Mechanisms of Senile Cataract Formation

Mechanisms of Senile Cataract Formation

Mechanisms of Senile Cataract Formation LEO T. CHYLACK, JR., MD Abstract: Research on the mechanisms of lens opacification during the past 20 years h...

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Mechanisms of Senile Cataract Formation LEO T. CHYLACK, JR., MD

Abstract: Research on the mechanisms of lens opacification during the past 20 years has revealed a number of individual, identifiable cataractogenic stresses in man. They include osmotic cataract formation (diabetic, galactosemic and hypoglycemic cataracts), radiation cataracts (X-irradiation, near U.V. radiation and microwave radiation) and in senile cataract the conversion of soluble low molecular weight cytoplasmic proteins to soluble high molecular weight aggregates, insoluble phases, and insoluble membrane-protein matrices. Oxidative stress has emerged as a common denominator of many changes in senile cataract. As we increase' our understanding of these mechanisms, we may be able to intervene therapeutically to delay or prevent human cataract formation in man. [Key Words: mechanisms of cataract formation, oxidative stress, sugar cataracts, sunlight, U.V. light.] Ophthalmology 91:596-602, 1984

Intrinsic to the work of most lens scientists is a wish to understand the mechanism(s) by which the human lens becomes opaque. Young scientists may be motivated simply by scientific curiosity, but the longer they remain in the field of cataract research the more they were struck by the enormousness of the public health problems posed by human senile cataract. In the United States alone, 31,000 people are legally blind from senile cataract or its complications. 1 At least 400,000 persons develop cataract each year, and cataract is responsible for 35% of existing visual impairment and for 53% of new visual impairment with human etiologies. 2 Worldwide, senile cataract is responsible for significant visual impairment in 30 to 45 million people. As scientists become aware of thesestatistics, their scientific curiosity about mechanisms of cataract formation is supplemented by an intense awareness that their work may ultimately diminish the public suffering from senile cataract. The ophthalmic surgeon is very familiar with the joy a patient experiences when surgical extraction of his lens opacity restores vision. The laboratory scientist may wish to experience this elation, albeit vicariously, through his efforts to understand the From the Harvard Medical School and Brigham and Women's Hospital, Boston. Presented at the Eighty·eighth Annual Meeting of the American Academy of Ophthalmology, October 3O-November 3, 1983. Supported by USPHS grant EY01276 and the Brigham Surgical Group Foundation. Reprint requests to Leo T. Chylack, Jr., MD, Howe Laboratory of Oph· thalmology, 243 Charles Street, Boston, MA 02114.

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mechanisms of and perhaps allow medical intervention in the process of cataract formation. That basic cataract research is a worthy endeavor is confirmed by the steady increase in federal and private funding supporting it. On purely economic grounds, NEI statisticians have estimated that "development of a therapy which could delay the need for cataract surgery by ten years would reduce the annual number of cataract operations by 45%. Assuming hospital and surgical costs of$2,500 per cataract operation, the reduction in cataract operations per year from 541,000 to 298,000 would save $608 million annually in medical care costs alone." At present, hospital and surgical costs per cataract operation may exceed $4500 per patient, and the savings resulting from a delay in cataract surgery by 10 years would be $1.094 billion/year. Therefore, it is not at all difficult to understand why cataract research in the U.S., Europe, and Japan has very recently increased the emphasis on mechanisms of human (as opposed to animal or experimental) cataract formation. The formation of the Cooperative Cataract Research Group (CCRG)3 in the U.S., European Cooperative Aging Research Program (EURAGE)4 and counterpart organizations in Japan are evidence of this increased dedication to human cataract research. As will become apparent later in this article, a single primary cause of senile cataract most likely does not exist. Research to date has revealed the multifactorial nature of cataract formation in man; several risk factors working concurrently lead to the loss oflens transparency. For example, it is generally accepted that age, corticosteroid use, ionizing radiation, and perhaps diabetes may all accelerate the rate of cataract formation in humans.

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MECHANISM OF SENILE CATARACT FORMATION

It remains to be seen what other risk factors can be identified and by what mechanisms their effects on the lens are cataractogenic. Risk factors have been listed in an elegant summary of the epidemiology of cataract: 5 1. Ultraviolet light long wavelength UV (UVL) a. Sunlight b. Occupational exposure (chemists, laundry workers, currency examiners, dentists, orthopedic technicians, dermatologists) 2. Ionizing radiation (therapeutic and diagnostic, CT scan) 3. Radiofrequency and microwave radiation (military, industrial, scientific) 4. Toxic drugs and chemicals (ie. corticosteroids, phenothiazines, miotic cholinergics, metals and others) 5. Diabetes (glucose-sorbitol; enzyme: aldose reductase) 6. Blood pressure (Framingham Eye Study; elevated blood pressure associated with presence of cataract) 7. Family history (families with high cataract prevalence) 8. Biochemical agents a. galactokinase deficiency b. elevated plasma tryptophan levels c. glucose-6-phosphate dehydrogenase deficiency d. proteins e. lipids

Although the identification of risk factors is an invaluable method of identifying, from among an infinite number of possible cataractogenic factors, those which are potentially significant, it is not an identification of mechanism(s) of cataractogenesis. In fact, only one mechanism (sorbitol pathway involvement in sugar cataract formation) has actually been identified. This will be discussed first; later, the possible mechanisms underlying senile cataract formation will be presented.

SUGAR CATARACfS In diabetes mellitus elevation of blood glucose is followed rapidly by an elevation of aqueous humor glucose to nearly 90% of the blood level. Glucose is transported from the aqueous humor into the lens via facilitated transport4 at a rate that is faster than would be predicted from the concentration gradient and the diffusion coefficient. Insulin is probably not required although some recent evidence6 suggests that insulin is present in the aqueous humor and may modify lenticular glucose uptake. 7 Once within the lens, glucose may be phosphorylated to (1) glucose-6-phosphate (G-6-P) by hexokinase (HK), (2) to sorbitol by aldose reductase (AR) and NADPH, or (3) to glycogen (a minor pathway). The Michaelis constants (Km) for hexokinase and aldose reductase in rat lens are .01 to .03 mM and 30 mM respectively.

The Km is the concentration at which the enzyme activity is half maximal, and from these Km's it is possible to see that the affinity of hexokinase for glucose is much greater than that of aldose reductase. In fact, in nondiabetic animals, very little if any glucose is converted to sorbitol by aldose reductase; it is all converted to G-6-P. However, in the diabetic animal, and perhaps in the nondiabetic after eating, the blood and aqueous humor levels rise sufficiently to activate aldose reductase and glucose is then reduced to sorbitol. The activity of AR depends on the availability of adequate glucose and NADPH, a product of the hexose monophosphate shunt (HMPS). Once formed, sorbitol, a sugar alcohol which cannot permeate cell membranes, accumulates within the cell rendering the internal cytoplasmic milieu hypertonic. To counteract this intracytoplasmic concentration, water enters the cell; this results in cellular swelling and lenticular opacification. This is the basic mechanism by which experimental diabetic cataracts are produced in animals. Galactosemia, a condition in humans caused by a deficiency of galactoseI-phosphate uridyl transferase, and induced in animals by feeding a galactose-rich diet, can lead to sugar cataract formation even more rapidly than in diabetes. A similar mechanism is involved: the conversion of galactose to dulcitol (sugar alcohol) by aldose reductase. The experimental work leading to these discoveries is outlined in an excellent review of mechanisms of cataract formation 7 and a detailed study of the cataract which forms in the rabbit lens exposed to high glucose. 8 While the osmotic swelling in response to sorbitol or galactitol accumulation was believed to be the primary mechanism of cataract formation, it was not until aldose reductase inhibitors (ARIs) were developed that more conclusive evidence was obtained. Tetramethylene glutaric acid (TMG)8 and Alrestatin 9 were able to inhibit AR and block sorbitol and galactitol accumulation. In vitro, lenses exposed to a medium containing high sugar levels and these drugs remained crystal clear even though there were very high levels of sugar within the lens. The ARls were not blocking sugar transport into the cell; rather they were exerting their effect by blocking AR. When animals were fed Alrestatin, the onset of sugar cataract formation was delayed by several weeks. Other data confirmed the importance of the sorbitol pathway in sugar cataract formation; a strain of congenitally hyperglycemic mice with very low or absent aldose reductase activity were found to be free of cataracts. 10 By reducing sugar intake and increasing the amount of fat, fructose and casein in the diet diabetic cataracts could be delayed. II • 12 However, it was not until the development of more potent ARIs (flavonoids I3 •14 and Sorbinil I5 •16) that diabetic and galactosemic cataracts could be prevented, not just delayed. Without Sorbinil, cataract formation appeared within 2 weeks; with Sorbinil no cataract appeared during the 4 months ofthe study. Recently reversal of advanced galactose cataracts in rats has been accomplished with sorbinil treatment l7 and by removing galactose from the diet. 18- 2o The remarkable ability of the animal lens to regenerate clear .cortex after a severe osmotic insult is 597

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testimony to the ability of the lens epithelium to survive or counteract the osmotic effects of sorbitol accumulation. Both diabetic and galactosemic animals have been used in experiments on the role of the sorbitol pathway in sugar cataract formation. The galactose cataract forms more rapidly than the diabetic because of the higher affinity of AR for galactose and the inability of the lens to further metabolize dulcitol (in contrast to sorbitol, which can be oxidized to fructose by the enzyme polyol delhydrogenase ). It was not until the mid to late 1970's that the importance of the sorbitol pathway in human lenses was explored. Human cataracts immediately after cataract extraction were incubated in TC199 medium containing 5.5 mM and 35.5 mM glucose. Sorbitol production doubled in the high glucose medium and could be blocked with an AR1. 21 ,22 It was difficult to correlate sorbitol production with the amount of lens swelling because even the control lenses swelled, but there was a statistically significant reduction in the number of lenses undergoing spontaneous rupture in high glucose medium containing the ARI. It was concluded that the human lens did have the potential to produce sorbitol in the presence of high glucose and that human lens AR was inhibitable by an ARI. A more detailed study of the sorbitol pathway enzymes and coenzymes in the human lens23 revealed that aldose reductase activity declines with age and even in the youngest lensesI S only about 16 to 30% as active as in rat and rabbit lens. The Km of human lens AR is 200 mM, a concentration suggesting that even in diabetes the enzyme will be much less active than in animal lenses. Seventy percent of the AR is in the lens epithelium and in the adult lens the AR activity is only two-thirds that of the juvenile lens epithelium. A major distinction between the sorbitol pathway in the human and animal is the very active polyol dehydrogenase (PD) activity in the former. In the fructose-sorbitol direction, it is 80 times more active in the human than the animal lens. In the sorbitol-fructose direction, it is several times more active than the animal lens. These data suggest that in the human lens, sorbitol may be oxidized to fructose at a more rapid rate than in the animal lens, which would decrease the likelihood of osmotically significant levels of sugar alcohol accumulating. On the other hand, if the lens has a source of fructose and NADH, sorbitol may be formed and augment that derived from glucose. Using the maximum enzyme activities of AR and optimum cofactor concentrations, it was calculated that the human lens could produce 6 to 7 JLmol of sorbitol to the lens in 24 hours. If all of this were confined to the epithelium, alarge osmotic stress could be developed (280-570 mOsm/24 hr/lens epithelium). Certainly this is enough of a stress to damage the epithelium directly and the underlying cortex indirectly. In addition to the osmotic theory of sugar cataract formation, other theories have been proposed; one study24 implicated nonenzymatic glycosylation of lens protein. The nonenzymatic addition of a glucose moiety to a lysine residue in lens proteins was suggested as a cause of protein 598



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aggregation, light scattering and cataract formation . However, another group of scientists2S showed that lenses from diabetic rats on Sorbinil (no cataracts) contained no fewer glucitol-Iysine residues than cataractous lenses from diabetic rats not treated with Sorbinil. If nonenzymatic glycosylation was an important cause of lens opacification, there should have been more glucitol-Iysine in the cataract than the clear lens. The biological function of the sorbitol pathway is unknown, however, two recent suggestions are intriguing. First the discovery of an enzyme in the lens capable of phosphorylating fructose (ketohexokinase)26 raises the possibility that the sorbitol pathway could be an alternative source of ATP (glucose ~ sorbitol ~ fructose ---+ fructose-I-P ~ glyceraldehyde-3-P ---+ glycolysis ~ ATP). Secondly, intracellular sorbitol production could counteract shifts in extracellular osmolarity due to rapid fluctuations in the level of blood and aqueous glucose.27Such shifts are known to be of major clinical importance in the condition called hyperosmolar hyperglycemic coma. If the second proposal is correct, then under normal conditions the sorbitol pathway would function cyclically to neutralize shifts in glucose-derived osmotic stress; sustained activity as in the diabetic galactosemic, would be abnormal and result in sugar cataract formation. In spite of the uncertainty regarding (1) the biological function of the sorbitol pathway, (2) the significance of the sorbitol pathway in human cataract formation in diabetics, and (3) the lack of knowledge of long term side effects of AR inhibition, many pharmaceutical houses in the U.S., Europe, and Japan are developing ARls for clinical use. Not only are these drugs likely to benefit diabetics at risk for cataract formation, but they have already been shown to maintain normal nerve conduction velocities in diabetic animals28 and are to be tested for beneficial effects on patients with diabetic retinopathy and nephropathy.

OXIDATIVE STRESS AND CATARACT FORMATION Although oxygen is of vital importance for most mammals, it is not necessary for the sustenance of the clarity of the young lens.29 The older lens produces more ATP from O2 than the younger lens,30 but the majority of the ATP comes from anaerobic glycolysis nonetheless. In fact, oxygen can be harmful to the lens. One or more of the oxidizing forms of oxygen can be harmful to the lens and possibly implicated in senile cataract formation . Oxygen can exist in one of four forms: I. molecular oxygen 0-0 hydroperoxide H-O-O 2. superoxide anion radical O23. hydrogen peroxide H-O-O-H (H 30 2) 4. hydroxide radical OH These forms are interrelated according to the following metabolic sequence:

CHYLACK

·0-0· (I)



~



MECHANISM OF SENILE CATARACT FORMATION

H-O-O· hydroperoxide

Jt 02" +H+



~

H-O-O-H hydrogen peroxide

superroxide anion radical

(III)



~

(H 30 2)' ! •

0H

hydroxide + H+ + OW radical (IV)

(II)

In 1976 31 it was shown that 10- 3 M H 20 2 partially inhibits 86Rb (and, therefore, K+ as well) uptake by the rat lens. A more recent study32 showed that 10- 3 M H20 2 in vitro leads to an increased Na +, Ca++ and water content and a lowered K+ content. NaK ATPase activity was reduced 37% and 86Rb uptake reduced by 32% by this concentrMion of H 20 2, and 86Rb efflux was increased nearly two-fold. Below 10-3M, H 20 2 had no effect. Garner et al 33 demonstrated an uncoupling of the NaK ATPase and the complete inhibition of rubidium uptake with 1 mM H 20 2. Apparently, H 20 2 modified the ability of the enzyme to bind its substrates; the low affinity site filling before the high affinity site. Evidence to date suggests that 10-3 M H 20 2 affects both the active and passive fluxes of Na+ and K+ in the lens. The concentration of H 20 2 in normal human aqueous humor is 1.4 - 3.1 X 10-5 M, approximately 30 times lower than the 10- 3 M concentration used above. Some cataract patients have markedly elevated H 20 2 levels in the aqueous humor. 34 The ability of the lens to detoxify H 20 2 was studied recently,35.36 and it was shown that the activity of the HMPS increases dramatically in response to H20 2. Presumably the increased shunt activity produces NADPH which allows GSSG (glutathione oxidized by H 20 2) to be reduced to GSH (glutathione). The capacity of the lens epithelium to detoxify H 20 2 is enormous; H 20 2 is reduced at a rate nearly 126 times that which could be accomplished by unregenerated lenticular GSH. To accomplish this, GSH must be regenerated at 126 times its normal rate of synthesis. If the H 20 2 level was raised even slightly above 10-3 M, the viability of the lens epithelial cells decreased. It appears that while there is the capacity in the lens to detoxify endogenous H 20 2 in aqueous humor, any rise above the normal level exceeds this capacity and results in damage to lens membranes and active transport mechanisms. Oxidation of lens proteins is more common in older than in younger lenses. 37 .38 This is evident in the accumulation of mixed disulfides, protein-protein disulfides, methionine sulfoxide, methionine sulfone and cysteic acid. Only in advanced cortical cataracts is sulfhydryl oxidation evident. 39-42 Oxidative changes are first seen in the membrane of the fiber cells. Furthermore, it is quite clear that oxidation, while being a necessary condition for cataract, is not sufficient, since cortical cataract involves extensive oxidation in the nuclear region but no high molecular weight disulfide-linked aggregates. Conversely, in nuclear cataract, oxidation is found in the cortex as well as the nuclear region but the aggregate formation occurs only in the nucleus. Evidence for the centrifugal progression of nuclear cataracts was obtained

from the analysis of nearly 2,000 classified human cataracts in the U.S. CCRG (Cooperative Cataract Research Group) study43 in which it was evident that if one arranged cataracts from pure nuclear to early and advanced corticonuclear cataracts there was a steady increase in age of the patient. The implication is that once a nuclear cataract begins it progresses with age centrifugally. The opposite was found for pure cortical cataracts which progress from corticonuclear to nuclear in a pattern suggesting centripetal development with age. Oxidative changes are believed to be partly responsible for the steady increase in insoluble lens protein that occur with cataractogenesis. However, the large increase in nonprotein disulfide found in the water-insoluble fraction of cataractous lens protein is not accounted for entirely by the decrease in nonprotein sulfhydryl. 39 Lens fiber cell membranes are made up of proteins and lipids. Methionine and cysteine, two amino acids in the membrane's protein fraction are extensively oxidized in the cataractous lens. Membrane proteins appear to be oxidized prior to cytoplasmic proteins. 37 One protein, a 43,000 dalton (molecular weight) extrinsic membrane protein, appears to link cytoplasmic high molecular weight, -S-S-linked protein aggregates to the membrane in nuclear cataracts. In cortical cataracts linkage to the membrane does not occur.45 The origin of the 43,000 dalton protein is still uncertain; however, recent work reported at the U.S.-EURAGE Cataract conference at Oakland University, Rochester, Michigan46 indicated that much of the 43,000 dalton fraction is the result of posttranslational changes; radioactive methionine is incorporated into only a part of the protein. An antibody to 43K reacts strongly with gamma crystallin and weakly with (3 crystallin. An antibody. to gamma crystallin (but not a or (3 crystallin) does react with the 43K protein. These data suggest that covalent polymerization of gamma crystallin is occurring; the mechanism by which this occurs is unknown. The disulfide-linked protein aggregates that are linked to the membrane by the 43K dalton protein may be enormous, with molecular weights of more than 5,000,000 daltons. 47 Proteins of this size are capable of scattering light and if sufficient in number (as in nuclear cataracts) degrading the quality of the retinal image and reducing visual acuity. The water-insoluble fraction of nuclear cataracts is made up of a urea-soluble fraction containing disulfide linked protein aggregates, and a urea-insoluble fraction linked by nondisulfide covalent bonds. The nature of these covalent bonds is unknown, but compounds con599

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sidered to be involved include kynurenine, anthranilic acid, beta carboline and bityosine. The inability to define this covalent linkage is one of the key obstacles to our understanding the mechanism of light scattering in nuclear cataracts. Many of the aforementioned processes are oxidative in nature; which oxidant(s) is/are responsible is not yet certain. However, several systems exist in the lens to protect it against oxidation. Glutathione is present in high concentration in the lens and it is a strong antioxidant. It is generated by glutathione reductase and the NADPH generated by the HMPS. It can maintain enzymatic and structural protein-SH groups in the reduced state, and it can reduce protein-protein and mixed disulfide linkages in advanced cataracts. 48 Since the activity of the HMPS depends on an adequate supply of G6P the decreased capacity of the aging lens to counteract oxidative stress may be due to a reduction in the activity of hexokinase, the enzyme which converts glucose to G6P.49 Another antioxidant in the lens is ascorbic acid. This vitamin which is present in the lens in very high concentration, actively scavenges free radicals and singlet oxygen and converts them to superoxide anion. 50 The superoxide anion, itself a powerful oxidant, can be converted to H 20 2 by the enzyme superoxide dismutase and H 20 2 eliminated by catalase or glutathione peroxidase. U ric acid and vitamin E can be identified in the lens and may also serve as antioxidants. The levels of these compounds was recently reported to be no higher in cataractous than normal lenses.51

SUNLIGHT AND ULTRA VIOLET RADIATION During the past decade there has been increasing evidence that sunlight and particularly long wavelength ultraviolet (300-400 nm) are positively associated with cataract.52,53 The mechanism by which UVL produces oxidative damage is uncertain but believed to involve the amino acid tryptophan. If human lenses are exposed to sunlight in the presence of tryptophan they turn yellow.5 4 It is believed that the production of colored pigments (fluorophors) in the lens is involved in the transfer of energy from light to receptor substances (unidentified) with resulting free radical formation. 55 UVL can also lead to H 20 2 and superoxide anion formation. Free radicals are strong oxidizers and together with H 20 2 and superoxide anion, may lead to the cataractous changes noted in section B. Visible light (>400 nm) also may be a deleterious stress if oxygen and a photosensitizer are present in the lens. Photosensitizers include riboflavin, N-formyl kynurenine and psoralens. The oxidant produced is singlet oxygen.

IONIZING RADIATION X-irradition has been a known cataractogenic stress for several decades. 56 In man the cataractogenic dose may 600



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be as low as 200 rads 57 and the onset of the cataract may be several years after the exposure. Cumulative low doses may be as harmful as a single high dose, but the evidence substantiating this is mainly anecdotal. Because of this, lead containing glasses are now used to shield the eyes of patients undergoing CT scanning in which the dose to the lens may be as high as 9 rad. 58,59 The x-ray cataract is typically located in the posterior subcapsular region. It is a collection of vacuoles and cellular debris. In many respects, it resembles the posterior subcapsular cataract that occurs with aging. The light scattering foci in these cataracts are interfibrillar water clefts which contain much less protein (and therefore have a much lower index of refraction) than the adjacent cytoplasm. Recent studies of x-ray cataract formation in animals has revealed that there are many biochemical similarities between the senile cataract in man and the x-ray cataract in animals: 60 ,61 I. High molecular weight protein aggregates are present (rabbit).62 2. Disulfide bonds are the binding links in these HMW aggregates.63 3. HMPS activity is reduced prior to mature cataract formation. 64 4. GSH and NADPH levels are also reduced. 64 5. Low molecular weight proteins leak out of the lens before the cataract appears. 65 Many of these changes may be due to x-ray induced, free radical formation and/or bond breakage (ionization). The importance of this model cataract to our understanding of the senile cataract in man cannot be overstated.

MECHANISMS OF LIGHT SCATTERING At present, several mechanisms are implicated in light scattering of nuclear and cortical cataracts: A, Nuclear I. High molecular weight, convalently linked, protein aggregate formation a) cytoplasmic b) membrane bound 2. Phase separation 66_a process by which soluble protein molecules associate without covalent bonding to form molecular clusters, large enough to scatter light. This is the pathogenetic mechanism of cold cataract formation. 3. Syneresis-the contraction of a gel-protein matrix or cytoskeletal matrix with resultant creation of zones with indices of refraction sufficiently different to scatter light. 67- 7o B. Cortical/Subcasular/Supranuclear I. Interfibrillar water cleft formation and membrane disintegration 71 2. Soluble, non-membrane bound, high molecular weight aggregate formation.

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CONCLUSION An understanding of the mechanisms of senile cataract formation must be derived from epidemiological studies which identify risk factors, biophysical studies which identify the foci of light scattering, biochemical and biophysical studies which identify, on a molecular basis, the difference between normal and cataractous lenses, and finally, on a molecular biological level, in which we are able to distinguish between pre- and posttranscriptional changes in the normal, aging clear, and cataractous lens. This last area has not been covered in this review, but relevant lens research has been stimulated by the creation of the Laboratory of Molecular Biology at the National Eye Institute and should be followed carefully in the next few years. It is likely that major advances in our understanding of the mechanisms of cataract formation will come from this laboratory and others like it.

REFERENCES 1. Statistics on Blindness in the Model Reporting Area, 1969-70. U.S. DHEW Publication No. (NIH) 73-427, 1973. 2. National Society to Prevent Blindness. Vision Problems in the U.S.; A Statistical Analysis Prepared by the Operational Research Department. New York: the Society, 1980. 3. Chylack LT Jr, Kinoshita JH. The Cooperative Cataract Research Group. Invest Ophthalmol Vis Sci 1978; 17:1131-4. 4. Giles KM , Harris JE. The accumulation of C14 from uniformly labeled glucose by the normal and diabetic rabbit lens. Am J Ophthalmol 1959; 48(Pt. 11):508-17. 5. National Advisory Eye Council. Vision Research; A National Plan 1983-1987. Vol. 2, pt. 3: Report of the Cataract Panel. Bethesda: U.S. Department of Health and Human Services (NIH Publication No_ 83-2473) 1983; 51-9. 6. Coulter JB III, Engelke JA, Eaton OK. Insulin concentrations in aqueous humor after paracentesis and feeding of rabbits. Invest Ophthalmol Vis Sci 1980; 19: 1524-6. 7. Kinoshita JH. Mechanisms initiating cataract formation. Invest Ophthalmol 1974; 13:713-24. 8. Chylack LT Jr, Kinoshita JH. A biochemical evaluation of a cataract induced in a high-glucose medium. Invest Ophthalmoll969; 8:40112. 9. Dvomik 0, Simard-Duquesne N, Krami M, et al. Polyol accumulation in galactosemic and diabetic rats: control by an aldose reductase inhibitor. Science 1973; 182: 1146-8. 10. Varma SO, Kinoshita JH. The absence of cataracts in mice with congenital hyperglycemia. Exp Eye Res 1974; 19:577-82. 11. Patterson JW. Effect of a high fat fructose and casein diet on diabetic cataracts. Proc Soc Exp Bioi Med 1955; 90:706- 8. 12. Patterson JW, Patterson ME, Kinsey EV, Reddy DVN. Lens assays on diabetic and galactosemic rats receiving diets that modify cataract development. Invest Ophthalmol 1965; 4:98-103. 13. Varma SO, Mikuni I, Kinoshita JH. Flavonoids as inhibitors of lens aldose reductase. Science 1975; 188:1215-6. 14. Varma SO, Kinoshita JH. Inhibition of lens aldose reductase by flavonoids-their possible role in the prevention of diabetic cataracts. Biochem Pharmacol 1976; 25:2505-13. 15. Fukushi S, Merola LO, Kinoshita JH. Altering the course of cataracts in diabetic rats. Invest Ophthalmol Vis Sci 1980; 19:313-5. 16. Peterson MJ, Sarges R, Aldinger CE, MacDonald DP. CP-45,634: a

novel aldose reductase inhibitor that inhibits poIyol pathway activity in diabetic and galactosemiC rats. Metabolism 1979; 28 (Suppl 1): 456-61. 17. Hu TS, Datiles M, Kinoshita JH. Reversal of galactose cataract with sorbinil in rats. Invest Ophthalmol Vis Sci 1983; 24:640-4. 18. Unakar NJ, Smart T, Reddan JR, Devlin I. Regression of cataracts in the offspring of galactose fed rats. Ophthalmic Res 1979; 11 :52-

64. 19. Reddy VN, Schwass 0, Chakrapani B, Lim CPo Biochemical changes associated with the development and reversal of galactose cataracts. Exp Eye Res 1976; 23:483-93. 20. Unakar NJ, Genyea C, Reddan JR, Reddy VN. Ultrastructural changes during the development and reversal of galactose cataracts. Exp Eye Res 1978; 26:123-33. 21. Chylack LT Jr, Henriques HF III, Cheng HM, Tung WHo Efficacy of Alrestatin, an aldose reductase inhibitor in human diabetic and nondiabetic lenses. Ophthalmology 1979; 86:1579-85. 22. Chylack LT Jr, Henriques HF III, Tung WH. Inhibition of sorbitol production in human lenses by an aldose reductase inhibitor. Doc Ophthalmol Proc Ser 1979; 18:65-75. 23. Jedziniak JA, Chylack LT Jr, Cheng HM. et al. The sorbitol pathway in the human lens: aldose reductase and polyol dehydrogenase. Invest Ophthalmol Vis Sci 1981; 20:314-26. 24. Stevens VJ, Rouzer CA, Monnier VM, Cerami A. Diabetic cataract formation: potential role of glycosylation of lens crystallins. Proc Nail Acad Sci USA 1978; 75:2918-22. 25. Chiou SH, Chylack LT Jr, Bunn HF, Kinoshita JH. Role of nonenzymatic glycosylation in experimental cataract formation. Biochem Biophys Res Commun 1980; 95:894-901. 26. Orhloff C, Zierz S, Hockwin O. Investigations of the enzymes involved in the fructose breakdown in the cattle lens. Ophthalmic Res 1982; 14:221-9. 27. Harding RH, Chylack LT Jr, Tung WHo The sorbitol pathway as protector of the lens against glucose-generated osmotic stress. ARVO Abstracts. Invest Ophthalmol Vis Sci 1981; 20(Suppl):34. 28. Gabbay KH. The sorbitol pathway and the complications of diabetes. N Engl J Med 1973; 288:831-6. 29. Kinoshita JH, Kem HL, Merola LO. Factors affecting the cation transport of calf lens. Biochem Biophys Acta 1961 ; 47:458-66. 30. Trayhum p, van Heyningen R. Aerobic metabolism in the bovine lens. Exp Eye Res 1971; 12:315-27. 31. Fukui HN. The effect of hydrogen peroxide on the rubidium transport of the rat lens. Exp Eye Res 1976; 23:595-9. 32. Delamere NA, Paterson CA, Cotton TR. Lens cation transport and permeability changes following exposure to hydrogen peroxide. Exp Eye Res 1983; 37:45-53. 33. Gamer WH, Gamer MH, Spector A. 1-W2-induced uncoupling of bovine lens Na+, K+-ATPase. Proc Nail Acad Sci USA 1983; 80:2044-8. 34. Spector A. Garner WHo Hydrogen peroxide and human cataract. Exp Eye Res 1981 ; 33:673-81 . 35. Giblin FJ, McCready JP, Reddy VN. The role of glutathione metabolism in the detoxification of H20 2 in the rabbit lens. Invest Ophthalmol Vis Sci 1982; 22:330-5. 36. Giblin FJ. Detoxification of H20 2 by cultured lens epithelial cells. U.S.EURAGE Cataract Conference, Oakland University, Rochester, Michigan, 1983. 37. Gamer MH, Spector A. Selective oxidation of cysteine and methionine in normal and senile cataractous lenses. Proc Nail Acad Sci USA 1980; 77:1274- 7. 38. Augusteyn RC. Protein modification in cataract: possible oxidative mechanisms. In: Duncan G, ed. Mechanisms of Cataract Formation in the Human Lens. London: Academic Press,1981; 71-115. 39. Anderson EI, Wright DO, Spector A. The state of sulfhydryl groups in normal and cataractous human lens proteins. II. Cortical and nuclear regions. Exp Eye Res 1979; 29:233-43.

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40. Truscott RJW, Augusteyn RC. Changes in human lens proteins during nuclear cataract formation. Exp Eye Res 1977; 24:159-70. 41. Truscott RJW, Augusteyn RC. The state of sulphydryl groups in normal and cataractous human lenses. Exp Eye Res 1977; 25:13948. 42. van Haard PMM, deMan BM, Hoenders HJ, Wollensak J. Sulfhydryl groups in individual normal and nuclear-cataractous human eye lenses; a study emphasizing age, cataractous state and cataract localization. Ophthalmic Res 1980; 12:118-27. 43. Ransil BJ, Chylack LT Jr. Classification of human senile cataractous change by the American Cooperative Cataract Research Group (CCRG). Method V. The lenticular distribution of cataract in an elderly extraction population and its association with sex and diabetes: combined region analysis. Exp Eye Res, Submitted. 44. Gamer MH, Spector A. Sulfur oxidation in selected human cortical cataracts and nuclear cataracts. Exp Eye Res 1980; 31:361-9. 45. Spector A. Oxidation and cataract development. U.S.-EURAGE Conference, Oakland University, Rochester, Michigan, 1983. 46. Spector A, Gamer MH, Gamer WH, et al. An extrinsic membrane polypeptide associated with high-molecular-weight protein aggregates in human cataract. Science 1979; 204:1323-6. 47. Augusteyn RC. On the possible role of glutathione in maintaining human lens protein sulphydryls. Exp Eye Res 1979; 28:665-71. 48. Cheng HM, Chylack LT Jr. Thiol oxidation in the crystalline lens. I. The rate-limiting role of hexokinase in aging rat and human lenses. Invest Ophthalmol Vis Sci 1980; 19:522-8. 49. Varma SO, Kumar S, Richards RD. Light-induced damage to ocular lens cation pump: prevention by vitamin C. Proc Natl Acad Sci USA 1979; 76:3504-6. 50. Bessens G. Antioxidants in the human lens. U.S.-EURAGE Cataract Conference, Oakland University, Rochester, Michigan, 1983. 51. Zigman S. Near UV light and cataracts. Photochem Photobiol 1977; 26:437-41. 52. Lerman S. Radiant Energy and the Eye. New York: MacMillan, 1980. 53. Zigman S. Eye lens color: formation and function. Science 1971; 171:807-9. 54. Weiter JJ, Sabramanian S. Free radicals produced in human lenses by a biphotonic process. Invest Ophthalmol Vis Sci 1978; 17:86973. 55. Chalupecky H. Ueber die Wirkung der Rontgenstrahlen. Centralbl Prakt Augenheilkd 1897; 21 :386-401.

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56. Merriam GR, Focht EF. A clinical study of radiation cataracts and the relationship to dose. Am J Roentgenol 1957; 77:759-85. 57. Littleton JT, Ourizch ML, Perry N. Radiation protection of the lens for patients and users. Radiology 1978; 129:795-8. 58. Schneider G, Sager WO, Spreizer H. Strahlenbelastung der Orbita bei der Computertomographie mit dem EMI-scanner CT 1010. ROFO 1978; 128:687-90. 59. Hockwin O. Early changes of lens metabolism after X-irradiation. Exp Eye Res 1962; 1:422-6. 60. Oische Z. Alterations of lens proteins as etiology in cataracts. In: Oardenne MU, Nordmann J, eds. Biochemistry of the Eye; Symposium atTutzing Castle, August 10-13,1966. Basel: S. Karger, 1968; 41328. 61. Liem-The KN, Stols ALH, Jap PHK, Hoenders HJ. X-ray induced cataract in rabbit lens. Exp Eye Res 1975; 20:317-28. 62. Giblin FJ, Chakrapani B, Reddy VN. High molecular weight protein aggregates in X-ray-induced cataract. Exp Eye Res 1978; 26:50719. 63. Giblin FJ, Chakrapani B, Reddy VN. The effects of X-irradiation on lens reducing systems. Invest Ophthalmol Vis Sci 1979; 18:468-75. 64. Brinkman CJJ, Broekhuyse RM. Lens permeability changes in relation to x-ray induced cataract as detected by cell-mediated immune activity. Ophthalmic Res 1980; 12:230-4. 65. Benedek GB, Clark JI, Serralack EN, et al. Light scattering and reversible cataracts in the calf and human lens. Philos Trans R Soc Lond [Bioi] 1979; 292:121-32. 66. Bettelheim FA. Syneresis and its possible role in cataractogenesis. Exp Eye Res 1979; 28:189-97. 67. Chylack LT Jr, Bettelheim FA, Tung WHo Studies on human cataracts. I. Evaluation of techniques of human cataract preservation after extraction. Invest Ophthalmol Vis Sci 1981; 20:327-33. 68. Siew EL, Bettelheim FA, Chylack LT Jr, Tung WHo Studies on human cataracts. II. Correlation between the clinical description and the lightscattering parameters of human cataracts. Invest Ophthalmol Vis Sci 1981; 20:334-47. 69. Bettleheim FA, Siew EL, Chylack LT Jr. Studies on human cataracts. III. Structural elements in nuclear cataracts and their contribution to the turbidity. Invest Ophthalmol Vis Sci 1981; 20:348-54. 70. Philipson B. Changes in the lens related to the reduction of transparency. Exp Eye Res 1973; 16:29-39.