oxiproducts of fatty acyl content of lenticular lipids was shown by direct gas chromatography technique obtaining fatty acid fluorine-substituted derivatives. The lens opacity degree is found to correlate with the level of the end LPO fluorescent product accumulation in its tissue, accompanied by SH group oxidation of crystallins due to decrease of reduced glutathione concentration in the lens. The injection of LPO products into the vitreous has been shown to induce cataract. It was concluded that peroxide damage of the lens fiber membranes may be the initiatory cause of cataract development.

Key words: Human lens; Cataract; Lipid peroxidation; Lipid layer damage; Aggre-

gates formation INTRODUCTION

Biochemical characteristics of cataract manifestation are the following: (1) formation of large high-molecular aggregates of low solubility in the lens tissue; (2) appearance of "blue" fluorescence of non-tryptophan nature; and (3) disintegration of the lens fiber plasma membranes. *To whom all correspondence and reprint requests should be addressed. 0047-6374/88/$03.50 Printed and Published in Ireland

© 1988 Elsevier Scientific Publishers Ireland Ltd.

70 Traditionally, the lens is considered as a "sac filled with proteins". That is why until now the majority of investigators are searching for the cause of lens highmolecular protein aggregate formation in crystallin physico-chemical properties alteration, in the reduction of the protein solubility, in the change of their amino acid content [1--3]. At the same time, it is well known that the protein aggregate formation in the lens is accompanied by an intrusion of lenticular fiber membrane fragments into cytoplasmic fraction [4]. Besides this, the content of the above-mentioned aggregates was revealed to contain phospholipids which can be found in the lens cytoplasmic protein fraction by phosphorus registration [5]. Since in transparent lens most lipids localize in membranes, we have suggested that processes inducing the damage of lenticular fiber plasma membrane lipid bilayer take part in the aggregation of crystallins. Among endogenous processes which can cause injury to membranous structures of cells and tissues one of the most important is lipid peroxidation (LPO) [6,7]. During aging and, in particular, during the development of senile cataract, activity of enzymic (superoxide dismutase, glutathione peroxidase, catalase) and nonenzymic (ascorbate, cysteine, glutathione) anti-oxidant systems in the lens and aqueous humor is reduced [8--10]. Accumulation of LPO products in the lens may be facilitated by the presence of compounds in the lens which are photosensitizers of free-radical oxidation reactions of the 3-hydroxykynurenine or N-formylkynurenine type, which absorb light in the near UV-region of the spectrum (360--400 mm). These photo-oxidation products of tryptophan under the influence of light can generate active forms of oxygen and products of its successive single-electron reduction (singlet oxygen, superoxide anion-radicals, hydrogen peroxide, hydroxyl radicals), which can be found in the lens tissue and also in the aqueous humor [11]. Several varieties of cataract have been described in the literature, whose mechanisms of development have been linked with the generation of active forms of oxygen. They include psoraline cataract, cataract induced by the action of hyperbaric oxygenation, and cataract arising in animals fed with the catalase inhibitor 3-amino1H, 1,2,4-triazole [ 12-- 14]. On the basis of these facts it has been postulated that LPO may play a role in the etiology and pathogenesis of cataract [15,16]. However, no direct proof of LPO activation has yet been obtained in cataract. Only insignificant amounts of malonyldialdehyde (one of LPO products) were found in human lens in its opacity development in 1981 [15]. However, the method used to determine malonyldialdehyde, based on interaction with 2-thiobarbituric acid, quite suitable in in vitro studies, is of absolutely no use, for quantitative analysis of LPO products in vivo, because of its low specificity. It is also necessary to consider that the malonyldialdehyde level in tissue can be reduced because the latter may be involved easily in cross-linking with phospholipids and can also be degraded by aldehyde dehydrogenase which has been found in the human lens [17]. As is well known, LPO is basically related to the presence of poly-unsaturated

71

fatty acids in the tissues, and it leads to mixtures of products: hydroperoxides with diene conjugated structures, low molecular weight hydrocarbons (ethane, pentane) being specific indicators of the LPO process in vivo. However, in mice with cataract no significant increase in expired ethane was revealed [18]. Absence of reliable differences in diene conjugate content was found at the later stages of the cataract development in comparison with transparent lenses by UV spectrophotometry of lipid extracts from the lens [19]. In the previous work [20] the authors did not demonstrate any significant differences in fatty polyunsaturated acids content in transparent and cataractous human lenses. On the basis of the above mentioned results a conclusion has been drawn that the LPO process involvement in cataract development is hardly probable. Furthermore, one should remember that the literature offers numerous examples of using inadequate methods of LPO product registration leading to false results [21]. The score of methods available for LPO product analysis at present, allows not only the reliable determination of the freeradical-induced oxidation process development in lipids, but also to quantitatively correlate the forming LPO product concentration with the value of their modifying effect upon structure-functional organization of the biomembranes. With regards to this the purpose of the present work was to find direct proofs of unsaturated fatty acid oxidation in cataract, as well as to study the mechanisms of the LPO process modifying effect on the structure-functional organization of the lens membranes in cataract. MATERIALS AND METHODS

The test material consisted of opaque human lenses at different cataract stages obtained during operation by intracapsular cryoextraction. Transparent human lenses were removed from the donor eyes provided by a bank. Before the operation, all the lenses were examined on a slit-lamp and were divided into groups according to clinical characteristic of opacity. The average age of the cataract patients and donors was 65 :t: 9 years. To evaluate objectively the lens opacity by means of biomicroscopy and further photoregistration, an image of the lens was obtained on "Leitz" TV-analyser. The values of the optic density in different lens zones were determined. The image was subsequently divided into zones with pre-set values of optic density. Their areas were measured, the redistribution of the zones over the lens surface was determined and the part of every zone in the whole capacity area of the lens was established. Two characteristics were introduced for quantitative evaluation of lens opacity degree: (1) the opacity intensity characterized with optic density value; and (2) the opacity extent defined as the opacity zone area. The lens image was produced on a TV-analyser. The intensity of the light incident on the lens was selected so as to maintain the light-flow quantity after passing the lens tissue constant. Then, optical density (OD) values were determined in different lens zones. The OD values were set

72 so as to be able to characterize the most dense, the less dense and transparent lens sections and to divide the lens image into zones of indicated OD values (so-called "equidensities"). Integral degree of the lens opacification is estimated as the ratio between the opacity zone area (which corresponds to maximum stages of the lens OD values) and the lens whole surface area, expressed as percentage.

Lipid extraction from the lens Immediately after the lens material had been obtained, lipids were extracted from the lens by Folch method [22]. The extraction was carried out by tissue homogenization in 20 vols. of chloroform/methanol mixture (2:1 by vol.) with 4-methyl-2,6ditert-butyl-phenol anti-oxidant addition (0.5 mg/100 ml) for 10 min. After filtration the sample obtained was put into a separating funnel for 5--8 h to stratify. Water was added in 7:1 ratio to promote the stratification. Temperature was maintained at 0 °C for all the operations. After separation of the phases and removal of the aqueous-methanol layer, the lower chloroform fraction was evaporated. Phospholipid content was assessed by the results of organic phosphorus evaluation [23]. Total lipid amount in the extract was determined gravimetrically, as well as by characteristic absorption in 206--210 nm area of the lipid sample after dissolution in 4 ml of methanol/heptane mixture (5:1 by vol.).

LPO product determination Accumulation of the lipid peroxidation primary products was estimated spectrophotometrically from characteristic absorbents of diene conjugates in the UV-region at 232 nm characterizing the level of hydroperoxides of polyunsaturated fatty acids, as well as by LPO secondary products absorbance at 274 nm, corresponding to the concentration of conjugated trienes and cetodienes [24] on "HITACHI-557" spectrophotometer (Japan). The content of end products of fluorescent LPO was determined from the lipid extract fluorescence intensity at 365 nm excitation and 420 --440 nm emission x~ave-lengths [25], measured on a "HITACHI-MPF-4" spectrofluorometer. The source material content in the samples was equalized by amount of phospholipids. The spectrofluorometer was calibrated at the beginning of every working day against a solution of quininesulfate (1 pg/ml in 0.1 N H2SO4)standard, at 435 nm fluorescence emission and 365 nm excitation wave-lengths.

Gas chromatography o f halogen-substituted derivatives o f the fatty acids Content of the polyunsaturated fatty acids in the lens is rather moderate, hence direct registration of their decrease in the course of LPO is quite difficult [20]. However, evidently to register directly an increase of oxiproducts in unsaturated fatty acids is of rather greater importance than to reveal a decrease in the acid content. In fact, it is the appearance of the fatty acid oxiderivatives as a LPO result in membrane lipid phase, that as is well-known, leads to their destruction [28]. In the applied gas-chromatography method we used not methyl esters of fatty acids but

73

their halogen-substituted derivatives. By fluorine atom introduction into fatty acid molecule we succeeded in selective labelling of its functional groups. Using this property, it is possible to determine the change of the number of oxigroups which gain in content in fatty acids in the LPO course both under in vitro and in vivo conditions. Electron-capture detector used in gas-chromatography of fluorine-substituted compounds is found to be more sensible than flame-ionization one: minimum detectable sample flow in substances with high affinity of the electron, such as fluorine-substituted compounds, for electron-capture detector, is of 10-~3g/s - - that is why this method is the optimum one to measure even lower levels of oxiderivative fatty acids in tissue lipid extracts. For selective determination in the lens lipid fraction of the substances containing oxigroups, fluorine-substituted derivatives of the fatty acids were obtained. For this purpose, after evaporation in the solvent nitrogen current, 100 ~1 of hexafluorine/ isopropanol-benzol mixture in proportion (1:4) and 40/~1 of penta-fluorinepropine acid anhydride were added to every sample of the lens lipids, the mixture was then sealed off and kept at 60°C for 30 min. After this, the ampoule content was evaporated in nitrogen current, the lipid residue was diluted in 150 ~I of isooctane and introduced into "Tracor-560" gas-chromatograph (U.S.A.) equipped with "Tracor770" autosampler (U.S.A.), "FSOT-I" capillary column (U.S.A.) of 50 m length with 0.25 mm inside diameter and also with an electron-capture detector. Hydrogen was used as carrier-gas, with 1:30 flow split. The following temperature conditions were maintained: initial temperature of 140°C (2 min), final temperature of 280°C (15 min), temperature gradient of 3 °C/min. Gas-chromatographic determination of halogen-substituted fatty acid derivatives was carried out by utilizing the method of inner standard by retention time comparison with the standards. "Sigma" fatty acid standards were used in the work.

Electron-microscopic study For electron microscopy, intact lenses were fixed with 4°7o glutaraldehyde prepared in phosphate buffer (0.1 M, pH 7.2u7.4), and then with 1~/00SO 4 solution. Afterwards, the samples were washed with 50 ° ethyl alcohol. Lens dehydration was performed in spirits of rising concentration: 60 °, then in 2010-uranylacetate on 70 ° spirit, 80 °, 96 °, 100 ° spirit. Further, the samples were put in absolute acetone, and then were kept in a mixture of acetone with epone resin (2:1) (1:2). The lenses kept in epone were maintained at 37°C (24 h), then at 60°C (48 h). The sections for electron-microscopy were performed perpendicularly to the surface in the medial lens zone area on a "LKB-200 11" ultramicrotome. The contrasting was done with lead nitrate by Reynolds method. The samples were examined on a "Jem 100B" electron microscope X 20 000. Cataract modelling with lipid peroxides was carried out in rabbits of chinchilla race of 4 months of age. The experiments were conducted in accordance with the ARVO Resolution on the Use of Animals in Research. After inducing anaesthesia

74 under aseptic conditions a microsection of the conjunctiva in 4 mm of the limbus was performed. Exposed sclera was pierced with a stilet-needle of 0.17 mm diameter, and, by means of a microsyringe, 0.05 ml of liposomal suspension containing 0.4 mg of phospholipids (dilinoleoyllecithin or dipalmitoyllecithin (Serva)) was injected into the posterior vitreous of rabbit eyes under indirect binocular ophthalmoscope control. In the case of highly oxidized phospholipids, malonyldialdehyde (MDA) content in the liposomes was 22.2 nmol MDA//amol of phosphorus, and in low oxidized phospholipids it was 2.0 nmol MDA//amol of phosphorus. The content of total and non-protein bound thiols in the lenses was determined with 5,5'-dithiobis-(2-nitrobenzoic acid) reaction [27]. RESULTS

Changes in membrane ultrastructural organization The electron microscopic observations have shown that, when the lens still preserves its transparency and protein aggregates cannot be detected in its tissue, the earliest changes in regular longitudinal lens fibers plasma membranes structure, did occur (Fig. la). They form typical " K n o b s " and "Sockets". Consequently, the lens opacification is preceded by deterioration of the lenticular fiber plasma membrane ultrastructure. Following opacification progression the lens fibers become more irregular, their electron density increases, the membranes form numerous diverticula, twistings, interdigitations, and fragments (Fig. lb). Then further membrane fragmentation and an increase of the membrane fragments' curvature are observed. Figure l c shows typical "wave-like" membrane structures forming multiple whorls around electron-dense centres. Such undulating structures differing by a number of different forms expand eccentrically from the lens fiber. Such formation has "string of pearls"-like shape. At the mature stage of cataract, Fig. ld, which is biochemically characterized by large high-molecular aggregate formation in the lens, ultrastructural characteristic of the membrane lesion is distinct with the lenticular fiber plasma membrane twisted fragments becoming the central mass of amorphous electron-grey debris and globules of different sizes (100m900 nm) and electron-dense granular contents. At the same time, fiber and membrane structures could no longer be identified and fibers appeared to have been converted to extensive masses of globular structures. First, smaller globules (100--200 nm) filled with granular material (probably of protein nature) of about the same density as the lens fiber cytoplasm, form, and then intermediate size globules (220--500 nm) containing aggregates of high density and dispersity appear. The loss of lenticular fiber-free membranes is probably connected with the mentioned membrane involvement in the protein aggregation. The larger globules (>1600 nm) seem to develop from the small via the intermediate globules (Fig. le). At the later stages of the lens opacification, the globules become more and more coiled and aggregated, intermediate filaments disappear. At this stage, the lens matter is filled mainly with protein aggregates. Significant scatter

.o,

76

of light accompanying the protein aggregate formation depends upon the incident wavelength of light in relation to the size and the concentration of globules. Accumulation o f the LPO products in the lens The results of determination of the LPO different molecular products revealed in the lipid extracts from the lens are given in Fig. 2. Typical UV absorption spectra of

Fig. lc

Fig. ld

77

Fig. le Fig. 1. Modification of the plasma membranes of the lens fiber cells by the lens opacification. Electron microphotographs of the midzonal area of the lens. Bar, 1/xa; a - - norm; b,c - - immature cataract; d nuclear cataract; e - - mature cataract.

lipids extracted from the lenses have their maximum in the 206 nm regions corresponding to absorption of isolated double bonds of hydrocarbon chains. In absorption spectra of lipid extracts from lenses with a cataract, there are two additional maxima at 230 and 274 nm. The first of them corresponds to absorption of diene conjugated structures. The maximum at 274 nm corresponds to triene conjugates secondary molecular LPO products. The results of the determination of diene conjugates in lipid extracts from the lenses at different stages of cataract maturation are '0.~

Optical density

Optical 0.sdenslty

I

II

/

Wavele~th

(rim)

Wavelength

(ilm)

Fig. 2. UV absorbtion spectra of lipid extracts from human lenses (methanol/heptane S:I) I - - norm; II - - cataract. The arrow points to the maximum characteristic of the diene conjugates absorbance.

78

presented in Fig. 3, and Table I. It is evident that the content of the hydroperoxides having conjugated double bonds and determined by characteristic maximum in UVspectrum at 230 nm, increases at the initial stages of the opacification up to almost mature stage of cataract. However, at the further stages of mature and hypermature cataract the LPO primary molecular product level drops a little. At the same time, determination of the content of end fluorescent products of the LPO-Schiff bases determined by lipid extract fluorescence intensity at 430 nm (fluorescence excitation, 365 nm) revealed an increase monotonously by cataract development (Fig. 4). Figure 3 shows that the maximum of the LPO primary product accumulation in cataract lens attains its maximum at the stage of 55--64% opacification degree. At the same time, the end fluorescent LPO product concentration in the lens correlates strongly with the opacity degree (r = + 0.956, P < 0.01). Meanwhile, an important regularity was revealed: accumulation of the LPO products depends on the cataract development stage, but does not depend on its kind, allowing to presume a universal role of the LPO in the lens opacification process.

The study of halogen-substituted derivatives of fatty acids Gas-chromatographic profiles as a function of the retention time of fluorinated derivatives of the fatty acid non-oxidized standards are presented in Fig. 5. Peak 1 corresponds to arachidonic non-oxidized acid yield from the column. Figure 6a

0

0.5

Optical density

1g

10-J5

L

JJ-6~

B

iiI~ ~

IiI

6~-1o0

~

N

/00

~

jC

B

v

vl

).25 ~

~ O

A

t

J3

,q.

WAVELEI~GTH

330-190

nJn

Fig. 3. The dynamics of the accumulation of the primary LPO molecular products in the lens opacification; a -- the lens opacity degree, We; I--VI -- cataract stages; A -- 190 nm, B -- 206 nm, C -- 230 nm (diene conjugates maximum), D -- 274 nm (triene conjugates maximum), E -- 330 nm.

64--100 9

36

1.9 4- 0.2 2.4 4- 0.2 P < 0.1 2.5 4- 0.1 P < 0.01 2.9 4- 0.2 P < 0.01 2.4 4- 0.1 P < 0.05 2.2 + 0.2 P > 0.1

C 0.30 4- 0.02 0.37 4- 0.03 P < 0.1 0.39 4- 0.015 P < 0.01 0.45 4- 0.03 P < 0.01 0.36 4- 0.025 P < 0.1 0.35 ± 0.04 P > 0.1

A~/A~ 0.45 4- 0.05 0.58 4- 0.10 P < 0.1 0.67 4- 0.05 P < 0.01 0.80 4- 0.06 P < 0.01 0.66 4- 0.06 P < 0.02 0.45 4- 0.07 P > 0.1

C' 0.07 4- 0.01 0.09 + 0.01 P < 0.1 0.13 4- 0.01 P < 0.01 0.16 4- 0.01 P < 0.01 0.11 4- 0.01 P < 0.I 0.08 4- 0.01 P > 0.1

184-5 194-7 P>0.1 454-8 P < 0.02 53.4 4- 12.0 P < 0.02 74.1 4- 13.2 P < 0.01 97.8 + 10.0 P < 0.01

F/

a, the lens opacity degree; C, the diene conjugates concentration, C~D~/CL; D~2, optical density; CL, lipid content, ms/ml; C', the triene conjugates (cetodienes) concentration, C'~D~,/CL; FI, fluorescence intensity of lipid extract in relative units; n, the number of examined lenses; P < 0.05, reliable values (compared with transparent lenses).

=100

53

55---64

Almost mature Mature

Hyper mature

26

10--55

Immature

18 6

0 < 10

Transparent lens Incipient

n

a, e:e

Cataract stage

CONTENT OF LIPID PEROXIDATION PRODUCTS IN HUMAN LENSES

TABLE 1

T

a

e~

6

0

o

B.

o

0

0

B

0 &

H

j-=

L

i

-

-

20:4

22:I

-

15:0

~-.- I 2 0 : 2 201~

18:0

18:3 18:2

16:1 16:0

T4:I

RESPONSE

-

18:1

17:0

B

0

E

~B ~2

B~

t~

u~J'n

mt~

81

Optical density units of optical

density)

~.0

0.5

23o

~o

Wavele r~ th (rim)

! i!IIi i p

II

i , J ~ i L ,,i iLL .l~l h,J~,~ ' ¸' R~TENTION TIME (rain)

Fig. 6. Investigation o f arachidonic acid oxidation products, a - - UV spectrum o f arachidonic acid lipid extract absorption ( m e t h a n o l / h e p t a n e 5:1), the arrows point to the oxidation products; b - - gas-chromatograms of oxidized arachidonic acid after the fluorination.

82

shows a typical UV absorption spectrum of arachidonic acid which has undergone ultrasound-induced oxidation. The maximum in 232 and 280 nm wavelength area corresponds to the absorption of diene and triene conjugated structures formed in the course of arachidonic acid peroxidative modification. Figure 6b presents chromatographic profile of the same sample of oxidized arachidonic acid. As is evident in Fig. 6b, as a result of the fluorination procedure carried out oxidized arachidonic acid, unlike the initial standard, is distinct with new chromatographic peak appearances, differing with intensity and retention time. Each of these parameters, consequently, characterizes the unsaturated fatty acid oxidation process. Typical chromatograms of lipid fraction from transparent and cataractous (mature cataract) human lenses are shown in Fig. 7. Although in mature cataract, on

I

II

6

RE]%!; ~0!, 'I'I:"

(' ~, ~

Fig. 7. Gas-chromatograms o f the lipid fraction from transparent (I) and cataractous (mature cataract) (II) lenses after the fluorination. A - - 18: I.

83

g a s - c h r o m a t o g r a p h i c prof'de o f t h e lens l i p i d s a m p l e , n o p r i n c i p a l l y n e w p e a k s a r e f o u n d t o a p p e a r , t h e i n t e n s i t y o f p e a k s m a r k e d l y d i f f e r s f r o m t h e n o r m as q u a n t i t a tive r a t i o . " A " p e a k c o r r e s p o n d s t o t h e f a t t y a c i d 18:1. A s this m e t h o d allows t h e d e t e r m i n a t i o n o f b o t h f a t t y a c i d s a n d t h e i r o x i d i z e d m e t a b o l i t e s , in t h e a b s e n c e o f a c o m p l e t e set o f t h e i r s t a n d a r d s , n o i d e n t i f i c a t i o n o f all t h e c h r o m a t o g r a p h i c p e a k s was p e r f o r m e d in t h e p r e s e n t s t u d y . H o w e v e r , a r e l i a b l e increase in t h e i n t e n s i t y o f the p e a k s w h o s e r e t e n t i o n t i m e d o e s n o t c o r r e s p o n d t o the f a t t y acids n o n - o x i d i z e d s t a n d a r d s ( T a b l e II), m o s t p r o b a b l y reflects t h e fact o f t h e increase in c o n t e n t o f o x i d e r i v a t i v e p o l y u n s a t u r a t e d f a t t y acids in c a t a r a c t . Cataract induction with LPO products

H i g h l y o x i d i z e d p h o s p h o l i p i d s ( M D A c o n t e n t o f 22.2 n m o l / t t m o l p h o s p h o r u s ) were f o u n d t o i n d u c e t h e lens o p a c i f i c a t i o n (Fig. 8a). L o w - o x i d i z e d p h o s p h o l i p i d s (2.0 n m o l M D A / t a n o l p h o s p h o r u s ) i n d u c e a lesser d e g r e e o f lens o p a c i f i c a t i o n (in a n u m b e r o f cases a l s o a n o p a c i f i c a t i o n o f t h e v i t r e o u s was m a r k e d ) . T o ensure t h a t t h e lens o p a c i f i c a t i o n i n d u c e d b y o x i d i z e d lipids is n o t d e t e r m i n e d b y t h e p e n e t r a t i o n in the lens o f w a t e r - i n s o l u b l e c o m p o u n d s ( s u s p e n s i o n s o f l i p o s o m e s ) , c o n t r o l experim e n t s were c a r r i e d o u t w i t h i n j e c t i o n o f l i p o s o m e s p r e p a r e d f r o m s a t u r a t e d p h o s -

TABLE II ANALYSIS OF FLUORINATED DERIVATIVES OF FATTY ACIDS IN TRANSPARENT AND CATARACTOUS HUMAN LENSES Retention time (min)

Concentration ( ~ )

P

Transparent lens (n = 5)

Mature cataract (n = 7)

17.32 35.26 35.76 45.61 59.83 63.67 67,68 68.7 73.93 74,35 78.07 82.17 84,95 85.37 86.1 94.58

2.08 + 0.10 2.17 4- 0.10 2.34 4- 0.10 42.6 4- 1.1 1.27 4- 0.10 19.56 4- 1.00 1.6 4- 0.1 2.61 4- 0.10 2.0 4- 0.1 9. I 4- O.1 1.8 4-0.1 1.8 4-0.2 1.4 4- 0.1 5.7 4- 0.1 2.1 4- 0.1 1.8 4- 0.2

0.65 4- 0.10 10.89 4- 1.10" 1.55 4- 1.00 40.7 4- 1.1 2.83 4- 0.10" 0.70 4- 0.10 0.6 4- 0.5 4.0 4- 0.1" 7.1 4- 0.1" 0.6 4- O.1 0.6 4- 0.2 0.6 4- 0.2 0.6 4- 0.2 0.6 4- 0.3 0.6 4- 0.2 2.2 4- 0.2* -

(1) Oxyproducts accumulation ratio in comparison with transparent lens is of 17.2"/0. (2) *Chromatographic peaks characterizing fatty acid oxyproducts accumulation.

<0.01 <0.01 >0.1 >0.1 <0.01 <0.01 >0.1 <0.01 <0.01 < 0.01 <0.01 <0.01 <0.01 <0.01 <0.01 >0.1

84

Fig. 8. Posterior subcapsular lens opacification induced by LPO products injection into rabbit vitreous; 0.4 mg injection o f (a) oxidized phospholipids; (b) saturated phospholipids.

TABLE III FLUORESCENT PRODUCTS OF LIPID P E R O X I D A T I O N IN EYE TISSUES BY CATARACT MODELLING WITH LIPOSOMES (Fluorescence relative units excitation/emission: 365/438 nm)

Tissue

Norm

Non-oxidized liposomes : 2.0 nmol M D A / ~mol o f phospholipids

Oxidized liposomes : 22.2 nmoI MDA / ~mol o f phospholipids

Saturated liposomes : 1 nmol MDA / ~mol o f phospholipids

l. Thelens

96.5 ± 23.1 (n = 10) 82.2 ± 28.5 176.3 +- 25.0

154.4 ± 17.6 (n = 7) 326.3 - 134.0 232.0 ± 55.1

173.0 ± 37.4 (n = 10) 173.3 ± 37.6 240.0 _+ 14.0

100.0 ± 11.2 (n = 6) 85.1 ± 11.6 171.0 _+ 7.9

2. The vitreous 3. Theaqueous humor

(1) The data presented are obtained by intraocular injection of 0.4 mg of phospholipids. (2) The material concentration in the samples was equalized by lipid concentration (calculated by characteristic lipid extract absorption at 206 nm). (3) n, the number of eyes examined.

85

TABLE IV THIOL CONTENT IN THE LENS BY CATARACT MODELLING WITH LIPOSOMES Total thiols, ttmol/protein ttmol; reduced glutathione, nmoi//tmol of the initial protein.

Liposomes

Phospholipids content (mg)

n

Total thiols

Reduced glutathione

(l)Non*oxidized unsaturated (2) Oxidized

0.4 1.5 0.4 1.5 0.4 1.5 --

~ 8 10 8 6 3 10

2.92 2.99 2.96 3.10 3.2 3.1 4.1

450 251 403 121 520 500 705

(3) Saturated (4) The controls

± ± ± ± ± ± ±

0.34 0.37 0.24 0.47 0.5 0.6 0.4

± 116 ± 75 ± 63 ± 52 ± 71 ± 102 ± 106

n, The number of eyes examined. Protein molecular weight was assumed to average 20 000.

pholipids (dipalmitoylphosphatidylcholine). It turned out that they do not induce any lens opacification at all (Fig. 8b). The cataract induction was accompanied by the accumulation of the LPO fluorescent products in the vitreous, aqueous humor and the lens (Table III). Table IV shows that, as a result of highly oxidized phospholipids injection, the reduced glutathione concentration in the lens decreases, however, high-molecular thiol reduction practically does not change. The obtained results indicate that in this cataract type the main cause of crystallin polymerization is their interaction with the LPO products, bifunctional compounds of dialdehyde type. DISCUSSION

Kinetic curves of the accumulation of the different LPO products in the lens by cataract development correspond to the scheme of their gradual transformation in biological membranes, their autooxidation being induced: phospholipids--hydroperoxides of phospholipids--carbonile-compounds (MDA)--intermolecular crosslinks of Schiff bases type. This sequence of formation of lens phospholipid autooxidation products in the course of cataract development, provides a demonstration of the lenticular membrane LPO activation being a leading factor in cataractogenesis. The variety of the LPO products, as well as of the processes developing as a result of autooxidation induction in lens fiber plasma membranes determines a large range of peroxidative effects on the lens opacification. The most characteristic cataract manifestations can be included in these effects: disintegration of lenticular fiber plasma membranes, formation of new fluorophores, forming of high-molecular aggregates leading to light-diffusion in lenticular matter.

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It is well known that disulfide bonds play a great part in high-molecular protein aggregate formation and stabilization in cataract [29]. The oxidation of proteinlinked thiols in the lens, in its turn, is explained by reduced glutathione level decrease (GHS) [30]. At the same time, it is also well known that the LPO process induction may be accompanied with oxidation of membrane protein SH-groups [31]. High correlations between the lens opacity degree, LPO end products level, GSH and TSH concentrations in the lens prove that lens transparency, lipid peroxide concentration and thiol level in it, are interrelated parameters of a single process [35]. Peroxidative reaction development in lipid membrane phase is accompanied by the accumulation of fluorescent products which have fluorescence excitation and emission spectra parameters similar to those of cataractous fluorophores [32]. In our previous study a tentative investigation of physico-chemical nature of the fluorophores accumulated in lenticular lipids by cataract development, was undertaken [36]. Fluorescent products found in chloroform-methanol extracts from human cataractous lenses, have lipid nature. Initial stages of the cataract development are characterized with lipid fluorophore appearance in the near UV and violet spectra (max. excitation, 302--330 urn; emission, 411 nm), of low polarity and low molecular weight. Such fluorescent products, evidently result from oxidative transformation of polyene fatty acids undergoing no polymerization. The formation of the latter takes place probably with endogenous phospholipases of A 2 type involvement which are able to attack both non-oxidized and oxidized phospholipids. Cataract maturation is characterized by an increase of long-wave fluorescence intensity in blue-green area (430---480 urn), with the formation of polymeric high-molecular fluorescent lipid products of high polarity. Evidently, they result from radical polymerization of phospholipids, fatty acids, Schiff bases formation, i.e. lipofuscin-like pigments. Together with polymerization of membrane-linked crystallins, the LPO product accumulation in lenticular membranes can lead to an inhibition of membrane-bound enzymes, an impairment of protein-lipid interactions in membrane, and also of molecular interactions of the membrane with cytosceleton and water-soluble lenticulax crystallins. LPO products can act as ion transfer inductors in the lens, increasing at corresponding stages of cataract development Na ÷ and Ca 2÷ions intracellular concentration enhancing the opacification progress [33]. Which properties of the products of lenticular membrane phospholipids peroxidation are responsible for the deterioration observed in lenticular fiber plasma membrane structure organization? Hydrophoby of phospholipid amphiphilic molecules is determined by their hydrocarbon tails, so their shortening by oxidation destruction of hydroperoxides or their chemical modification leading to polar (hydroperoxide, carbonile) groups appearance in fatty acyls, will inevitably effect their hydrophilic-hydrophobic balance. Such modified molecules acquire properties of detergents similar to lisophospholipids forming under the influence of A 2 phospho-

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lipase. Consequently, as an LPO result, phospholipids modified with oxygen appear in lenticular fiber membrane which are able to increase the plasma membrane hydrophilyty and to impair lipid-lipid and protein-lipid interactions. Detergent action of the phospholipid oxidation products accumulated in cataract lenticular fiber membranes offers an explanation of the observed deterioration of the lens plasma membrane organization. Phospholipid molecules modified with oxygen, incorporating themselves into the bilayer, change its geometry, diminishing the area of its hydrophobic portion, increasing its surface curvature owing to shortened radius of membrane particle. This effect is obviously the basis of the membrane fragmentation and smaller vesicle formation in cataract. It is also possible that the change of lenticular membrane bilayer geometry can occur under the influence of phospholipid formation with Ca 2÷ion complexes. The study of cataract formation molecular mechanisms raises a question: whether the modification of the lipids which are the minor (only 2% of lens wet mass) component, can lead to conformation and solubility change of proteins constituting 35~/0 of the lens wet mass7 In general, the question may be formulated as follows: whether the LPO process is a factor which, together with other metabolic shifts, only accompanies the cataract development, or, in a number of cases, the LPO activation can be an initiatory cause of the lens opacification? Earlier it was shown that injection of 22:6 into the rabbit posterior vitreous can induce cataract [37]. The onset of posterior subcapsular cataract formation was temporally correlated with the rapid decline in vitreous MDA levels. This suggests, but does not prove, that MDA or some other toxic product of lipid peroxidation may have diffused anteriorly and reacted with the posterior lens surface, resulting in posterior subcapsular cataracts. In the present investigation a possibility of cataract induction with phospholipid peroxidation products' injection into rabbit vitreous was studied, and, simultaneously, the lens thiol system state was controlled as well as the level of the LPO fluorescent product accumulation in the vitreous, aqueous humour and the lens. Autooxidized phospholipids did induce cataract after their injection with the concomitant accumulation of the LPO products in the lens and the reduction of the lenticular glutathione concentration. Injection of saturated phospholipids resistant for peroxidation in the same concentrations did not induce any lens opacification. That is the evidence for the initiatory effect of the LPO products to the cataract formation. Which are the mechanisms of intermolecular protein cross-links formation in cataract? Dialdehyde-bifunctional reagents are characteristic of LPO products, and their interaction with free aminogroups entails Schiff base forming and, subsequently, inter- and intramolecular sutures. It should be pointed out that phospholipid intermolecular cross-links formation has been registered in this study by characteristic fluorescence of lipid extract from cataractous lens. Covalent crosslinking of lenticular proteins in cataract may be possible also as a result of their interaction with the lipid-free radicals appearing in the course of LPO process. Con-

88

sidering the a b o v e

stated,

it is o f i m p o r t a n c e that the a c c u m u l a t i o n o f rather small

a m o u n t s o f oxidized lipids i n the m e m b r a n e s is a b s o l u t e l y sufficient to i n d u c e further p r o t e i n o x i d a t i o n [34]. R e d u c e d thiol c o n c e n t r a t i o n decrease is o n e o f the characteristic indices o f redoxb a l a n c e d e t e r i o r a t i o n in c a t a r a c t o u s lens. As it a p p e a r s f r o m the results o b t a i n e d , lenticular thiols are r a p i d l y i n a c t i v a t e d b y a n increase o f lipid f r e e - r a d i c a l - i n d u c e d o x i d a t i o n reactions i n the lens. These processes p r o m o t e h i g h - m o l e c u l a r aggregate f o r m a t i o n i n the lens. T h u s the presented d a t a allow us to suggest that peroxide d a m a g e to lenticular fiber p l a s m a m e m b r a n e s in cataract n o t o n l y a c c o m p a n i e s its d e v e l o p m e n t b u t is the i n i t i a t o r y cause o f the disease. This b e i n g true, b y p r e v e n t i n g peroxide c o m p o u n d s f r o m a c c u m u l a t i n g in the lens, a n d also by m a i n t a i n i n g a high level o f reduced g l u t a t h i o n e in it, we c a n susp e n d f u r t h e r d e v e l o p m e n t o f cataract a n d , i n a n u m b e r o f cases, even partially restore the t r a n s p a r e n c y o f a l r e a d y opacified lens. REFERENCES I

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