Inhibition of lens crystallin glycation and high molecular weight ...

Investigative Ophthalmology & Visual Science, Vol. 30, No. 6, June 1989Copyright © Association for Research in Vision and Ophthalmology

Inhibition of Lens Crystallin Glycotion ond HighMolecular Weight Aggregate Formation

by Aspirin In Vitro and In VivoM. S. Swamy ond E. C. Abraham

Previous studies have shown that glycation of lens proteins could be a contributory factor in thedevelopment of diabetic and senile cataracts. Acetylation by aspirin (acetylsalicylic acid or ASA) hasbeen used as an inhibitor of glycation which blocks the potential glycation sites (c-NH2 groups). Ifglycation is a contributory factor, inhibition of glycation by acetylation should bring about a corre-sponding decrease in cataractogenic changes. We relied on in vitro glycation system and streptozoto-cin-diabetic rats to study the effects of ASA on lens crystallin glycation, thiol oxidation and aggrega-tion. For in vitro studies, sterile lens soluble crystallin preparations from 1-month-old rats wereincubated, under nitrogen, with 50 mM glucose and 20 mM ASA up to 15 days at 37°C. To study thein vivo effect in diabetic rats, ASA feeding (200 mg/kg body wt/day) was initiated 1 week prior tostreptozotocin administration, and sacrificed on 15,30, 60 and 90 days after injection. The in vitro datashow the inhibitory effect on glycation of ASA with all concentrations that were tested (5,10, 20 mMASA); the percentage inhibition increased with increasing ASA concentration and time. For example,with 50 mM glucose and 20 mM ASA incubated for 15 days, there was a significant decrease inglycation (P < 0.05), thiol oxidation (P < 0.05) and aggregation (P < 0.02). Similarly, in in vivoexperiments ASA feeding delayed lens opacification by about 30 days with a significant inhibition oflens protein glycation (P < 0.02), while the levels of glucose remaining almost the same (P ^ 0.5), witha corresponding decrease in HMW aggregates (P < 0.02) and an increase in free protein thiols (P< 0.02) in 90 day diabetic animals. These results strongly suggest that enhanced protein modificationby glycation followed by protein unfolding and sulfhydryl oxidation is a critical factor in diabeticcataractogenesis. Invest Ophthalmol Vis Sci 30:1120-1126,1989

Previous studies in our laboratory12 and else-where3"7 have indicated that crystallin glycationcould be a contributory factor in the formation ofhigh molecular weight (HMW) protein aggregatesthat are either disulfide-linked, cross-linked by ad-vanced glycation products, or noncovalently linked.It is believed that glycation initiates or enhances pro-tein unfolding, thus exposing sulfhydryl groups toundergo oxidation especially under increased oxida-tive challenge.3 Although this is a reasonable hypoth-esis for the development of cataract, diabetic cataract

From the Department of Cell and Molecular Biology, MedicalCollege of Georgia, Augusta, Georgia.

Preliminary results were presented at the ARVO Annual Meet-ing, Sarasota, Florida, May, 1988 and at the International Congressof Eye Research, San Francisco, California, September, 1988.

Supported by a grant from National Institutes of Health(EY-07394).

Submitted for publication: August 29, 1988; accepted December13, 1988.

Reprint requests: E. C. Abraham, PhD, Department of Cell andMolecular Biology, Medical College of Georgia, Augusta, GA30912-2100.

in particular, direct involvement of lens protein gly-cation in the events leading to cataractogenesis is yetto be established. One way of achieving this goal is toinhibit glycation by blocking the glycation sites with-out affecting the level of glucose. Post-translationalmodifications are believed to play a significant role inpathogenesis of several tissues including lens pro-teins.7 Acetylation by acetylsalicylic acid (aspirin orASA) of lysine residues inhibits glycation of plasmaproteins,8"10 hemoglobin," and lens proteins.1213

Acetylation has also been shown to prevent carba-mylation, another post-translational modification oflens crystallins.14 Rao et al15 reported that acetylationof e-amino groups of lens crystallins by ASA mayprotect against aggregation by blocking glycation,carbamylation or isopeptide bond formation. Use ofaspirin as therapeutic agent to reduce diabetic com-plications was earlier reported by Reid et al.'6 Cotlierand colleagues,17"19 and van Heyningen and Hard-ing20 proposed possible protection against cataract byaspirin, although the evidence for aspirin was dis-puted by others.21"24 We have used acetylation byASA of potential glycation sites to study its effect on

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No. 6 LENS CRYSTALUN GLYCATION AND AGGREGATION / Swomy and Abraham 1121

protein glycation, thiol oxidation and aggregation inan in vitro glycation system and in streptozotocin-di-abetic rats.

Materials and Methods

In Vitro Glycation Studies

The purpose of these studies was to demonstratethat in vitro glycation also can lead to HMW proteinaggregation and that acetylation by ASA inhibits lenscrystallin glycation and prevents HMW aggregation.The in vitro incubation of lens proteins was doneaccording to the method described by Stevens et al.3

Briefly, lenses were removed from 1-month-old maleSpague-Dawley rats, homogenized under nitrogen in50 mM sodium phosphate buffer, pH 7.0, centrifugedat 6000 g for 60 min, the supernatant dialyzed overnight against the same buffer and sterile filteredthrough a series of 5,0.8, and 0.45 nm filters (GelmanSciences, Ann Arbor, MI). The initial experimentswere designed to establish the time and ASA concen-tration dependent inhibition of glycation. For thispurpose incubations were done with [14C]glucose andthe cpm incorporated per milligram of protein wasmonitored. Incubation mixture contained 50 mMglucose and 10 /*Ci [14C]glucose (specific activity 250mCi/mmol; ICN Radiochemical, Irvine, CA), 50 y\streptomycin-penicillin, and 2.5 mg of lens crystal-lins per milliliter. To determine the time-dependentchanges aliquots of 0.5 ml were removed after 1, 3, 5,10 and 15 days of incubation at 37°C in the presenceand absence of 20 mM ASA. After reduction with100 /il of 1 M NaBH4 (10 min at room temperatureand an additional 50 min in ice) the samples wereextensively dialyzed against water (with at least twochanges) overnight at 4°C. The radioactivity wasmeasured with toleune-based scintillation cocktail(containing triton X-100, POPOP and PPO). To de-termine the ASA concentration dependence of glyca-tion inhibition, incubations were done for a fixed pe-riod of 15 days with 0, 5, 10 and 20 mM ASA. ASAconcentrations beyond 20 mM resulted in proteinprecipitation and were not further pursued. Afterthese initial studies, additional lens crystallin incuba-tions were done with 50 mM glucose alone (contain-ing no [14C]glucose), 50 mM glucose and 20 mMASA, 20 mM ASA alone, and with no additives (ieprotein alone) to determine the effect of ASA onHMW aggregation and free thiols. In these experi-ments aliquots were removed on the 5th and 15th dayof incubation and mixed with equal volume of 14 Murea to ensure complete solubilization. Sulfhydryl ti-tration with parachloromercuribenzoate (PCMB) wasperformed immediately whereas affinity chromato-graphic quantitation of glycated protein and high

pressure liquid chromatographic (HPLC) separationof HMW aggregates were done after reduction withNaBH4 (as described elsewhere in the methods).

Studies in Streptozotocin-Diabetic Rats

Seventy male Sprague-Dawley rats, age 1 month,were divided into four groups. Group 1 (15 rats)served as controls. Group 2 (20 rats) were made dia-betic by intravenous injection of streptozotocin (65mg/kg body weight) through tail vein. Group 3(15rats) were aspirin controls; these animals were fedpowdered rat chow mixed with ASA (MallinckrodtChemical Works, St. Louis, MO), 200 mg/kg bodyweight/day, according to Rendell et al." Group 4 (20rats) were aspirin diabetic; these animals were fedsimilar to group 3 but were made diabetic 1 weekafter starting the aspirin diet. Three to four animalsfrom each group were sacrificed on the 15, 30, 60 and90 days after injection. Blood was collected for thedetermination of plasma glucose and glycated hemo-globin (GHb). Lenses were removed and homoge-nized and separated into water-soluble (WS) andwater-insoluble (urea-soluble or US) fractions for theseparation of HMW protein aggregates and the deter-minations of glycated protein and free thiols.

Animal experiments were performed in compli-ance with ARVO Resolution on the Use of Animalsin Research and of the Institutional Committee forAnimal Care.

Crystallin Preparation

The water-soluble and the urea-soluble crystallinswere prepared according to the modified procedureof Herbrink and Bloemendal25 as described in detailbefore.1

Quantification of Glycated Crystallins

The glycated proteins in the WS and US fractionswere determined by affinity chromatography as de-scribed in previous communications1'2 using Glyc-Affin microcolumns from Isolab Inc. (Akron, OH).The columns prepacked with phenylboronate agaroseand the developers needed for the chromatographywere supplied by the manufacturer. About 0.5 mg ofthe WS or the US fraction (or the urea-solubilizedtotal protein from the in vitro studies) was used foreach determination. For the US fractions all thebuffers contained 7 M urea.

Sulfhydryl Titration

The concentration of free protein thiols was deter-mined by titration with PCMB according to the mod-ified method of Boyer.26


1122 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1989 Vol. 30

Incubation period (days) Aspirin concentration <mM>

Fig. 1. (A) Time-dependence of the inhibition of [l4C]glucoseincorporation into lens crystallins by aspirin, in vitro. Lens crystal-lins were incubated with 50 mM glucose containing [14C]glucosewith 20 mM aspirin (•) or without 20 mM aspirin (•) up to 15days. Before counting for radioactivity, aliquots were reduced withNaBH4 and dialyzed extensively. (B) Aspirin concentration depen-dence of the inhibition of [l4C]glucose incorporation. Lens crystal-lins were incubated with 50 mM glucose and 5-20 mM aspirin for15 days.

Molecular Sieve HPLC Separation of HMWAggregates

For separation of the HMW aggregates in the WSand US fractions previously reported molecular sieveHPLC methodologies were used.27 The basic set-upconsisted of a Beckman HPLC system, having model421 controller, dual 110 pumps and model 160 de-tector with 20 jtl flow cell and Hewlett-Packard3390-A recording integrator. For WS proteins TSK3000 SW (7.5 X 600 mm) column coupled in serieswith a TSK 4000 SW (7.5 X 300 mm) column wasused. The isocratic mobile phase consisted of 50 mMsodium phosphate, 50 mM NaCl, pH 6.8 with a flowrate of 1 ml min"1. The crystallin components fromthe urea-solubilized fractions were separated withTSK 2000 SW (7.5 X 600 mm) column coupled with

a TSK 3000 SW (7.5 X 300 mm) column. The iso-cratic mobile phase consisted of 7 M urea, 100 mMsodium phosphate, 100 mM NaCl and 5 mM EDTAat pH 6.5. All chromatograms were run at ambienttemperature and absorbance was monitored at280 nm.

Other Methods

Plasma glucose levels were determined by the glu-cose oxidase assay of Raabo and Terkildsen28 pro-vided in kit from Sigma Chemical Company (St.Louis, MO). GHb levels were determined with theIsolab Glyc-Amn System and the method describedby the manufacturer was strictly followed. Statisticalanalysis was done using the student t-test where com-parisons were made between untreated diabeticgroup and aspirin treated diabetic group and also be-tween controls and aspirin treated controls.

Results

Inhibition of Glycation and Aggregation ofCrystallins by Aspirin in the In Vitro System

Figure 1A shows the time dependence of in vitroglycation, assessed by cpm incorporated per milli-gram protein of lens crystallins with 50 mM glucose(containing 10 fid of 14C glucose) in the presence andabsence of 20 mM ASA. The presence of ASA inhib-ited glycation substantially; the percentage inhibitionincreased from 35% on day 1 to 46% on day 15 indi-cating increased modification of potential glycationsites with increasing time. The inhibitory effect ofASA was evident at all concentrations that weretested (Fig. IB); again, percentage inhibition in-creased with increasing ASA concentration. Analysisof the incubation mixture for glycated protein, HMWaggregates, and thiol groups on the 5th and 15th dayof incubations is shown in Table 1 (also see Fig. 2 forthe separation of HMW aggregates in the urea-solubi-

Table 1. Effect of aspirin (ASA) on glycation, thiol oxidation and HMW aggregation of lens proteins in vitro

Incubationconditions

0 mM glucose50 mM glucose0 mM glucose

+ 20 mM ASA50 mM glucose

+ 20 mM ASA

Glycatedproteins

(%)

7.1 ± 1.519.9 ±4.0

7.1 ±0.9

12.9 ± 1.9

5 day

Thiols(n moles -SH/mg

protein)

48 ± 5.025 ± 8.0

42 ± 5.6

41 ±6.0

Incubation period (days)

HMWaggregate

(%)

1.3 ±0.43.6 ± 0.9

0.8 ± 0.2

2.7 + 0.5

Glycatedproteins

(%)

7.4 ± 0.924.7 ±5.1

7.9 ±0.8

14.9 ± 1.9

15 day

Thiols(n moles —SH/mg

protein)

43 ±4.518 ±6.6

42 ± 4.2

28 ± 6.5

HMWaggregate

(%)

1.2 ±0.520.5 ± 5.0

3.5 ± 1.0

8.4 ± 2.0

0 day values: glycated proteins: 3.0 ± 1.0; thiols 54 ± 4.0; HMW: 1.3 ± 0.2


No. 6 LENS CRYSTALUN GLYCATION AND AGGREGATION / Swamy and Abraham 1123

lized incubation mixture). The glycation data, ob-tained by the affinity column technique also con-firmed that ASA indeed inhibits glycation. Increasedglycation has led to increased HMW aggregation andthiol oxidation whereas the inhibition of glycation byASA produced corresponding inhibition of proteinaggregation and protection of protein thiols (15 dayincubation: glycated proteins, P < 0.05; thiols, P< 0.05; HMW aggregates, P < 0.02). Incubation ofprotein alone did not generate much HMW aggre-gates. On the contrary, incubation of a mixture of theprotein and ASA (without glucose) showed an in-crease in the protein aggregates, but the increase wasrelatively small (less than 3-fold) compared to about17-fold increase in aggregation observed with 50 mMglucose. It is difficult to establish the source of oxida-tive challenge in our in vitro system. Although theincubation tubes were sealed under nitrogen (follow-ing the method of Stevens et al3), the residual oxygenin the incubation buffers may oxidize the exposedthiols or oxidation may have occurred by hithertounknown mechanism.

0.05-1

••5 0.025-

10 20 30 40 10 20

Retention Time (in min)

Fig. 2. Molecular sieve HPLC separation of lens proteins. After15 days incubation lens crystallins were mixed with equal volumesof 14 M urea, reduced with NaBH4, dialyzed and separated on TSK2000 SW column coupled in series to TSK 3000 SW column withan isocratic mobile phase containing 7 M urea and developed witha flow rate of I ml min"1.

In Vivo Effect of Aspirin in Streptozotocin-Diabetic Rats

Cataract development: Among the 40 rats that weremade diabetic, 20 received ASA-containing diet. Inabout 90 days, lens opacity was obvious in all thediabetic animals (about 2+), whereas ASA-treatedones were close to normal (Fig. 3). However, by 120days the ASA-treated diabetic animals developed cat-aract (about 2+), whereas the untreated ones showedextremely dense opacity (4+ or higher) (data notshown). Thus, acetylation with ASA seems to havedelayed the glycation induced lens opacification.

Lens protein glycation: As expected, the plasmaglucose levels were higher in the diabetic rats andthese levels were unaffected by aspirin treatment (P2: 0.5) (Fig. 4A). GHb levels also responded to hyper-glycemia in the diabetic animals not receiving ASA;however, a significant decrease in GHb was noticedin the ASA-treated diabetic animals (P < 0.002 for30-90 day values), indicating inhibition of hemoglo-bin glycation (Fig. 4B). Glycated protein levels of theWS and US fractions were significantly reduced byASA treatment (P < 0.05 for 60 day values) (Fig. 4C,D). The results of the glycated protein determinationsof the control animals were rather inconsistent; how-ever, the general trend was a decrease in glycationwith ASA treatment.

HMW aggregate formation: Figure 5B and C showthe effect of ASA treatment on lens protein aggrega-tion. As expected from our previous studies1 therewas a steady increase of the HMW aggregate forma-

tion in the WS and US fractions of the diabetic andcontrol animals, the former consistently havinghigher levels of aggregates than the latter. The forma-

Fig. 3. Delay of lens opacification by aspirin. (A) 90 day un-treated diabetic rat; (B) 90 day diabetic rat fed with aspirin.



isoo-0)

0 10 20 30 40 SO 60 70 80 90 100 70 B0 90 100

Fig. 4. Effect of aspirin feeding on thelevels of: (A) plasma glucose; (B) glycatedhemoglobin; (C) lens soluble glycated pro-teins; and (D) lens insoluble glycated pro-teins. • , age-matched controls, X con-trols fed with aspirin, •, diabetic, and A,aspirin-fed diabetics. Controls n = 3 anddiabetics n = 4. Values are mean ± SD.

0 10 20 30 40 SO 60 70 B0 90 100

Diabetic duration (In days)

0 10 20 30 40 50 60 70 80 90 100

Diabetic duration (in days)

tion of both these types of aggregates was significantlyreduced (10-40%) in the diabetic animals receivingASA (soluble, P < 0.02 for 30-90 day values; insolu-ble, 60 day, P < 0.02; 90 day, P < 0.002). In thecontrols, on the other hand, after 90 days of ASAadministration an increase in the HMW aggregateswas noticed (soluble, P < 0.02; insoluble, P < 0.05),which was consistent with what was seen in the invitro system (Fig. 2, Table 1).

ASA treatment also resulted in convincingly higherlevel of free protein thiols than those not receivingthis drug (for 60 and 90 day, P < 0.02) (Fig. 5A).

DiscussionIt is well known that ASA can acetylate at the

amino groups, such as a-NH2 and e-NH2 groups, onproteins.8""15 A number of reports in the past haveindicated ASA therapy having a beneficial effect in

preventing cataract in human,17 19 presumably by in-hibiting glycation, whereas others contradicted suchfindings by showing increased risk of having cataractby long-term ASA treatment.2'-24 Most of these stud-ies focused on senile cataract formation. It wouldhave been more meaningful to look at the effect ofASA on the development diabetic cataract, where ex-cessive glycation seems to play a significant role. Thiswas one of the reasons for us to focus on in vitroglycation and on a diabetic animal model. The cur-rent studies also were expected to establish a relation-ship between lens protein glycation and aggregationin cataract development. Each system had a certainadvantage. The in vitro system lacked all other fac-tors except the presence of high levels glucose and theresultant increased glycation could be inhibited byASA to study its effect on protein aggregation. In thein vivo studies, on the other hand, in addition to


No. 6 LENS CRYSTALUN GLYCATION AND AGGREGATION / Swamy and Abraham 1125

hyperglycemia all their metabolic activities were pres-ent and the effect of inhibition of glycation by ASAcould be studied in a true in vivo model.

The results of the in vitro studies have convincinglyshown that glycation precedes protein unfolding andaggregation. By inhibiting glycation, a correspondinglevel of inhibition of HMW aggregate formation wasseen (Fig. 2, Table 1). The results of the in vivo stud-ies in streptozotocin-diabetic rats complementedthose of the in vitro studies. An ASA diet had a signif-icant inhibitory effect on crystallin glycation in thediabetic animals which in turn inhibited the forma-tion of both nondisulfide-linked soluble aggregatesand disulfide-linked insoluble aggregates (Figs. 4, 5).This may mean that glycation directly influencesprotein unfolding, and sulfhydryl oxidation and ag-gregation is only a secondary event (because the solu-ble aggregates contain no disulfides). However, signif-icant depletion of thiol groups occurred even duringthe early period (within 5 days) of incubation in thepresence of 50 mM glucose (P < 0.01) and also duringearly days (within 15 days) of hyperglycemia in thediabetic rats (P < 0.05). During this period, however,the increase in HMW aggregates was only minimal(Table 1, Fig. 5). Thus, it is possible that even beforeHMW aggregates become detectable, disulfide-linked, low molecular weight aggregates could beformed.

The inhibitory effect of acetylation by ASA appearsto be mediated through a blockage of amino groupsthat prevents glycation. In fact, the direct effect ofacetylation, apart from protecting these aminogroups, seems to be a slight increase in the unfoldingof the proteins as seen in the control incubations oflens crystallins with ASA (without glucose) and incontrol rats receiving ASA (Table 1 or Fig. 5). On thecontrary, in the diabetic animals where excessive gly-cation is the main problem, this "adverse effect" ofacetylation is overcome by the apparent beneficialeffect of glycation inhibition. In essence, it appearsthat acetylation is less harmful and has a less destabi-lizing effect on lens protein organization than glyca-tion. These findings may have some bearing on thevarious contradictory reports, based on clinical expe-rience, on the effect of ASA therapy on cataractogen-esis.21-24 Glycated lens proteins accumulates ratherslowly over the entire life of a nondiabetic rat (orhuman) and to effectively inhibit glycation ASA hasto be administered over a long period of time, whichmay be undesirable because the adverse effects mayoverwhelm the beneficial effects. This increases therisk of senile cataract. In the diabetic population, onthe other hand, glucose level increases rapidly andcataract develops over a relatively short period oftime, which could be delayed by ASA treatment.

c

ote

i

a.aEiW(A

0Ec

70 -

6 0 -

5 0 -

40 -

3 0 -

20 -

10 -0 10 20 30 40 50 60 70 80 90 100

10 20 30 40 50 60 70 80 90 100

0 10 20 30 40 50 60 70 80 90 100

Diabetic duration (in days)

Fig. 5. Effect of aspirin feeding on: (A) soluble lens protein thiols;(B) soluble HMW aggregates; and (C) insoluble HMW aggregates.Line legends are same as in Figure 4. Controls n = 3 and diabetics n= 4. Values are mean ± SD.

However, the dosage used in the current study is ex-cessively high, as is the blood glucose level. ASA ishydrolyzed to salicylic acid, which is not an acetylat-ing agent.2930 Therefore, higher doses are required tobring about protection of amino groups by acetyla-tion, which may also be one of the reasons for only30-40% protection observed in these in vivo studies(Fig. 5).

It seems that there is a lot of analogy between theeffect of ASA on glycation and its effect on carbamy-lation of lens proteins as investigated by Harding andcoworkers.14 The carbamylation of lens proteins also



causes conformational changes and cataract. It wasshown that ASA reduces the rate of carbamylation ofall the lens crystallins whereby decreasing the opacifi-cation.

Interestingly, in a recent report Huby and Hard-ing'3 showed protection of nonenzymatic galactosy-lation of lens crystallins by ASA in an in vitro system,which is in agreement with our findings concerningnonenzymatic glycosylation both in vitro and in vivo.We have chosen to run appropriate controls for allthe experiments, which made the interpretation ofthe data much easier and the conclusions moremeaningful.

Key words: diabetes, glycation, acetylation, lens crystallins,protein aggregation

AcknowledgmentWe are indebted to Mrs. A. Abraham for excellent tech-

nical assistance.

References1. Perry RE, Swamy MS, and Abraham EC: Progressive changes

in lens crystallin glycation and high-molecular weight aggre-gate formation leading to cataract development in streptozot-ocin-diabetic rats. Exp Eye Res 44:269, 1987.

2. Swamy MS and Abraham EC: Lens protein composition, gly-cation and high molecular weight aggregation in aging rats.Invest Ophthalmol Vis Sci 28:1693, 1987.

3. Stevens VJ, Rouzer CA, Monnier VM, and Cerami A: Dia-betic cataract formation: Potential role of glycosylation of lenscrystallins. Proc Natl Acad Sci USA 75:2918, 1978.

4. Monnier VM, Stevens VJ, and Cerami A: Nonenzymatic gly-cosylation, sulfhydryl oxidation, and aggregation of lens pro-teins in experimental sugar cataracts. J Exp Med 150:1098,1979.

5. Monnier VM and Cerami A: Non-enzymatic browning invivo: Possible process for aging of long-lived proteins. Science211:491, 1981.

6. Pongor S, Ulrich PC, Bencsath FA, and Cerami A: Aging ofproteins: Isolation and identification of fluorescent chromo-phore from the reaction of polypeptides with glucose. ProcNatl Acad Sci USA 81:2684, 1984.

7. Harding JJ: Nonenzyamatic covalent posttranslational modifi-cation of proteins in vivo. Adv Prot Chem 37:247, 1985.

8. Day JF, Thorpe SR, and Baynes JW: Nonenzymatically glu-cosylated albumin: In vitro preparation and isolation fromnormal human serum. J Biol Chem 254:595, 1979.

9. Day JF, Thornbug RW, Thorpe SR, and Baynes JW: Nonen-zymatic glucosylation of rat albumin: Studies in vitro and invivo. J Biol Chem 254:9394, 1979.

10. Yue DK, McLennan S, Handelsman DJ, Delbridge L, ReeveT, and Turtle JR: The effect of salicylates on nonenzymaticglycosylation and thermal stability of collagen in diabetic rats.Diabetes 33:745, 1984.

11. Rendell M, Nierenberg J, Brannan C, Valentine JL, StephenPM, Dodds S, Mercer PO, Smith PK, and Walder J: Inhibitionof glycation of albumin and hemoglobin by acetylation in vitroand in vivo. J Lab Clin Med 108:286, 1986.

12. Swamy MS and Abraham EC: Influence of glycation inhibitorson thiol oxidation and aggregation of lens crystallins in vitro.ARVO Abstracts. Invest Ophthalmol Vis Sci 29(Suppl):30,1988.

13. Huby R and Harding JJ: Non-enzymic glycosylation (glyca-tion) of lens proteins by galactose and protection by aspirinand reduced glutathione. Exp Eye Res 47:53, 1988.

14. Crompton M, Rixon K.C, and Harding JJ: Aspirin preventscarbamylation of soluble lens proteins and prevents cyanate-induced phase separation opacities in vitro: A possible mecha-nism by which aspirin could prevent cataract. Exp Eye Res40:297, 1985.

15. Rao GN, Lardis MP, and Cotlier E: Acetylation of lens crys-tallins: A possible mechanism by which aspirin could preventcataract formation. Biochem Biophys Res Comm 128:1125,1985.

16. Reid J, MacDougall AJ, and Andrews MM: Aspirin and dia-betes mellitus. Br Med J ii:1071, 1957.

17. Cotlier E: Senile cataracts: Evidence for acceleration by dia-betes and deceleration by salicylate. Can J Ophthalmol 16:113,1981.

18. Cotlier E and Sharma YR: Aspirin and senile cataracts inrheumatoid arthritis. Lancet i:338, 1981.

19. Cotlier E, Sharma YR, Niven T, and Brescia M: Distributionof salicylates in lens and intraocular fluids and its effects oncataract formation. Am J Med 74:83, 1983.

20. van Heyningen R and Harding JJ: Do aspirin-like analgesicsprotect against cataract? A case-control study. Lancet i:l 111,1986.

21. Seigel D, Speraduto RD, and Ferris FL: Is ASP therapy forcataracts justified? Can J Ophthalmol 17:135, 1982.

22. Bignall JC: Aspirin, nifedipine, and cataract. Lancet ii:42,1986.

23. Williams DG: Aspirin, nifedipine, and cataract. Lancet ii:43,1986.

24. Kewitz H, Nitz M, and Gaus V: Aspirin and cataract. Lancetii:689, 1986.

25. Herbrink P and Bloemendal H: Studies on /3-crystallin: I. Iso-lation and partial characterization of the principal polypeptidechain. Biochim Biophys Acta 336:370, 1974.

26. Boyer PD: Spectrophotometric study of the reaction of proteinsulfhydryl groups with organic mercurials. J Am Chem Soc76:4331, 1954.

27. Perry RE and Abraham EC: High performance liquid chro-matographic separation of lens crystallin and their subunits. JChromatogr 351:103, 1986.

28. Raabo E and Terkildsen TC: In the enzymatic determinationof blood glucose. Scand J Clin Lab Invest 12:402, 1960.

29. Rowland M and Riegelman S: Pharmacokinetics of acetyl sali-cylic acid and salicylic acid after intravenous administration inman. J Pharm Sci 57:1313, 1968.

30. Costello PB and Green FA: Aspirin survival in human bloodmodulated by the concentration of erythrocytes. ArthritisRheum 25:550, 1982.


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