Phototransformations of advanced glycation end products in the human eye lens due to ultraviolet A light irradiation.

Previous studies from this laboratory have shown that ultraviolet A (UVA) light can bleach the yellow advanced glycation end products (AGEs) of aged and cataractous human lenses. The AGEs OP-lysine and argpyrimidine are two UVA-absorbing posttranslational modifications that are abundant in the eye lens. The purpose of this study was to outline the changes in these two AGEs due to UVA irradiation. The changes of OP-lysine, OP-phenethylamine (a phenethylamine analogue of OP-lysine), and argpyrimidine due to irradiation with UVA light in the presence or absence of air and ascorbic acid were followed by different spectral methods. Aged human lenses were similarly irradiated in artificial aqueous humor. The amounts of OP-lysine in the irradiated lenses and in the corresponding dark controls were determined by HPLC. Both OP-lysine and argpyrimidine decreased 20% when irradiated with UVA light in the absence of ascorbic acid. Under the same conditions, OP-lysine was bleached 80% in the presence of ascorbic acid during irradiation experiments. In contrast, argpyrimidine UVA light bleaching was not affected by the presence of ascorbic acid. Interestingly the major product of OP-phenethylamine after UVA irradiation in the presence of ascorbic acid was phenethylamine, which indicates that the entire heterocycle of this AGE was cleaved and the initial amino group was restored. Some AGEs in the human eye lens can be transformed by UVA light.

Similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid-modified calf lens proteins: evidence for ascorbic acid glycation during cataract formation.

Chromatographic evidence supporting the similarity of the yellow chromophores isolated from aged human and brunescent cataract lenses and calf lens proteins ascorbylated in vitro is presented. The water-insoluble fraction from early stage brunescent cataract lenses was solubilized by sonication (WISS) and digested with a battery of proteolytic enzymes under argon to prevent oxidation. Also, calf lens proteins were incubated with ascorbic acid for 4 weeks in air and submitted to the same digestion. The percent hydrolysis of the proteins to amino acids was approximately 90% in every case. The content of yellow chromophores was 90, 130 and 250 A(330) units/g protein for normal human WISS, cataract WISS and ascorbate-modified bovine lens proteins respectively. Aliquots equivalent to 2.0 g of digested protein were subjected to size-exclusion chromatography on a Bio-Gel P-2 column. Six peaks were obtained for both preparations and pooled. Side by side thin-layer chromatography (TLC) of each peak showed very similar R(f) values for the long wavelength-absorbing fluorophores. Glycation with [U-(14)C]ascorbic acid, followed by digestion and Bio-Gel P-2 chromatography, showed that the incorporated radioactivity co-eluted with the A(330)-absorbing peaks, and that most of the fluorescent bands were labeled after TLC. Peaks 2 and 3 from the P-2 were further fractionated by preparative Prodigy C-18 reversed-phase high-performance liquid chromatography. Two major A(330)-absorbing peaks were seen in peak 2 isolated from human cataract lenses and 5 peaks in fraction 3, all of which eluted at the same retention times as those from ascorbic acid glycated calf lens proteins. HPLC fractionation of P-2 peaks 4, 5 and 6 showed many A(330)-absorbing peaks from the cataract WISS, only some of which were identical to the asorbylated proteins. The major fluorophores, however, were present in both preparations. These data provide new evidence to support the hypothesis that the yellow chromophores in brunescent lenses represent advanced glycation endproducts (AGEs) probably due to ascorbic acid glycation in vivo.

Sugar-mediated crosslinking of alpha-biotinylated-Lys to cysteamine-agarose support: a method to isolate Maillard Lys-Lys-like crosslinks.

Advanced glycation end products (AGEs) and, specifically, protein-protein AGE crosslinks have long been studied for their potential role in aging, diabetic complications and Alzheimer disease. With few exceptions, the chemical nature of these structures remains unknown. We report here a simple approach that allows the preparation and isolation of milligram quantities of sugar-mediated AGE Lys-Lys-like crosslinks from glycation mixtures. The method is based on a sugar-dependent incorporation of N(alpha)-biotinyl-L-Lys into cysteaminyldisulfide Sepharose 6B (AE-S-S-Sepharose 6B). Glycation mixtures with six different sugars showed a time- and sugar-dependent decrease in the concentration of the support-bound primary amino groups and accounted for almost 90% loss of cysteaminyl amino groups at the end of the various incubation periods. 4-Hydroxyazobenzene-2-carboxylic acid-avidin assays indicated the incorporation of N(alpha)-biotinyl-L-Lys equal to 8% of the total support amino groups with methylglyoxal after 7 d and 1% with fructose and glucose after 1 mo of incubation. Treatment of the washed, sugar-modified supports with 2-mercaptoethanol released the bulk of the bound AGE modifications and the crosslinks. Subsequent fractionation of these preparations over a monomeric avidin column afforded a complete separation of sugar-mediated AGE modifications and the crosslinks. Depending on the sugar employed, micromolar amounts of biotinylated Lys-Lys-like crosslinks were generated by this two-step procedure from 8 mL of the original AE-S-S-Sepharose 6B.

Inhibitors of advanced glycation end product-associated protein cross-linking.

The reaction of lens proteins with sugars over time results in the formation of protein-bound advanced glycation end products (AGEs). The most damaging element of AGE formation may be the synthesis of protein-protein cross-links in long-lived proteins, such as collagen or lens crystallins. A quantitative cross-linking assay, involving the sugar-dependent incorporation of [U-(14)C]lysine into protein, was employed to determine the efficacy of a variety of potential cross-linking inhibitors. Reaction mixtures contained 5.0 mM L-threose, 2.5 microCi [(14)C]lysine (1.0 mCi/mmole), 5.0 mg/ml bovine lens proteins, 0-10 mM inhibitor and 1.0 mM DTPA in 100 mM phosphate buffer, pH 7.0. Of 17 potential inhibitors tested, 11 showed 50% inhibition or less at 10 mM. The dicarbonyl-reactive compounds 2-aminoguanidine, semicarbazide and o-phenylenediamine inhibited 50% at 2.0 mM, whereas 10 mM dimethylguanidine had no effect. Several amino acids failed to compete effectively with [(14)C]lysine in the cross-linking assay; however, cysteine inhibited 50% at 1.0 mM. This was likely due to the sulfhydryl group of cysteine, because 3-mercaptopropionic acid and reduced glutathione exhibited similar activity. Sodium metabisulfite had the highest activity, inhibiting 50% at only 0.1-0.2 mM. Protein dimer formation, as determined by SDS-PAGE, was inhibited in a quantitatively similar manner. The dicarbonyl-reactive inhibitors and the sulfur-containing compounds produced similar inhibition curves for [(14)C]lysine incorporation over a 3 week assay with 250 mM glucose. A much lesser effect was observed on either the incorporation of [(14)C]glucose, or on fluorophore formation (360/420 nm), suggesting that non-cross-link fluorophores were also formed. The inhibitor data were consistent with cross-linking by a dicarbonyl intermediate. This was supported by the fact that the inhibitors were uniformly less effective when the 5.0 mM threose was replaced by either 3.0 mM 3-deoxythreosone or 3.0 mM threosone.

The non-oxidative degradation of ascorbic acid at physiological conditions.

The degradation of L-ascorbate (AsA) and its primary oxidation products, L-dehydroascorbate (DHA) and 2,3-L-diketogulonate (2, 3-DKG) were studied under physiological conditions. Analysis determined that L-erythrulose (ERU) and oxalate were the primary degradation products of ASA regardless of which compound was used as the starting material. The identification of ERU was determined by proton decoupled (13)C-nuclear magnetic resonance spectroscopy, and was quantified by high performance liquid chromatography, and enzymatic analysis. The molar yield of ERU from 2,3-DKG at pH 7.0 37 degrees C and limiting O(2)97%. This novel ketose product of AsA degradation, was additionally qualitatively identified by gas-liquid chromatography, and by thin layer chromatography. ERU is an extremely reactive ketose, which rapidly glycates and crosslinks proteins, and therefore may mediate the AsA-dependent modification of protein (ascorbylation) seen in vitro, and also proposed to occur in vivo in human lens during diabetic and age-onset cataract formation.

Chromatographic comparison of the UVA sensitizers present in brunescent cataracts and in calf lens proteins ascorbylated in vitro.

The water-insoluble (WI) fraction from aged human lenses contains yellow chromophoric sensitizers, which generate reactive oxygen species (ROS) when irradiated with UVA light. The WI proteins from type I to V brunescent cataract lenses were assayed for UVA-dependent superoxide anion synthesis. Rates varied from 8.4-15 nMol h(-1)mg protein(-1), but there was no significant difference in specific activity between cataract types. When calf lens soluble proteins were incubated with ascorbic acid for 4 weeks and dialyzed, they were capable of generating 30-40 nMol h(-1)mg protein(-1)superoxide anion when irradiated with UVA light. Two preparations each of brunescent cataract WI proteins and bovine lens proteins ascorbylated in vitro were extensively digested with proteolytic enzymes and the released amino acids separated by normal phase HPLC. The elution profiles of the digests were very similar based upon the absorbance at 330 nm and fluorescence at 350 nm excitation/450 nm emission. Each peak was pooled and analyzed for the UVA-dependent generation of both superoxide anion and singlet oxygen. Every peak exhibited sensitizer activity, and the UVA-dependent ROS generation was roughly proportional to the absorbance at 330 nm. In addition, the ratio of superoxide anion to singlet oxygen generated was similar with both preparations. These data argue that it is the brown, fluorescent compounds which accumulate during aging and cataract formation that are responsible for the UVA-dependent ROS formation, and that these browning products may be similar to the advanced glycation endproducts produced by ascorbylation of lens proteins under oxidative conditions. This work also presents an initial report of a chromatographic method to separate the UVA-sensitizers present in each of these protein preparations without the use of acid or base hydrolysis.

Spontaneous generation of superoxide anion by human lens proteins and by calf lens proteins ascorbylated in vitro.

The proteins isolated from aged human lenses and brunescent cataracts exhibit extensive disulfide bond formation. Diabetic rat lenses similarly contain disulfide-bonded protein aggregates. These observations are consistent with the known link between diabetes, glycation and oxidative damage, and suggest a role for reactive oxygen species (ROS) in this process. To assess whether the glycation-related modifications in human lens proteins spontaneously generate ROS, superoxide anion formation was measured using both cataractous lens proteins and calf lens proteins glycated in vitro with ascorbic acid (ascorbylated). The water-insoluble fraction from aged normal human lenses generated 0.3-0.6 nmol superoxide h(-1)mg protein(-1), whereas the activity increased to 0.5-1.8 nmol h(-1)mg protein(-1)with the WI fraction from brunescent cataracts, and 2.3 nmol h(-1)mg protein(-1)with calf lens proteins ascorbylated for 4 weeks in vitro. The activity in the human lens proteins was observed in both the water-soluble and water-insoluble fractions, and was completely dependent upon the presence of oxygen. The pH optimum curve for superoxide formation increased from pH 6.5 to 10 with both the cataract and ascorbylated proteins. The superoxide-generating activity in human lens was completely bound to a boronate affinity column, but only partially bound with the ascorbylated proteins. The superoxide anion produced by a 5 m m solution of purified N(epsilon)-fructosyl-lysine was barely detectable, and therefore, could not account for the superoxide formed by any of the lens protein preparations. Also, superoxide formation increased 10-fold at pH 8.8 with fructosyl-lysine, but only 1.3-1.8-fold with human lens proteins. The addition of copper-stimulated superoxide formation with glycated bovine serum albumin, but no stimulation was seen with cataractous proteins. Assays of specific compounds showed that catechol, hydroquinone, 3-OH kynurenine and 3-OH anthranylic acid exhibited the greatest activity for superoxide generation, but had a very short halflife. 2,3-Dihydroxypyridine and 4,5 dihydroxynaphthalene were one and two orders of magnitude less reactive. In long-term incubations at 37 degrees, cataractous proteins retained the potential to produce superoxide anion, losing only half of the initial activity after 6-7 days. Therefore, the water-insoluble fraction from aged human lenses and dark brown cataracts are potentially capable of generating >100 nmol mg protein(-1)and >170 nmol mg protein(-1)of superoxide anion respectively, likely due to the presence of advanced glycation endproducts in human lens proteins. This spontaneous generation of superoxide anion in vivo could account for a major portion of the oxidation of sulfur amino acids seen during aging and cataract formation.

Lens proteins block the copper-mediated formation of reactive oxygen species during glycation reactions in vitro.

The formation of advanced glycation endproducts (AGEs) from glucose in vitro requires both oxygen and a transition metal ion, usually copper. These elements combine to produce reactive oxygen species (ROS) which degrade glucose to AGE-forming compounds. We measured the ability of Cu(2+) to accelerate ROS formation, and the effect of added lens proteins on these reactions. Increasing levels of Cu(2+) accelerated the formation of superoxide anion with glucose and fructosyl-lysine, but the addition of 2.0 mg/ml calf lens proteins completely blocked superoxide formation up to 100 microM of added Cu(2+). Lens proteins, however, had no effect on superoxide generated by the hypoxanthine/xanthine oxidase system. The oxidation of ascorbic acid was increased 170-fold by the addition of 10 microM Cu(2+), but was also completely prevented by added lens proteins. Hydroxyl radical formation, as measured by the conversion of benzoate to salicylate, was increased to 30 nmoles/ml after 18 h by the addition of 100 microM Cu(2+) and 2.5 mM H2O2. This increase was also blocked by the addition of lens proteins. However, hydroxyl radical formation, as estimated by the crosslinking and fragmentation of lens proteins, was observed in the presence of 100 microM Cu(2+), likely at the sites of Cu(2+) binding. Since the ratio of lens proteins to Cu(2+) in human lens is at least 1000-fold higher than those used here, the data argue that Cu(2+) in the lens would be tightly bound to protein, preventing ROS-mediated AGE formation from glucose in vivo.

Superoxide free radical generation by Amadori compounds: the role of acyclic forms and metal ions.

Generation of oxygen free radicals by glycated proteins is widely believed to be one of the causes of oxidative stress in diabetes and aging. Metal ion catalysis is regarded as an essential part of the oxidative mechanism. In this work, we also considered an alternative "metal-free" superoxide radical formation by a number of fructose-amino acids (Amadori compounds) derived from glycine and lysine, which represent the simplest models for early glycated proteins. In the superoxide dismutase-dependent cytochrome c assay, 1 mM Chelex-treated aqueous solutions of monofructose-amino acids 4-6 generated 0.9-3.6 x 10(-10) M s-1 O2*- at pH 7. Surprisingly, the rates of superoxide radical formation in the solutions of difructose-amino acids 7-9 were significantly higher (0.75-5.8 x 10(-9) M s-1 O2*-). The percentage of acyclic sugar anomers (

Electron paramagnetic resonance and spin trapping investigations of the photoreactivity of human lens proteins.

Oxidation of cysteine, glutathione and ascorbate by photoexcited proteins from normal and cataractous lenses was investigated using electron paramagnetic resonance in combination with spin trapping. We report that illumination of these proteins in pH 7 buffer with light > 300 nm in the presence of thiols (RSH) and a spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), afforded DMPO/S-cysteine and DMPO/SG adducts, suggesting the formation of the corresponding thiyl radicals. In a nonbuffered aqueous solution, illumination of the proteins and glutathione also produced superoxide detected as a DMPO/O2H adduct. Irradiation of these proteins in the presence of ascorbate generated ascorbate radical. We conclude that chromophores present in the natural normal and cataractous lenses are capable of initiating photooxidative processes involving endogenous thiols and ascorbic acid. This observation may be pertinent to UV-induced development of cataract.

The relative ability of glucose and ascorbate to glycate and crosslink lens proteins in vitro. off.

Nonenzymatic glycation by glucose and/or ascorbate leads to the formation of advanced glycation end products (AGEs), which are thought to be a critical element in lens protein aging and cataract formation. The relative participation of these two glycating agents was evaluated in vitro. The incubation of 100 mM [U-14C]-D-glucose and 10 mM [U-14C]-L-ascorbate with lens proteins resulted in an increasing incorporation over 3 weeks, reaching a maximum of 100 nMol mg-1 protein and 160 nMol mg-1 protein with ascorbate. Glycation was proportional to carbohydrate concentration with both reagents, however ascorbate was 18-fold more reactive with lens proteins than glucose. Protein crosslinking was not obvious with 250 mM glucose as measured by SDS-PAGE, however, ascorbate caused extensive crosslinking even at 3.0 mM. The sugar-dependent incorporation of N alpha-formyl-[U-14C]-L-lysine ([U-14C]Nfl) into proteins, gave values of 1.5 nMol mg-1 protein after 3 weeks with 100 mM glucose compared to 11 nMol mg-1 protein with 10 mM ascorbate. On a molar basis, ascorbate was 70-fold more active than glucose and 100-fold more active than fructose in the crosslinking assay. N alpha-formyl-N epsilon-fructosyllysine (1.0 mM) dissociated to cause the incorporation of 1.2 nMol of [U-14C]NfL, but 1.0 mM 3-deoxyglucosone, the putative active dissociation product of fructosyl-lysine, produced only 1.5 nMol mg-1 protein of crosslinks. The chelator, DTPA, had little or no effect on crosslinking in our assay except at the highest carbohydrate level. These data argue that glucose crosslinking can be shown in vitro with lens proteins, however, it does not proceed significantly via 3-deoxyglucosone, and does not require transition metal ion-mediated oxidation to occur. Quantitatively, however, it is almost two orders of magnitude less than the crosslinking by ascorbate oxidation products in vitro.

The generation of superoxide anions in glycation reactions with sugars, osones, and 3-deoxyosones.

Glycoxidation is a process whereby glycated proteins chemically generate oxygen free radicals. Superoxide anion formation was measured by the superoxide dismutase-dependent reduction of ferricytochrome C in glycation reactions at pH 7.0 in the absence of transition metal ions. Assays were linear over 1 h, and most activity was seen after a 2 d incubation of 5 mM L-threose and 10 mM alpha-N-acetyl-lysine (N-Ac-Lys) or 10 mg/mL RNase A. Trioses, tetroses and their corresponding osones and 3-deoxyosones had the highest activity (12-16 nmoles O.-2/hr/ml) with N-Ac-Lys. Osones and 3-deoxyosones alone generated considerable O.-2, whereas aldose sugars largely did not. Xylosone and 3-deoxyxylosone produced 6 and 10 nmoles O.-2/hr/ml respectively with N-Ac-Lys, however, xylose was inactive, as were glucose and fructose. Glycation assays with 3-deoxyglucosone and glyoxal showed no activity, however, methyl glyoxal generated 1.7 and 2.0 nmoles O.-2/hr/ml with N-Ac-Lys and N-Ac-Arg, respectively. Therefore, Amadori compounds composed of lysine and short chain sugars can rapidly generate superoxide anion in the absence of metal ions.

UVA irradiation of human lens proteins produces residual oxidation of ascorbic acid even in the presence of high levels of glutathione.

The oxidation products of ascorbic acid (AscH-) can rapidly glycate and crosslink lens proteins in vitro, producing fluorophores and browning products similar to those present in cataractous lenses. The accumulation of AscH- oxidation products, however, would largely be prevented by the millimolar levels of glutathione (GSH) present in human lens. Here we investigate whether protein aggregation could allow the oxidation of AscH- by UVA-induced reactive oxygen species in the presence of physiological levels of GSH. The metal-catalyzed oxidation of 1.0 mM AscH- by 50 microM Cu(II) was almost complete after 1 h, but no oxidation was seen in the presence of GSH concentrations as low as 0.5 mM. UVA irradiation of protein aggregates from human lens, which accumulated more than 2.0 mM singlet oxygen after 1 h, caused a 50-60% oxidation of 1.0 mM AscH-. The addition of 204 mM GSH, however, decreased AscH- oxidation by less than half, and 30% of the AscH- was oxidized even in the presence of 15 mM GSH. This diminished protection may be due, in part, to the ability of AscH-, but not GSH, to penetrate to the sites of singlet oxygen generation located within the protein. Consistent with this hypothesis, greater GSH protection was seen when a proteolytic digest of the human proteins was subjected to the same irradiation or when singlet oxygen was chemically generated from 3-(4-methyl-1-naphthyl)propionic acid endoperoxide (MNPAE) at 37 degrees C in the medium. The addition of 50 microM Cu(II) had no effect on the rate of degradation of dehydroascorbic acid (DHA). Singlet oxygen, either UVA- or MNPAE-generated, increased the rate of DHA loss. This secondary oxidation of DHA by singlet oxygen would allow the accumulation of AscH- oxidation products was not reducible by GSH. Therefore, the data presented here argue that the protein aggregation seen in older human lenses may permit oxidized AscH--induced crosslinking to occur even at physiological GSH levels.

The aggregation in human lens proteins blocks the scavenging of UVA-generated singlet oxygen by ascorbic acid and glutathione.

One hour of UVA irradiation of air-saturated solutions of 2 mg/mL solubilized lens protein aggregates from aged human lens is able to produce on accumulated concentration of more than 2mM 1O2, along with oxidation of 120 nmol/mL of both Trp and His amino acid residues. Increasing concentrations of either sodium azide or ascorbic acid (up to 10 mM) during the irradiation decreased th His destruction by no more than 50-60% with the intact aggregates, but completely prevented the His loss with proteolyzed aggregates. Glutathione (up to 10 mM) was able to protect less than 10% of the aggregate His residues from oxidative damage, whereas His loss was almost completely prevented in the proteolyzed aggregates. Similar data were obtained for teh UVA photolysis of the Trp residues. This finding led us to study the role a protein conformation of these aggregates plays in the diminishing of antioxidant ability to prevent UVA-mediated photolysis of 1O2-sensitive amino acid residues. We found that Trp, His, and Cys are buried in the aggregates and cannot be oxidized by a relatively high concentration of 1O2 generated externally to the protein. Increasing urea denaturation of the aggregates caused exposure of the buried Trp residues as determined by the red shift of the fluorescence maximum and by a marked increase in the acrylamide and iodide fluorescence quenching. The ability of glutathione to prevent Trp oxidation by UVA light correlated directly with the extent of Trp exposure. These data suggest that the aggregation of the lens crystallins during aging produces a barrier, which prevents the access of water-soluble antioxidants to the sites of UVA-dependent singlet oxygen generation. In this case UVA proteolysis of the lens proteins can proceed even in the presence of physiological levels of antioxidants.

The relative UV sensitizer activity of purified advanced glycation endproducts.

The oxidation products of ascorbic acid react with lens proteins to form advanced glycation endproducts (AGE) that are capable of generating reactive oxygen species when irradiated with UVA light. L-Threose, the most active of these oxidation products, was reacted with N-acetyl lysine and six AGE peaks were isolated by RP-HPLC. Each peak exhibited fluorescence and generated superoxide anion and singlet oxygen in response to UV light. Solutions of these AGE peaks (50 micrograms/mL) generated 5-10 nmol/mL of superoxide anion during a 30 min irradiation. This activity was 100-fold less than the superoxide anion generated by kynurenic acid and 400-fold less than riboflavin. Ultraviolet irradiation generated from 1.2 to 2.7 mumol/mL of singlet oxygen with the purified threose AGE compounds. This activity was similar to that seen with other purified AGE compounds (pentosidine, LM-1 and Ac-FTP) and with kynurenine and 3-OH kynurenine. This considerable singlet oxygen formation, however, was still 40-fold less than that obtained with kynurenic acid and 100-fold less than riboflavin under the same irradiation conditions. In spite of this lower sensitizer efficiency, the purified AGE generated 20-60-fold more singlet oxygen on a weight basis than either crude ascorbic acid glycated proteins or a preparation of water-insoluble proteins from aged normal human lenses. On a molar basis, therefore, AGE could account for the sensitizer activity in these protein preparations if they represented less than 1% of the total amino acids.

Quantitation of the singlet oxygen produced by UVA irradiation of human lens proteins.

Ultraviolet irradiation of aged human lens proteins in vitro causes extensive photolytic damage of His and Trp residues. Protection by sodium azide argues for a process mediated by singlet oxygen (1O2). In the work described here, the synthesis of 1O2 was measured by the bleaching of N,N-dimethyl-4-nitrosoaniline (RNO), the oxidation of added histidine and the oxidation of furfuryl alcohol. To obtain a more accurate value for 1O2 generation, a known quantity of 1O2 was generated by the thermal dissociation of 3-(4-methyl-naphthyl)propionic acid endoperoxide, and the efficiency of each assay method to report on the 1O2 generated was determined. The values obtained were 0.003 mol of RNO bleached/mol of 1O2 generated, 0.55 mol of furfuryl alcohol oxidized/mol 1O2 and 0.5 mol of His oxidized/mol 1O2 generated. Irradiation of the human lens proteins with UVA light produced from 2.1 to 2.4 mM of 1O2 by RNO bleaching, 2.6-2.8 mM 1O2 by furfuryl alcohol oxidation and up to 1.9 mM of 1O2 by histidine oxidation during a 1 h irradiation period. The average value (2.2 mM of 1O2) corresponds to the theoretical production of 30 nmol of singlet oxygen at UVA light intensities equivalent to a 1 h exposure to sunlight at noon in the northern hemisphere.

Rapid assessment of early glycation products by mass spectrometry.

In order to detect the early glycation products, we have reacted a model peptide (t-boc-lys-ala-ala) with L-threose (a degradation product of ascorbic acid) and analyzed the reaction products by a combination of HPLC and mass spectrometry. Amino group modification, as observed by a fluorescamine assay, indicated complete modification after 3 days of incubation with a 10-fold excess of threose. As much as 60% of the adducts were acid labile and only 4% of the adducts could be observed by amino acid analysis. However, Fast atom bombardment mass spectrometry (FABMS) of the samples incubated for 6 hr showed relative molecular masses consistent with the formation of adducts corresponding to the addition of one and two molecules of L-threose to the peptide. Likewise, samples incubated for 12 hr showed peptide adducts with two and three L-threoses. The number of threose molecules added to the peptide was also confirmed from the FABMS analysis by using [1-13C]-threose as the glycating agent.

Glycation mediated crosslinking between alpha-crystallin and MP26 in intact lens membranes.

With advancing age, progressive crosslinking occurs between lens crystallin proteins and other lenticular components. This crosslinking may be involved in the development of senile cataracts. Experiments were conducted to determine whether non-enzymatic glycation could be involved in the crosslinking between lens alpha-crystallin and MP26, an abundant lens fiber cell membrane intrinsic protein. In vitro crosslinking of alpha-crystallin and MP26 of bovine lens membranes was observed in presence of two degradation products of ascorbic acid (ASA), dehydroascorbic acid (DHA) and threose. Alkali-washed bovine lens membranes, isolated after glycation with DHA and threose, contained both alpha-crystallin and MP26, as determined by immunoblot and double immunocytochemical labeling studies. In contrast, membranes incubated without these glycating compounds contained only MP26. SDS-PAGE analysis of [125I] alpha-crystallin incubated with lens membranes in the presence of threose showed a higher amount of radioactivity in high molecular weight aggregates than in the aggregates produced when alpha-crystallin and threose were incubated without membranes. A slot-blot immunoassay of alkali-washed human lens membranes showed a higher amount of covalently bound alpha-crystallin in aged, cataractous or diabetic lens membranes than was present in lens membranes from young normal donors. Based on the in vitro results, we hypothesize that non-enzymatic glycation is one of the vivo mechanisms in the crosslinking of alpha-crystallin to lens membrane proteins, such as MP26. This crosslinking may contribute significantly to the development of age-related and diabetic cataracts.