Malladi Prabhakaram

The relative UV sensitizer activity of purified advanced glycation endproducts.

Abstract— 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 μg/mL) generated5–10 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.

Suppression of Pentosidine Formation in Galactosemic Rat Lens by an Inhibitor of Aldose Reductase

Recent work from our laboratory revealed a correlation between the degree of protein pigmentation in human cataractous lens and the advanced Maillard reaction as reflected by pentosidine formation. Although the data suggested a role for ascorbate in pentosidine formation in senile cataractous lenses, elevated pentosidine levels in diabetic cataracts suggested that glucosylation may be involved directly in pentosidine biosynthesis. To clarify this issue, we quantified pentosidine in lenses from rats with experimental galactosemia with and without aldose reductase inhibitor treatment. At 12 months, pentosidine-like fluorescence (335/385 nm) was three to six times higher (P < 0.0001) in water soluble and insoluble crystallins of galactosemic compared with nongalactosemic rats. Actual pentosidine levels increased shortly after onset of galactosemia. Contents in water-insoluble crystallins were 6.32 ± 2.2 and 1.40 ± 0.66 pmol/mg protein in galactosemic and control lenses, respectively (P < 0.001). Fluorescence and pentosidine were suppressed to almost control levels upon treatment with sorbinil. Incubation experiments showed that pentosidine could form slowly from galactose, but much more rapidly from ascorbate and its oxidation products. Its formation could be inhibited partly by both reduced and oxidized glutathione or ε-aminocaproic acid. The requirement of oxygen for pentosidine formation suggests that oxidative stress associated with glutathione depletion and ascorbate oxidation are plausible mechanisms for rapid pentosidine formation upon onset of galactosemia. In contrast, Maillard reaction by glycoxidation products may account for the sustained increase in pentosidine. Both these events may be linked to the newly recognized pseudohypoxic state of cells exposed to high sugar concentrations.

Site-specific glycation of lens crystallins by ascorbic acid

The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1-14C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH3. This rate of incorporation was 6-7-fold more rapid than that obtained with [14C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the crystallin subunits. [1-14C]Ascorbic acid-label led alpha-crystallin was separated into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N epsilon-(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.

The glycation-associated crosslinking of lens proteins by ascorbic acid is not mediated by oxygen free radicals

The reaction by which ascorbic acid (ASA) causes the glycation and crosslinking of lens proteins displays a rigid requirement for the presence of oxygen, and is inhibited by the presence of glutathione. Oxygen is required to oxidize ASA to dehydroascorbic acid (DHA) and other products which are the active glycating species. No evidence could be found to support a role for oxidative protein crosslinking by a free radical mechanism. Crosslinking was not inhibited by blocking protein sulfhydryl groups with iodoacetamide, nor were the protein crosslinks dissociated by boiling with 2% mercaptoethanol prior to SDS-PAGE. The addition of a variety of oxygen free radical quenchers had no effect on the extent of protein crosslinking. In fact, the removal of oxygen from the reaction mixture had no effect on either protein glycation, protein crosslinking or the modification of lysine residues, provided DHA was used as the glycating agent. All of these activities were inhibited, however, if ASA was the glycating agent. This confirms that oxygen is required only to convert ASA to DHA.

The glycation and cross-linking of isolated lens crystallins by ascorbic acid

Individual lens crystallins were isolated from calf lens extracts and incubated in the presence of ascorbic acid for 3 weeks under aerobic conditions. Both alpha-crystallin and beta H-crystallin rapidly cross-linked to form high molecular weight proteins, which did not enter the resolving gel on SDS-PAGE. Beta L-crystallin was somewhat less reactive, but gamma-crystallin showed little or no crosslinking. Gamma-crystallin, however, was almost equivalent to the other crystallins as a substrate for glycation. This was measured by: (a) the binding of protein to a boronate affinity column; (b) the incorporation of 3H from NaB3H4 into protein; (c) amino acid analysis of the modified proteins to estimate the extent of lysine modification; and (d) the incorporation of [1-14C]ASA into individual crystallins. When the separated crystallins were combined with [125I]gamma-crystallin and incubated with ascorbic acid, radioactivity was readily incorporated into the cross-linked products with other crystallins, but again not with gamma-crystallin itself. Gel filtration chromatography of a mixture of [125I]gamma-crystallin and alpha-crystallin showed the formation of a complex between gamma- and alpha-crystallins. These data suggest that all crystallins are glycated, but that cross-linking occurs preferentially between proteins, which are already bound together non-covalently.

The extent of Nϵ-(carboxymethyl)lysine formation in lens proteins and polylysine by the autoxidation products of ascorbic acid

The autoxidation of ascorbic acid (ASA) leads to the formation of compounds which are capable of glycating and crosslinking proteins in vitro. When the soluble crystallins from bovine lens were incubated with ASA in the presence of sodium cyanoborohydride, a single major adduct was observed, whose appearance correlated with the loss of lysine. When polylysine was reacted with equivalent amounts of ASA under the same conditions, this product represented half of the total lysine content after four weeks of incubation at 37 degrees C. This adduct was isolated and identified as N epsilon-(carboxymethyl)lysine (CML) by TLC, GC/MS and amino acid analysis. Several oxidation products of ASA were each reacted with polylysine in the presence of sodium cyanoborohydride to identify the reactive species. CML was the major adduct formed with either ASA and dehydroascorbic acid (DHA). Markedly diminished amounts were seen with L-2,3-diketogulonic acid (DKG), and L-threose, while no CML was formed with L-threo-pentos-2-ulose (L-xylosone). In the absence of sodium cyanoborohydride the yield of CML was similar with each of the ASA autoxidation products and required oxygen. Reactions with [1-14C]ASA gave rise to [14C]CML, but only with NaCNBH3 present. At least two routes of CML formation appear to be operating depending upon whether NaCNBH3 is present to reduce the putative Schiff base formed between lysine and DHA.

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

Abstract 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 α-crystallin and MP26, an abundant lens fiber cell membrane intrinsic protein. In vitro crosslinking of α-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 α-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 [ 125 I]α-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 α-crystallin and threose were incubated without membranes. A slot-blot immunoassay of alkali-washed human lens membranes showed a higher amount of covalently bound α-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 in vivo mechanisms in the crosslinking of α-crystallin to lens membrane proteins, such as MP26. This crosslinking may contribute significantly to the development of age-related and diabetic cataracts.

Characterization of a Blue Fluorophore Isolated from in Vitro Reaction of N-α-Acetyllysine and 3-Deoxyglucosone

With the objective to investigate 3-deoxyglucosone (3-DG) mediated lysine crosslinks in vivo, we have isolated a lysine-3-DG-lysine crosslink from in vitro reaction of 3-DG and N-proportional to-acetyllysine (NAL). This crosslink, named as furopyrrolopyridine crosslink (FPPC), has intense blue fluorescence with absorption maxima at 235, 270 and 370 nm and emission maximum at 470 nm. The absorption and fluorescence spectra of FPPC were not altered in pHs ranging from 2-12, but the characteristic spectrum of FPPC (at pH 7.0) disappeared when it was reduced with sodium borohydride. FAB-MS showed that FPPC has a molecular mass of 611, equivalent to the reaction of two molecules each of NAL and 3-DG with the concomitant loss of 5 molecules of water. NMR data showed that FPPC has a pyridinium ring and four free hydroxy groups. Since acid hydrolyzed FPPC can be detected by amino acid analysis, we have determined its levels in the acid hydrolyzates of proteins glycated by 3-DG or in the acid hydrolyzates of normal aged, cataractous, diabetic and brunescent human lens proteins as well as in the acid hydrolyzed glycated hemoglobin, A0.

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 of MP26 and MP22 in bovine lens membranes

Alkali treated membranes were isolated from mature bovine lenses and incubated with different sugars for 3 weeks to study the effect of glycation on the lens intrinsic membrane proteins, MP26 and MP22. The obtained results show that a) [1-14C] ascorbic acid (ASA) was able to glycate the intrinsic membrane proteins as rapidly as soluble lens proteins; b) on 15% acrylamide gels in SDS, glucose, fructose, galactose and ribose exhibited low activity for crosslinking membrane proteins; whereas ASA, dehydroascorbate (DHA), diketogulonate (DKG), xylosone and threose, all showed not only the formation of protein multimers, but also highly crosslinked products, which did not enter the spacer gel; c) except glycated MP22, all of the crosslinks of MP26 or MP22, and also the glycated MP26, showed cross reactivity with polyclonal MP26 antibody; d) the extent of crosslinking correlated with an equal loss of lysine and arginine contents by amino acid analysis.