Characterisation of a novel AGE-compound derived from lysine and 3-deoxyglucosone

Dicarbonyl compounds are supposed to be reactive intermediates in the non-enzymatic glycation of proteins. A process that eventually could lead to the development of late diabetic complications. Glyoxal lysine dimer (GOLD) and methylglyoxal lysine dimer (MOLD) have previously been described as such reactive dicarbonyls. Here a new compound 3-deoxyglucosone lysine dimer (DOLD), a cross-link resulting from the reaction between hippuryl-lysine and 3-deoxyglucosone, has been isolated by HPLC and the structure determined by mass spectrometry and NMR.     

Age-dependent increase in ortho-tyrosine and methionine sulfoxide in human skin collagen is not accelerated in diabetes. Evidence against a generalized increase in oxidative stress in diabetes

The glycoxidation products Nepsilon-(carboxymethyl)lysine and pentosidine increase in skin collagen with age and at an accelerated rate in diabetes. Their age-adjusted concentrations in skin collagen are correlated with the severity of diabetic complications. To determine the relative roles of increased glycation and/or oxidation in the accelerated formation of glycoxidation products in diabetes, we measured levels of amino acid oxidation products, distinct from glycoxidative modifications of amino acids, as independent indicators of oxidative stress and damage to collagen in aging and diabetes. We show that ortho-tyrosine and methionine sulfoxide are formed in concert with Nepsilon-(carboxymethyl)lysine and pentosidine during glycoxidation of collagen in vitro, and that they also increase with age in human skin collagen. The age-adjusted levels of these oxidized amino acids in collagen was the same in diabetic and nondiabetic subjects, arguing that diabetes per se does not cause an increase in oxidative stress or damage to extracellular matrix proteins. These results provide evidence for an age-dependent increase in oxidative damage to collagen and support previous conclusions that the increase in glycoxidation products in skin collagen in diabetes can be explained by the increase in glycemia alone, without invoking a generalized, diabetes-dependent increase in oxidative stress.     

Role of NADH/NADPH oxidase-derived H2O2 in angiotensin II-induced vascular hypertrophy

The Maillard reaction between sugar and protein has been postulated as the cause for the browning and arrestment of caries lesions. This reaction has been implicated as the cause for decreased degradability of collagen in vivo. The aim of the present study was to verify the occurrence of the reaction in vivo. Carious and sound dentin samples were taken from extracted human teeth and analyzed for the fluorescence characteristic of the Maillard reaction and oxidation and, by HPLC, for Maillard products. In addition, physiological cross-links were analyzed by HPLC. Oxidation- and Maillard reaction-related fluorescence increased in collagenase digests from carious dentin. Advanced Maillard products (carboxymethyllysine and pentosidine) increased, whereas furosine, a marker for the initial reaction, was not observed consistently. This implies no direct addition of sugars to protein, but rather the addi-tion of smaller metabolites and glycoxidation products. In addition, the physiological cross-links hydroxylysinonorleucine and dihydroxylysinonorleucine decreased in carious dentin. Also for hydroxylysylpyridinoline, a decrease was observed, but not consistently. In conclusion, the caries process modifies amino acids in dentin collagen, which can lead to increased resistance against proteolysis and ultimately to caries arrestment.     

Age-related increase in an advanced glycation end product in penile tissue

Nonenzymatic glycosylation (glycation) of proteins, often referred to as the Maillard reaction, has been proposed to play a role in age and diabetes-related processes by forming protein and DNA adducts and cross-links. These cross-links may contribute to erectile dysfunction by scavenging nitric oxide, which is needed for erection. As the basis for a possible role of the advanced Maillard reaction in age-related erectile dysfunction, we investigated the presence of the specific advanced glycation endproduct (AGE) pentosidine in penile corpus cavernosum tissue and penile tunica albuginea tissue as a function of age. A total of 23 penile tissue specimens were obtained at autopsy, from which 19 samples of tunica albuginea and 21 samples of corpus cavernosum were derived. In addition, 13 penile corporal and tunical specimens were procured at the time of insertion of a penile prosthesis, from which 12 tunica albugineal specimens and 10 samples of corpus cavernosum were derived. Collagen was extracted with acetic acid and pepsin digestion, and the final insoluble collagen product was acid-hydrolyzed with 6 N HCL for 24 h at 110 degrees C. Pentosidine was quantified by high-performance liquid chromatography using a reverse-phase column. The level of pentosidine (expressed in picomoles per milligram of insoluble collagen) was found to increase with age in cadaver as well as living penile corporal and tunical albugineal tissues. Best-fit analysis revealed an exponential increase in both types of cadaver penile tissue, with regression equations of y = 15.29 x 10(9.9e-3x), R2 = 0.79, being obtained in the tunica and y = 13.2 x 10(7.63e-3x), R2 = 0.56, in the corpora. These correspond to 6- and 4-fold increases in pentosidine levels from puberty to the age of 100 years (P < 0.05), respectively. Mean pentosidine levels were higher in the tunica than in the corpora. Comparison of pentosidine levels in the tunica versus the corpora revealed a weakly linear correlation (y = 24.88 + 1.08x, R2 = 0.32). Levels in the tunical and corporal specimens from the living human specimens fell with the predicted confidence intervals of the cadaveric tissue. Tunical specimens from patients who underwent repair or revision of a previously inserted penile prosthesis had very low levels of pentosidine. The exponential age-related increase in pentosidine observed in both types of penile tissue suggests an impairment of collagen turnover, which could be related to the advanced glycation reaction in aging. It is not known whether pentosidine itself is directly associated with erectile dysfunction, but its formation is usually accompanied by extensive tissue modification. Formation of advanced Maillard reaction products, which is greatly accelerated in aging, diabetes, and uremia, could contribute to erectile dysfunction in these syndromes.     

Are aldehydes in heat-sterilized peritoneal dialysis fluids toxic in vitro?

OBJECTIVE: Chemical analysis of several brands of peritoneal dialysis fluids (PD fluids) has revealed the presence of 2-furaldehyde, 5-HMF (5-hydroxymethylfuraldehyde), acetaldehyde, formaldehyde, glyoxal, and methylglyoxal. The aim of this study was to investigate if the in vitro side effects caused by glucose degradation products, mainly formed during heat sterilization, are due to any of these recently identified aldehydes. DESIGN: Cell growth media or sterile filtered PD fluids were spiked with different concentrations of thealdehydes. MEASUREMENTS: In vitro side effects were determined as the inhibition of cell growth of cultured mouse fibroblasts or stimulated superoxide radical release from human peritoneal cells. RESULTS: Our results demonstrate that the occurrences of 2-furaldehyde, 5-HMF, acetaldehyde, formaldehyde, glyoxal, or methylglyoxal in heat-sterilized PD fluids are probably not the direct cause of in vitro side effects. In order to induce the same magnitude of cell growth inhibition as the heat-sterilized PD fluids, the concentrations of 2-furaldehyde, glyoxal, and 5-HMF had to be 50 to 350 times higher than those quantified in the PD fluids. The concentrations of acetaldehyde, formaldehyde, and methylglyoxal observed in the heat-sterilized PD fluids were closer to the cytotoxic concentrations although still 3 to 7 times lower. CONCLUSION: Since none of these aldehydes caused in vitro toxicity at the tested concentrations, the toxicity found in PD fluids is likely to be due to another glucose degradation product, not yet identified. However, it is possible that these aldehydes may still have adverse effects for patients on peritoneal dialysis.     

Cross-linking of proteins by Maillard processes--model reactions of D-glucose or methylglyoxal with butylamine and guanidine derivatives

Advanced Maillard reaction in proteins leads to formation of covalently cross-linked aggregates the chemical nature of which is largely unknown. From model reactions of methylglyoxal and butylamine with creatine or alpha-N-acetyl-L-arginine, one main product each was isolated. These two compounds were identified, on the basis of unequivocal spectroscopic evidence, as 2-[(5-butylimino-4-methyl-4,5-dihydro-1H-2-imidazolyl)(methyl)amin o]acetic acid and 2-acetylamino-5-[(5-butylimino-4-methyl-4,5-dihydro-1H-2-imidazoly l)amino]pentanoic acid, respectively. Using D-glucose instead of methylglyoxal, two main products each were obtained from reaction with the respective guanidine derivative. The spectroscopic data definitively establish the formation of the diastereoisomeric 2-[(4-butyl-6,7-dihydroxy-4,5,6,7,8,8a-hexahydroimidazo[4,5-b]a zepin-2-yl)(methyl)amino]acetic acid from the reaction with creatine, and of the diastereoisomeric 2-acetylamino-5-[(4-butyl-6,7-dihydroxy-4,5,6,7,8,8a-hexahydroimidazo [4,5-b]azepin-2-yl)amino]pentanoic acid from the reaction with alpha-N-acetyl-L-arginine. All products were isolated in fairly good yield and represent 1:1:1 adducts of the respective reaction partners. Formation of these compounds thus constitutes an efficient reaction pathway for linking primary amines to guanidine derivatives. It seems justified, therefore, to expect cross-linking of proteins by action of reducing carbohydrates to proceed analogously.     

Free lysine, glycine, alanine, glutamic acid and aspartic acid reduce the glycation of human lens proteins by galactose

The amino acids lysine, glycine, alanine, glutamate and aspartate formed adducts with galactose at physiological pH and temperature as shown by incorporation of U[14C] galactose. The percentage of galactose reacting with lysine, glycine, alanine, glutamate and aspartate was 4.5 to 7.8, 7.9 to 10.8, 3.2 to 4.6, 2.8 to 4.8 and 3 to 5.2, respectively. Studies with lysine showed that the extent of glycation of the free amino acid increased with time. Incubation of lens homogenate with galactose, effected glycation of proteins. Addition of lysine in concentrations of 5 and 10 mM to equimolar concentrations of galactose decreased the glycation of lens proteins by 64% to 71%; glycine, alanine, glutamate and aspartate decreased glycation by 23 to 68%, 32 to 61%, 35 to 56% and 26 to 61% respectively. Under similar conditions, glycine reacts to a greater extent than lysine, alanine, glutamic and aspartic acids. However, lysine was more effective than glycine, alanine, aspartic and glutamic acids in decreasing glycation of lens proteins by galactose. The decrease of glycation with added lysine increased with time. In general increase of amino acid concentration rather than that of sugar augmented the decrease of glycation of lens proteins.     

On the occurence of isopeptide bonds in the chorion of the rainbow trout (Salmo gairdneri Rich.) (author's transl)

Chorions of unfertilized and fertilized eggs of the rainbow trout were isolated from tissue and yolk components and investigated for isopeptide bonds. The complete hydrolysis of the alpha-amide bonds was obtained by a system of four proteases. A chromatographic ion exchange system was used to separate Nepsilon-(beta-aspartyl)lysine as well as Nepsilon-(gamma-glutamyl)lysine from single amino acids. Chorions of unfertilized eggs contain neither Asp Lys nor Glu Lys bonds. Only chorions of fertilized eggs contain Glu Lys isopeptide. It is probably the high content of this isopeptide that is responsible for the greater mechanical stability of the fertilized teleostean egg chorion.     

Hydrolysis of gamma:epsilon isopeptides by cytosolic transglutaminases and by coagulation factor XIIIa

Nepsilon-(gamma-glutamyl)lysine cross-links, connecting various peptide chain segments, are frequently the major products in transglutaminase-catalyzed reactions. We have now investigated the effectiveness of these enzymes for hydrolyzing the gamma:epsilon linkage. Branched compounds were synthesized, in which the backbone on the gamma-side of the cross-bridge was labeled with a fluorophor (5-(dimethylamino)-1-naphthalenesulfonyl or 2-aminobenzoyl) attached through an epsilon-aminocaproyl linker in the N-terminal position, and the other branch of the bridge was constructed with Lys methylamide or diaminopentane blocked by 2,4-dinitrophenyl at the Nalpha position. Hydrolysis of the cross-link could be followed in these internally quenched substrates by an increase in fluorescence. In addition to the thrombin and Ca2+-activated human coagulation Factor XIIIa, cytosolic transglutaminases from human red cells and from guinea pig liver were tested. All three enzymes were found to display good isopeptidase activities, with Km values of 10(-4) to 10(-5) M. Inhibitors of transamidation were effective in blocking the hydrolysis by the enzymes, indicating that expression of isopeptidase activity did not require unusual protein conformations. We suggest that transglutaminases may play a dynamic role in biology not only by promoting the formation but also the breaking of Nepsilon-(gamma-glutamyl)lysine isopeptides.     

The level of the collagen cross-link pyridinoline reflects the improvement of cutaneous lesions in one case of skin alveolar echinococcosis

Cutaneous parasitic lesions, associated with a dense fibrous reaction, markedly improved under albendazole treatment in one case of supraumbilical skin localization of alveolar echinococcosis. Since collagen cross-linking increases during fibrogenesis and contributes to the stability of fibrotic lesions, we monitored the level of the cross-links pyridinoline and pentosidine in skin lesions from this patient to determine if they would reflect the changes occurring during treatment. We looked at the deposition of cross-linked type I collagen by immunohistochemistry and also measured the serum concentrations of pentosidine and of a fragment of type I collagen (ICTP), which contains a site of pyridinoline formation. Albendazole treatment did not affect either the collagen content of skin lesions or the serum concentrations of ICTP and pentosidine, but it led to a pronounced decrease in pyridinoline level concomitant with the disappearance, observed by immunohistochemistry, of extensively cross-linked fibrotic type I collagen. The follow-up of collagen cross-linking by pyridinoline in skin tissue thus appears to be useful in reflecting the improvement of fibrotic skin diseases during therapy.     

Immunohistochemical detection of imidazolone and N(epsilon)-(carboxymethyl)lysine in aortas of hemodialysis patients

The modification of long-lived proteins with advanced glycation endproducts (AGEs) has been hypothesised to contribute to the development of pathologies associated with uremia. Imidazolone and N(epsilon)-(carboxymethyl)lysine (CML) are common epitopes of AGE-modified proteins. Imidazolone is a reaction product of arginine with 3-deoxyglucosone (3-DG) which is markedly accumulated in uremic serum. CML is produced by glycoxidation, and represents a marker of oxidative stress. The specificity of anti-imidazolone antibody that we had developed was further examined using ELISA. The antibody reacted only with imidazolone derived from 3-DG and arginine, but did not react at all with the other imidazolone-like compounds such as reaction products of glyoxal, methylglyoxal, glucosone with arginine or a reaction product of 3-DG with creatine. Further, to determine if AGEs are involved in the development of atherosclerosis in hemodialysis (HD) patients, we studied the localisation of imidazolone and CML in the aortas obtained from HD patients by immunohistochemistry using the anti-imidazolone and anti-CML antibodies. Imidazolone and CML were localised in all atherosclerotic aortic walls of the HD patients. In conclusion, imidazolone and CML are localised in the characteristic lesions of atherosclerosis in HD patients. These results strongly suggest that imidazolone produced by 3-DG, and CML produced by glycoxidation may contribute to the development of atherosclerosis in uremic patients.     

The outbreak of an epidemic of tick-borne encephalitis in Kielec province induced by milk ingestion

New liquid UHT milks supplemented with iron (0.9-1.4 mg/100 ml), vitamin C (1-7 mg/100 ml), lactose (2-4 g/100 ml) and linoleic acid (200-400 mg/100 ml), named growth milks, have recently become available to satisfy the specific nutritional needs of children aged 1-3 years. But the iron-vitamin C mixture could activate the lactose-induced Maillard reaction and tryptophan (Trp) oxidation in proteins. We have therefore examined the Amadori product and Trp concentrations of these milks. Forty-two commercial growth milks from five firms were analysed for the Maillard reaction and the soluble protein Trp content and compared with 64 UHT milks. The furosine concentration of total proteins was two to four times higher in 'growth' milks than in standard UHT milks, indicating a proportional loss of available lysine. The Trp fluorescence of undenatured proteins soluble at pH 4.6 was almost three times lower in 'growth' than in standard milks and Trp concentration 36% lower suggesting destruction of this oxidation-sensitive amino-acid. The mechanism of Trp destruction remains to be elucidated, and the roles of iron and Amadori products determined.     

Age-dependent structural changes in intact human lenses detected by synchrotron radiation X-ray scattering. Correlation with Maillard reaction protein fluorescence

To understand alterations in the spatial organization of the crystallins, the major determinant of lens transparency, the x-ray scattering by intact normal human lenses from individuals 6-82 years of age was measured using synchrotron radiation. The angular dependence of the integrated scattering intensity is consistent with short-range order within the crystallin assemblies. A significant change in the scattering patterns of the lenses occurs after 55 years of age, in parallel with an increase of the fluorescence of the urea-insoluble crystallin fraction. This correlation suggests a gradual derangement of the short-range order as a result of cross-linking of the crystallin subunits by advanced Maillard reaction products that are generated by the continuous reaction of sugars, such as glucose or fructose, with proteins.     

Tissue ascorbic acid and polyol pathway metabolism in experimental diabetes

Previous studies demonstrating reduced plasma concentrations of ascorbic acid (AA) in diabetes and interactions between this vitamin and biochemical mechanisms such as synthesis of structural proteins, oxidative stress, polyol pathway and nonenzymatic glycation of proteins suggest that disturbed AA metabolism may be important in the pathogenesis of diabetic microangiopathy. However, limited information is available on the concentration of AA in tissues which develop diabetic complications. This study demonstrates reduced renal but not sciatic nerve or plasma AA concentration in two animal models of insulin-dependent diabetes mellitus, namely the STZ-diabetic rat and the spontaneously diabetic BB rat. Decreased lens AA concentration was also observed in STZ-diabetic rats. Improvement of glycaemic control by insulin treatment (albeit insufficient to achieve normoglycaemia) partially corrected lens and renal AA concentration in STZ-diabetic rats. AA treatment increased kidney and lens AA concentrations of STZ-diabetic and non-diabetic rats and corrected the abnormalities observed for untreated diabetic rats. Sciatic nerve AA concentration was not increased by AA treatment in any group. Tissue ratios of dehydroascorbic acid (DHAA)/AA, one index of oxidative stress, were not different between the diabetic and non-diabetic groups and were unaltered by AA supplementation. AA treatment of STZ-diabetic rats had no effect on elevated tissue concentrations of glucose, sorbitol and fructose or reduced myo-inositol concentration. The effect of reduced tissue AA levels in diabetes on either collagen synthesis or ability to combat increased free radical production is not known. However, correction of abnormal kidney and lens AA concentrations in experimental diabetes by AA supplementation suggests that if AA does have a role in the development or progression of the renal and ocular complications of diabetes, this treatment could be beneficial.     

The intermolecular cross-links in uterine collagens of guinea pig, pig, cow, and human beings

The intermolecular cross-links have been studied in the uterine insoluble collagen of guinea pig, pig, cow, and human beings with a single given procedure. After NaB3H4 reduction, there are three intermolecular cross-links; namely, dihydroxylysinonorleucine, hydroxylysinonorleucine, and histidinohydroxymerodesmosine. In human uterine collagen samples these reduced cross-links are present in equal amounts. The reduced intermolecular collagen cross-links of uterine leiomyoma are very similar to those of the normal uterine tissue. Dihydroxylysinonorleucine is the principal reduced cross-link in uterine collagen of guinea pig, pig, and cow. Alkaline hydrolysis reveals that dehydrodihydroxylysinonorleucine and dehydrohydroxylysinonorleucine occur in vivo as glycosylated derivatives.     

Reaction of N-acetylglycyllysine methyl ester with 2-alkenals: an alternative model for covalent modification of proteins

Among the various reactions of lipid peroxidation products with proteins, 2-alkenals have been shown to react extensively with the epsilon-amino group of lysine residues [Zídek et al. (1997) Chem. Res. Toxicol. 10, 702-710]. To obtain additional information about the kinetic and mechanistic aspects of this modification, a model peptide (N-acetylglycyllysine O-methyl ester) was reacted with 2-hexenal. The reaction products were characterized through a combination of NMR and MS techniques. The structural elucidation efforts have shown the formation of pyridinium salts through the reaction of two or more alkenals with one amino group. Kinetic data were obtained using a continuous infusion of the reaction mixture into an electrospray ionization mass spectrometer. A mechanism is proposed that offers an alternative model for the formation of stable protein cross-links. The reaction progresses through a Schiff base intermediate to form a dihydropyridine species which can be alternatively reduced to form various 3,4- or 2,5-substituted pyridinium species or react with another Schiff base to form a trialkyl-substituted pyridinium structure. The stoichiometry of this structure (aldehyde/amine) is 3:2, in contrast to the widely accepted 1:2. Therefore, it represents another possible cross-linking mechanism for bifunctional products of lipid peroxidation.     

Variability of choline acetyltransferase in ocular tissues of rabbits, cats, cattle and humans

It has been proved in three rodent species that in the insoluble collagen fraction which accumulates in skin collagen with age of the two categories of collagen present (collagen type I and collagen type III), their proportion alters in favour of collagen type I with the advancing age. Since it has also been shown that collagen type I is less resistant towards proteolytic cleavage than is collagen type III its accumulation can be explained either by rapidly advancing cross-linking of this collagen type or more likely by different proteosynthesis. The second alternative is preferred since a step-wise polymerization of collagen type III was also observed. No information revealing to what extent the lysine derived cross-links can combine both collagen types is at present available. On the basis of this information the rapid decrease in insoluble collagen in very early ontogeny (rats below 8 weeks of age) is explained.     

Oxidation-reduction properties of methylglyoxal-modified protein in relation to free radical generation

Oxidation-reduction properties of methylglyoxal-modified protein in relation to free radical generation were investigated. Glycation of bovine serum albumin by methylglyoxal generated the protein-bound free radical, probably the cation radical of the cross-linked Schiff base, as observed in the reaction of methylglyoxal with L-alanine (Yim, H.-S., Kang, S.-O., Hah, Y. C., Chock, P. B., and Yim, M. B. (1995) J. Biol. Chem. 270, 28228-28233) or with Nalpha-acetyl-L-lysine. The glycated bovine serum albumin showed increased electrophoretic mobility suggesting that the basic residues, such as lysine, were modified by methylglyoxal. The glycated protein reduced ferricytochrome c to ferrocytochrome c in the absence of oxygen or added metal ions. This reduction of cytochrome c was accompanied by a large increase in the amplitude of the electron paramagnetic resonance signal originated from the protein-bound free radical. In addition, the glycated protein catalyzed the oxidation of ascorbate in the presence of oxygen, whereas the protein free radical signal disappeared. These results indicate that glycation of protein generates active centers for catalyzing one-electron oxidation-reduction reactions. This active center, which exhibits enzyme-like characteristic, was suggested to be the cross-linked Schiff base/the cross-linked Schiff base radical cation of the protein. It mimics the characteristics of the metal-catalyzed oxidation system. The glycated bovine serum albumin cross-linked further to the cytochrome c in the absence of methylglyoxal. The cross-linked cytochrome c maintains its oxidation-reduction properties. These results together indicate that glycated proteins accumulated in vivo provide stable active sites for catalyzing the formation of free radicals.     

High galactose levels in vitro and in vivo impair ascorbate regeneration and increase ascorbate-mediated glycation in cultured rat lens

In contrast to conventional view that glucose is the sole glycating agent, ascorbate has now emerged as a potential precursor of advanced glycation products in lenses during cataractogenesis, owing to the high concentration present in human lens. The effects of high hexose environment in vitro and in vivo on the disruption of redox equilibrium of ascorbate (ASA) to dehydroascorbate (DHA), which is required for ascorbate-mediated crystallin modification by the Maillard reaction during cataractogenesis were examined. Organ culture experiments were performed with rat lenses that were first exposed to high galactose levels in vitro and in vivo and then incubated with 1-14C-labeled ASA, DHA or DKG (2,3-diketogulonic acid). Formation of ASA degradation products as a function of time was assessed by radiometric TLC method. Upon incubation with ASA or DHA, an elevated level of the degradation product, DKG, was detected in lenses exposed to galactose in vivo and in vitro. ASA uptake was significantly enhanced in the galactosemic lenses as compared to controls (P = 0.01). Regeneration of ASA from DHA in both galactose treated and galactosemic lenses was impaired when compared to control lens which completely converted DHA from the medium into ASA. Surprisingly, the galactose exposed lenses showed enhanced permeability to DKG which was picked up readily from the medium in contrast to normal healthy lenses which remained impermeable to DKG. Galactose exposed lenses both in vitro and in vivo showed a 5-9-fold increase in crystallin bound Schiff base-linked radioactivity when incubated with 1-14C-labeled ASA or DHA. As a preamble to the question of whether lens pigmentation predisposes towards ascorbate oxidation, lens homogenate from normal young and old pigmented cataractous lenses were incubated with [1-14C]ASA. After 2 days, ASA levels were found to have decreased by 74% and DKG levels increased by 48% in brunescent lens as compared to the young lens. These data demonstrated that profound abnormalities in ASA metabolism exist in lenses exposed to a high sugar environment suggestive of a breakdown of the redox equilibrium of ASA to DHA and a loss of membrane permeability barrier for DKG. The latter would further contribute toward a ASA-catalysed Maillard reaction in the redox impaired lens.