Sugar and sugar alcohol levels in the aging rat lens

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1 "T Sugar and sugar alcohol levels in the aging rat lens John F. R. Kuck, Jr." Fructose and sorbitol accumulate in the aging rat lens, the levels rising from values of 7 and 17 mg. per 0 Gin. wet lens weight, respectively, in weanlings, to 27 and 3 mg. per 0 Gm. in 200-day-old animals. The formation of fructose and sorbitol from glucose via the sorbitol pathway apparently decreases with age. Penetration of the lens in vitro by fructose-u-c-1 decreases with age. Furthermore, since the levels of glucose in the lens and aqueous humor and of fructose in the aqueous humor do not increase with age, the increased levels of fructose and sorbitol in older lenses must be a consequence of the failure of fructose to escape as readily from older lenses as from younger lenses. \ m.m JLhe discovery that diabetic rat lenses have high concentrations of fructose 1 and that all types of cataractogenic sugar diets,^»_ produce high lenticular concentrations of sugar alcohols 2 led to the suggestion that,/* ' the sorbitol pathway 3 - * might be involved >., in cataractogenesis. A weakness of this explanation is that only a relatively small '**' percentage of the glucose consumed by the J..*.. lens r ' may be diverted through the sorbitol pathway. The suggestion of Kinoshita 0 that k *- the accumulation of dulcitol in lenses of \ galactose-fed rats causes disruption of lens fibers by endosmosis has again inspired ^ interest in the sorbitol pathway as a mecha- From the Kresge Eye Institute, Detroit, Mich. This study was supported in part by Research Grant NB from the National Institute of Neurological Diseases and Blindness, United States Public Health Service, Bethesda, Md., and by the United States Atomic Energy Commission Contract No. AT(11-1)-152. "Present address: Laboratory for Ophthalmic Research, Emory University School of Medicine, Atlanta, Ga nism, the abnormal operation of which may initiate cataract formation. The present investigation stems from the discovery that lenses of 1-month-old rats have a level of fructose which is three times that of lenses of 6-week-old rats. The rate at which the fructose level changes with age has been determined and some of the factors which would be expected to affect its formation have been studied. Measurements have also been made of the rates of sorbitol and fructose accumulation by lens dispersions in vitro. Methods Rats of the Yale strain were decapitated, a blood sample obtained by drainage from the carcass, and the eyes removed. Samples of aqueous humor were obtained by nicking the orb with the point of a sharp scalpel just posterior to the limbus, inserting the needle of a Hamilton syringe past the iris into the anterior chamber, and aspirating gently as long as liquid flow continued. The eye was then opened beginning at this nick to expose the lens from the posterior. After removal the lens was rinsed in Ringer's solution, the zonules teased off, if possible, and the lens blotted free of clinging humors by rolling on dry tissue. Occasion- Downloaded From: on 0/17/201

2 60 Kuck Investigath : Ophthalmology December 19C3 r ally, concentrated zinc sulfate was added to the Ringer's solution to fix the protein at the lens surface to prevent loss of glucose. After weighing, the lens was heated in 0.2 ml. 5 per cent zinc sulfate at 0 C. for 5 minutes, crushed with a glass rod, and after cooling treated with barium hydroxide solution to prepare a Somogyi proteinfree extract, Samples of laked blood and aqueous humor were likewise converted to Somogyi extracts. All analyses were performed upon such extracts which were then frozen for storage. Analytical methods in part have been described previously, > 5 glucose by glucose oxidase and fructose by the Roe method. Sorbitol was analyzed by Faulkner's 7 modification of the West and Rapoport method for polyols, with the substitution of arsenate for stannous chloride as the reagent for destroying excess periodate. Simultaneous glucose and fructose standards were run to determine corrections for these sugars where present in appreciable amounts. In the incubation experiments, when fructose accumulation rates are compared in old and young lenses, the lens (for small lenses two were pooled) was crushed in a lens mineral solution Tris PCX medium 5 containing mg. per 0 ml. glucose or 360 mg. per 0 ml. glucose and 36 mg. sorbitol, and incubated at 37 C. for 3 hours. The contralateral lens was converted at once to a Somogyi extract to give initial values for the sugars and sorbitol. The penetration of fructose-u-c-1 into intact lenses was determined by incubating them with a medium devised by Kinoshita, s which contained 0 mg. per 0 ml. glucose and tracer fructose with or without 165 or 265 mg. per 0 ml. of carrier fructose. Counts were made on the medium and on Somogyi extracts of the lenses. Results The solid line of Fig. 1 shows the increase in fructose concentration in the lens as a function of the age of the animal. The lens weights were accurately measured; the ages are approximate as indicated by the breeder. The open circles of Fig. 1 show the values for glucose concentration in the lenses of the same animals. The glucose level is independent of age. Likewise the glucose level of the blood and aqueous humor and the level of fructose in the aqueous humor as shown in Table I appear to be independent of age. These data are from many of the same animals as those of Fig. 1. LENS WEIGHT (mg) Fig. 1. Levels of fructose and glucose in the aging rat lens. Later work has shown that the values for lens fructose in the two oldest groups of rats are abnormally high, probably as a result of an altered nutritional state. In Table II are the results from incubations that compare the fructose levels and accumulation rates of lens dispersions from young and old rats. There appears to be a slightly greater rate of fructose accumulation by old rats. However, these experiments did not take into account the initially greater amounts of sorbitol present in older lenses (Table V). In Table III are the results for incubations in which the accumulation rate of sorbitol was studied in lenses of various ages incubated in mg. per 0 ml. glucose. There is a significantly lower rate of sorbitol accumulation in older lenses. The results of experiments showing the penetration of tracer fructose into young and old lenses are given in Table IV. The concentration ratio lens fructose-c-1/ medium fructose-c-1 is larger for young lenses, i.e., is inversely proportional to the lenticular surface area. Table V sumarizes data for lenses of various ages including analyses for sorbitol (polyols). The values for the polyol have been corrected for interference caused by the presence of sugars. The correction in the case of fructose is large, therefore, the sorbitol values are less accurate than the sugar values. K K- Downloaded From: on 0/17/201

3 Volum, Numht Sugar and sugar alcohol levels in aging rat lens 609 Table I. The effect of aging (lens weight) on the levels of glucose in blood and aqueous humor and fructose in aqueous humor Blood sugar (mg./loo ml.) Glucose (me./0 ml.) Aqueous humor Fructose (mg./looml.) Table II. Fructose accumulation by dispersions of rat lenses of different ages incubated for 3 hours in LMS-Tris at initial concentrations of 360 mg./loo ml. glucose and 36 mg./loo ml. sorbitol Final fructose level Fructose accumulation (mg./j 00 ml.) rate (mg./gm./hr.) Table III. Sorbitol accumulation by dispersions of rat lenses of different ages incubated for hours in LMS-Tris at an initial glucose concentration of mg./loo ml ,9 Final glucose level (mg./loo ml.) Sorbitol accumulation rate (mg./gm./hr.) Table IV. Accumulation of fructose-u-c-1 by various sized lenses in vitro limitation is expressed as the ratio of 6 7 Time (hrs.) 2 2 CL/CM" of C-1 in the lens water to C-1 in the Lens surface (mm. 1 ) Downloaded From: on 0/17/201

6 Kuck Investigative Ophthalmology December 1963 Table V. Fructose, sorbitol, and glucose levels in lenses from rats of different ages 0 75 150 200 17.3 25.3 32.5 9. 5.0 15 2 2 2 Fructose (mg./loo Cm.

4 6 Kuck Investigative Ophthalmology December 1963 Table V. Fructose, sorbitol, and glucose levels in lenses from rats of different ages Fructose (mg./loo Cm.) Sorhitol (mg./loo Gm.) Glucose (mg./loogm.) Discussion In view of current interest in the hypothesis that the accumulation of dulcitol in the lenses of galactose-fed rats is the precipitating event in cataract formation, it appears timely to explore other situations where similar accumulations of polyols occur. The increasing levels of fructose and sorbitol in the aging rat lens are to be contrasted with the decreasing activity of the hexosemonophosphate shunt with age,"' Iu since it is only this pathway which can yield the supply of TPNH needed for the reduction of glucose to sorbitol. The increased accumulation of fructose and sorbitol in old lenses may be a result of any combination of three possibilities: (1) increased synthesis from glucose, (2) decreased consumption of fructose, or (3) decreased loss of fructose into the aqueous humor. Several points have been argued against the first explanation: the well-documented 0 ' decrease of hexosemonophosphate shunt activity with age, the absence of any increase in substrate levels with age (Table I), the decrease with age of the sorbitol accumulation rate in vitro (Table III), and the lack of a significant increase in the fructose accumulation rate in old lenses (Table II). When the second explanation is considered, the consumption of fructose by the lens in the presence of a normal glucose level is minimal and any decrease could hardly be significant. The evidence in favor of the third explanation includes the constancy of aqueous humor fructose levels with age despite an increased lens, aqueous concentration gradient (Table I), and the relative impermeability of old lenses to tracer fructose in the surrounding medium (Table V). Thus it appears that the most important factor leading to the accumulation of sorbitol and fructose is the failure of fructose to escape from the lens. The total amount of sorbitol and fructose in the lenses of 1-month-old rats approaches 0.1 per cent of the wet weight, which is less than 20 per cent of the mineral constituents of the lens. Salit and associates 11 reported that 9.5 mg. rat lenses contained per cent ash. The polyhydroxy compounds are thus a minor part of the osmotically active lens constituents, even in old rat lenses, and this fact correlates with the complete clarity of such lenses. A supposed connection between cataract formation and the accumulation of polyhydroxy compounds must involve secondary postulates, the first being that transient hyperglycemia will produce a great enhancement of the accumulation, the second being that the magnitude of this accumulation may be great enough to cause a serious loss of essential ions (K +, PO ), or else the rapidity with which it occurs may not be matched by the ability of the lens to adjust to the accompanying osmotic change. If osmotic damage occurs, the older lenses are less capable of making repairs because of their lower metabolic rate. The three important factors operating in this situation are: (1) a mechanism exists which appears capable of producing osmotic injury in hyperglycemia, (2) the accumulation of osmotically active products of this media- Downloaded From: on 0/17/201

5 Volume 2 Number 6 Sugar and sugar alcohol levels in aging rat lens 611 nism is increased in the aging lens, and (3) the necessary osmotic adjustments and the repairs to any injury cannot be carried out as easily in an older lens. Cataractogenesis of this type in older lenses is quite unlike that in young, rapidly growing lenses where it is usually assumed that there is an interference with metabolic processes. Summary The increased levels of fructose and sorbitol in older lenses are thought to result from a failure of fructose to escape from older lenses as readily as from younger lenses, rather than from increased synthesis or decreased utilization. This belief is supported by evidence that the formation of sorbitol from glucose decreases with age while the subsequent conversion of sorbitol to fructose does not increase with age. Furthermore, the levels of glucose in the lens and aqueous humor and of fructose in the aqueous humor do not increase with age. Finally, the accumulation of exogenous fructose, studied by incubating young and old lenses in fructose-u-c-1, has shown that older lenses are significantly less permeable to fructose. I wish to thank V. Everett Kinsey for his support of this investigation and Dianne Kokowicz for excellent technical assistance. REFERENCES 1. Kinsey, V. E., Wachtl, C, Kuck, J. F. R., Jr., and Reddy, D. V. N.; Current research on the culture of lenses, Acta Cone. 1: 65, VanHeyningen, R.: Formation of Polyols by the lens of the rat with "sugar" cataract, Nature 1: 19, VanHeyningen, R.: Metabolism of xylose by the lens. 2. Rat lens in vivo and in vitro, Biochem. J. 73: 197, Kuck, J., Jr.: The formation of fructose by the ocular lens, A. M. A. Arch. Ophth. 65: 0, Kuck, J., Jr.: Glucose metabolism and fructose synthesis in the diabetic rat lens, INVEST. OPHTH. 1: 390, Kinoshita, J. H., Merola, L. O., Satoh, K, and Dikmak, E.: Osmotic changes caused by the accumulation of dulcitol in the lenses of rats fed with galactose, Nature 19: 5, Faulkner, P.: Enzymic reduction of sugar phosphates in insect blood, Biochem. J. 6: 36, Kinoshita, J., and Merola, L. O.: Personal communication. 9. Lerman, S.: Carbohydrate metabolism in the rat leas as related to the age of the animal, A. M. A. Arch. Ophth. 65: 11, Kinoshita, J. H., and Dillen, I.: The effect of age on the carbohydrate metabolism of bovine lens, Acta Cone. 1: 7, Salit, P. W., Swan, K. C, and Paul, W. D.: Changes in the mineral composition of rat lenses with galactose cataract, Am. J. Ophth. 25: 12, 192. Downloaded From: on 0/17/201

A theory for explaining the etiology of

A theory for explaining the etiology of Lens assays on diabetic and galactosemic rats receiving diets that modify cataract development John W. Patterson, Margaretta E. Patterson, V. Everett Kinsey, and D. V. N. Reddy Cataract formation is delayed