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1.  Citric acid inhibits development of cataracts, proteinuria and ketosis in streptozotocin (type1) diabetic rats 
Although many fruits such as lemon and orange contain citric acid, little is known about beneficial effects of citric acid on health. Here we measured the effect of citric acid on the pathogenesis of diabetic complications in streptozotocin-induced diabetic rats. Although oral administration of citric acid to diabetic rats did not affect blood glucose concentration, it delayed the development of cataracts, inhibited accumulation of advanced glycation end products (AGEs) such as Nε-(carboxyethyl)lysine (CEL) and Nε-(carboxymethyl)lysine (CML) in lens proteins, and protected against albuminuria and ketosis . We also show that incubation of protein with acetol, a metabolite formed from acetone by acetone monooxygenase, generate CEL, suggesting that inhibition of ketosis by citric acid may lead to the decrease in CEL in lens proteins. These results demonstrate that the oral administration of citric acid ameliorates ketosis and protects against the development of diabetic complications in an animal model of type 1 diabetes.
doi:10.1016/j.bbrc.2010.01.095
PMCID: PMC2917331  PMID: 20117096
Advanced glycation end-product (AGEs); Nε-(carboxyethyl)lysine (CEL); cataract; diabetes; ketosis; nephropathy
2.  Diagnostic potential of plasma carboxymethyllysine and carboxyethyllysine in multiple sclerosis 
Background
This study compared the level of advanced glycation end products (AGEs), N-(Carboxymethyl)lysine (CML) and N-(Carboxyethyl)lysine (CEL), in patients with multiple sclerosis (MS) and healthy controls (HCs), correlating these markers with clinical indicators of MS disease severity.
Methods
CML and CEL plasma levels were analyzed in 99 MS patients and 43 HCs by tandem mass spectrometry (LC/MS/MS). Patients were stratified based on drug modifying therapies (DMTs) including interferon beta, glatiramer acetate and natalizumab.
Results
The level of plasma CEL, but not CML, was significantly higher in DMT-naïve MS patients when compared to HCs (P < 0.001). Among MS patients, 91% had higher than mean plasma CEL observed in HCs. DMTs reduced CML and CEL plasma levels by approximately 13% and 40% respectively. CML and CEL plasma levels correlated with the rate of MS clinical relapse.
Conclusion
Our results suggest that AGEs in general and CEL in particular could be useful biomarkers in MS clinical practice. Longitudinal studies are warranted to determine any causal relationship between changes in plasma level of AGEs and MS disease pathology. These studies will pave the way for use of AGE inhibitors and AGE-breaking agents as new therapeutic modalities in MS.
doi:10.1186/1742-2094-7-72
PMCID: PMC2984414  PMID: 21034482
3.  Advanced glycation end product (AGE) recognition by the receptor for AGEs (RAGE) 
SUMMARY
Nonenzymatic protein glycation results in the formation of advanced glycation end products (AGEs) that were implicated in the pathology of diabetes, chronic inflammation, Alzheimer’s disease, and cancer. AGEs mediate their effects primarily through a receptor-dependent pathway in which AGEs bind to a specific cell surface associated receptor, the Receptor for AGEs (RAGE). Nε-carboxy-methyl-lysine (CML) and Nε-carboxy-ethyl-lysine (CEL), constitute two of the major AGE structures found in tissue and blood plasma, and are physiological ligands of RAGE. The solution structure of a CEL containing peptide-RAGE V domain complex reveals that the carboxyethyl moiety fits inside a positively charged cavity of the V domain. Peptide backbone atoms make specific contacts with the V domain. The geometry of the bound CEL peptide is compatible with many CML (CEL) modified sites found in plasma proteins. The structure explains how such patterned ligands as CML (CEL)-proteins bind to RAGE and contribute to RAGE signaling.
doi:10.1016/j.str.2011.02.013
PMCID: PMC3150472  PMID: 21565706
glycation; receptor for glycation end products; pattern recognition receptor; Nε-carboxy-methyl-lysine; Nε-carboxy-ethyl-lysine
4.  Accumulation of argpyrimidine, a methylglyoxal-derived advanced glycation end product, increases apoptosis of lens epithelial cells both in vitro and in vivo 
Experimental & Molecular Medicine  2011;44(2):167-175.
The formation of advanced glycation end products (AGEs) has been considered to be a potential causative factor of injury to lens epithelial cells (LECs). Damage of LECs is believed to contribute to cataract formation. The purpose of this study was to investigate the cytotoxic effect of AGEs on LECs both in vitro and in vivo. We examined the accumulation of argpyrimidine, a methylglyoxal-derived AGE, and the expression of apoptosis-related molecules including nuclear factor-kappaB (NF-κB), Bax, and Bcl-2 in the human LEC line HLE-B3 and in cataractous lenses of Zucker diabetic fatty (ZDF) rats, an animal model of type 2 diabetes. In cataractous lenses from twenty-one-week-old ZDF rats, LEC apoptosis was markedly increased, and the accumulation of argpyrimidine as well as subsequent activation of NF-κB in LECs were significantly enhanced. The ratio of Bax to Bcl-2 protein levels was also increased. In addition, the accumulation of argpyrimidine triggered apoptosis in methylglyoxal-treated HLE-B3 cells. However, the presence of pyridoxamine (an AGEs inhibitor) and pyrrolidine dithiocarbamate (a NF-κB inhibitor) prevented apoptosis in HLE-B3 cells through the inhibition of argpyrimidine formation and the blockage of NF-κB nuclear translocalization, respectively. These results suggest that the cellular accumulation of argpyrimidine in LECs is NF-κB-dependent and pro-apoptotic.
doi:10.3858/emm.2012.44.2.012
PMCID: PMC3296813  PMID: 22139526
apoptosis; argpyrimidine; cataract; diabetes mellitus, type 2; epithelial cells; glycosylation end products, advanced; lens, crystalline; NF-κB
5.  Analysis of nuclear fiber cell compaction in transparent and cataractous diabetic human lenses by scanning electron microscopy 
BMC Ophthalmology  2003;3:1.
Background
Compaction of human ocular lens fiber cells as a function of both aging and cataractogenesis has been demonstrated previously using scanning electron microscopy. The purpose of this investigation is to quantify morphological differences in the inner nuclear regions of cataractous and non-cataractous human lenses from individuals with diabetes. The hypothesis is that, even in the presence of the osmotic stress caused by diabetes, compaction rather than swelling occurs in the nucleus of diabetic lenses.
Methods
Transparent and nuclear cataractous lenses from diabetic patients were examined by scanning electron microscopy (SEM). Measurements of the fetal nuclear (FN) elliptical angles (anterior and posterior), embryonic nuclear (EN) anterior-posterior (A-P) axial thickness, and the number of EN fiber cell membrane folds over 20 μm were compared.
Results
Diabetic lenses with nuclear cataract exhibited smaller FN elliptical angles, smaller EN axial thicknesses, and larger numbers of EN compaction folds than their non-cataractous diabetic counterparts.
Conclusion
As in non-diabetic lenses, the inner nuclei of cataractous lenses from diabetics were significantly more compacted than those of non-cataractous diabetics. Little difference between diabetic and non-diabetic compaction levels was found, suggesting that diabetes does not affect the degree of compaction. However, consistent with previous proposals, diabetes does appear to accelerate the formation of cataracts that are similar to age-related nuclear cataracts in non-diabetics. We conclude that as scattering increases in the diabetic lens with cataract formation, fiber cell compaction is significant.
doi:10.1186/1471-2415-3-1
PMCID: PMC140319  PMID: 12515578
6.  ANAEROBIC VS. AEROBIC PATHWAYS OF CARBONYL AND OXIDANT STRESS IN HUMAN LENS AND SKIN DURING AGING AND IN DIABETES: A COMPARATIVE ANALYSIS 
Free radical biology & medicine  2010;49(5):847-856.
The effects of anaerobic (lens) vs aerobic (skin) environment on carbonyl and oxidant stress are compared using de novo and existing data on advanced glycation and oxidation products in human crystallins and collagen. Almost all modifications increase with age. Methylglyoxal hydroimidazolones (MG-H1), carboxymethyl-lysine (CML), and carboxyethyl-lysine (CEL) are several folds higher in lens than skin, and markedly increase upon incubation of lens crystallins with 5 mM ascorbic acid. Vice-versa, fructose-lysine, glucosepane crosslinks, glyoxal hydroimidazolones (G-H1), metal catalyzed oxidation (allysine) and H2O2 dependent modifications (2-aminoapidic acid and methionine sulfoxide) are markedly elevated in skin, but relatively suppressed in the aging lens. In both tissues ornithine is the dominant modification, implicating arginine residues as the principal target of the Maillard reaction in vivo. Diabetes (here mostly type 2 studied) increases significantly fructose-lysine and glucosepane in both tissues (P<0.001) but has surprisingly little effect on the absolute level of most other advanced glycation end products (AGEs) . However, diabetes strengthens the Spearman correlation coefficients for age-related accumulation of hydrogen peroxide mediated modifications in the lens. Overall, the data suggest oxoaldehyde stress involving methylglyoxal from either glucose or ascorbate is predominant in the aging non-cataractous lens, while aging skin collagen undergoes combined attack by non-oxidative glucose mediated modifications, as well as those from metal catalyzed oxidation and H2O2.
doi:10.1016/j.freeradbiomed.2010.06.003
PMCID: PMC2910832  PMID: 20541005
crystallins; collagen; glycation; oxidative stress; methylglyoxal; metals
7.  Validation study to compare effects of processing protocols on measured Nε-(carboxymethyl)lysine and Nε-(carboxyethyl)lysine in blood 
Epidemiological studies show that elevated plasma levels of advanced glycation end products (AGEs) are associated with diabetes, kidney disease, and heart disease. Thus AGEs have been used as disease progression markers. However, the effects of variations in biological sample processing procedures on the level of AGEs in plasma/serum samples have not been investigated. The objective of this investigation was to assess the effect of variations in blood sample collection on measured Nε-(carboxymethyl)lysine (CML), the best characterised AGE, and its homolog, Nε-(carboxyethyl)lysine (CEL). The investigation examined the effect on CML and CEL of different blood collection tubes, inclusion of a stabilising cocktail, effect of freeze thaw cycles, different storage times and temperatures, and effects of delaying centrifugation on a pooled sample from healthy volunteers. CML and CEL were measured in extracted samples by ultra-performance liquid chromatography-tandem mass spectrometry. Median CML and CEL ranged from 0.132 to 0.140 mM/M lys and from 0.053 to 0.060 mM/M lys, respectively. No significant difference was shown CML or CEL in plasma/serum samples. Therefore samples collected as part of epidemiological studies that do not undergo specific sample treatment at collection are suitable for measuring CML and CEL.
doi:10.3164/jcbn.13-5
PMCID: PMC3818270  PMID: 24249965
advanced glycation end-products; Nε-(carboxymethyl)lysine; Nε-(carboxyethyl)lysine; epidemiology; blood sampling
8.  Advanced glycation end product deposits in climatic droplet keratopathy 
Background
Climatic droplet keratopathy (CDK), known as spheroid degeneration of the cornea, is one of the most frequent degenerative corneal disorders affecting visual function. However, the histochemical nature of the deposits seen in CDK is still unclear.
Aim
To investigate the pathogenesis of CDK, we investigated the immunohistochemical localisation of advanced glycation end products (AGEs) in surgical specimens of CDK.
Methods
Immunohistochemical localisation of Nε‐(carboxymethyl)‐L‐lysine (CML), Nε‐(carboxyethyl)‐L‐lysine (CEL), pyrraline, pentosidine and imidazolone was examined in three corneas with CDK, six corneas with bullous keratopathy and three corneas without any corneal diseases.
Results
In all the specimens with CDK, immunoreactivity was strong in CML, moderate in pyrraline and pentosidine, and weak in imidazolone. Immunoreactivity was absent in CEL. In contrast, no immunoreactivity to CML, pyrraline, pentosidine, imidazolone or CEL was detected in corneas with bullous keratopathy, or in corneas without any corneal diseases.
Conclusions
CDK is caused by an aggregation of AGE‐modified proteins. The result is consistent with etiological findings that ultraviolet irradiation and ageing, both of which are accelerators of AGE formation, are closely related to the development of CDK.
doi:10.1136/bjo.2006.099812
PMCID: PMC1857579  PMID: 16973666
9.  Nonenzymatic glycosylation, sulfhydryl oxidation, and aggregation of lens proteins in experimental sugar cataracts 
The Journal of Experimental Medicine  1979;150(5):1098-1107.
The formation of sugar-cataracts has been hypothesized to involve the nonenzymatic glycosylation, sulfhydryl oxidation, and aggregation of lens proteins. Cataractous lenses of diabetic and galactosemic rats were analyzed for glycosylated lysine residues in crystallins. A five- and a ten-fold increase in glycosylated lysine residues was measured in galactose and diabetic cataracts, respectively. The modification was predominant in the insoluble fraction of the lens homogenate. The proteins were further examined for the presence of disulfide bonds and high molecular weight aggregates. After careful disruption of the lens in a nitrogen environment, a cloudy solution was obtained from cataractous lenses whereas a clear solution was obtained from normal lenses. The absorbance at 550 nm of the solution of both the galactosemic and the diabetic cataracts could be decreased by approximately 50% with the addition of dithioerythritol (50 mM). The presence of high molecular weight aggregates was ascertained by sucrose gradient centrifugation and gel filtration chromatography. The proteins were heterogenous in size and showed a mol wt range of 36 to greater than 176 million daltons. Treatment with dithioerythritol induced a marked decrease in the amount of high molecular weight proteins. These data suggest that sugar cataracts of experimental animals have, in common with human cataracts, the presence of high molecular weight aggregates which are in part linked by disulfide bonds.
PMCID: PMC2185697  PMID: 501285
10.  KIOM-79 Prevents Lens Epithelial Cell Apoptosis and Lens Opacification in Zucker Diabetic Fatty Rats 
Damage of lens epithelial cells (LECs) has been implicated in cataract formation. The aim of this study was to investigate the protective effect of KIOM-79, a combination of four plant extracts, on LECs. We examined the levels of advanced glycation end products (AGEs), nuclear factor-kappaB (NF-κB) activation and inducible nitric oxide synthase (iNOS) expression in LECs during cataract development using the Zucker diabetic fatty (ZDF) rat, an animal model of type 2 diabetes. KIOM-79 was orally administered by gavage to ZDF rats once a day for 13 weeks. Apoptosis was detected by TUNEL assay, and NF-κB activation and iNOS expression were studied by southwestern histochemistry and immunohistochemistry, respectively. In diabetic cataractous lenses, TUNEL-positive LECs were markedly increased 20-fold, and AGEs were highly accumulated (2.7-fold) in LECs. In addition, both NF-κB activation, and iNOS expression were significantly enhanced 3- to 5-fold, respectively, compared to levels found in normal ZL rats. However, the administration of KIOM-79 delayed the development of diabetic cataracts and prevented LEC apoptosis (70%) through the inhibition of AGEs, NF-κB-activation and iNOS expression. These observations suggest that KIOM-79 is useful in inhibiting diabetic cataractogenesis and acts through an antiapoptotic mechanism to protect LECs from injury.
doi:10.1155/2011/717921
PMCID: PMC2952320  PMID: 20953387
11.  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.
doi:10.1196/annals.1333.021
PMCID: PMC1564128  PMID: 16037236
ascorbic acid; OP-lysine; UVA light; eye lens; glycation
12.  The effects of low-dose Nepsilon-(carboxymethyl)lysine (CML) and Nepsilon-(carboxyethyl)lysine (CEL), two main glycation free adducts considered as potential uremic toxins, on endothelial progenitor cell function 
Background
Patients with chronic kidney disease (CKD) are at high risk of cardiovascular disease (CVD). Endothelial progenitor cell (EPCs) dysfunction plays a key role in this pathogenesis. Uremic retention toxins have been reported to be in associated with EPC dysfunction. Advanced glycation end-products (AGEs) free adducts, including Nepsilon-(carboxymethyl)lysine (CML) and Nepsilon-(carboxyethyl)lysine (CEL), are formed by physiological proteolysis of AGEs and released into plasma for urinary excretion. They are retained in CKD patients and are considered to be potential uremic toxins. Though AGEs have been demonstrated to impair EPC function in various ways, the effect of AGE free adducts on EPC function has not been studied. Thus, we examined the role of CML and CEL in the regulation of growth-factor-dependent function in cultured human EPCs and the mechanisms by which they may affect EPC function.
Methods
Late outgrowth EPCs were incubated with different concentrations of CML or CEL for up to 72 hours. Cell proliferation was determined using WST-1 and BrdU assays. Cell apoptosis was tested with annexin V staining. Migration and tube formation assays were used to evaluate EPC function.
Results
Though CML and CEL were determined to have anti-proliferative effects on EPCs, cells treated with concentrations of CML and CEL in the range found in CKD patients had no observable impairment on migration or tube formation. CML and CEL did not induce EPC apoptosis. The reduced growth response was accompanied by significantly less phosphorylation of mitogen-activated protein kinases (MAPKs).
Conclusions
Our study revealed that CML and CEL at uremic concentrations have low biological toxicity when separately tested. The biologic effects of AGE free adducts on the cardiovascular system merit further study.
doi:10.1186/1475-2840-11-90
PMCID: PMC3471041  PMID: 22853433
Endothelial progenitor cells; Mitogen-activated protein kinases; Nϵ-(carboxyethyl)lysine; Nϵ-(carboxymethyl)lysine; Uremic toxins
13.  Spectroscopic and biochemical correlations during the course of human lens aging 
BMC Ophthalmology  2006;6:10.
Background
With age, the human lens accumulates variety of substances that absorbs and fluorescence, which explains the color of yellow, brunescent and nigrescent cataract in terms of aging. The aim of this study was to assess lens fluorophores with properties comparable to those of advanced glycated end products (AGEs) in relation to age in human lenses. These fluorescent compounds are believed to be involved in the development of cataract.
Methods
Spectroscopic (UV-Vis-NIR) and fluorescence photography (CCD-Digital based image analysis) studies were carried out in randomly selected intact human lenses (2–85 years). AGE-like fluorophores were also measured in water soluble and insoluble (alkali soluble) fractions of human lenses (20–80 years).
Results
Our experimental findings suggest that there was a progressive shift in the absorbance characteristic of intact lens in the range of λ210 nm-λ470 nm. A relative increase in the absorptivity at λ(511–520 nm), with age, was also observed. In addition, the ratio of absorptivity at λ(511–520 nm) versus the maximum absorbance recorded at blue-end cut-off (210–470 nm) was also found to increase, with age. The fluorescent intensity in the intact lens at both UV-B (λEx312 nm) and UV-A (λEx365 nm) were found to be positively correlated (r2 = 0.91 & 0.94, respectively; Confidence interval 95%) upto 50 years of age. In addition, a concomitant changes in AGE- like fluorophores were also observed in the processed lens samples (soluble and insoluble fractions) along the age. A significant increase in the concentration of AGE- like fluorophores, both in intact and processed lens was observed during the period of 40 – 50 years.
Conclusion
Based on the present investigation, it was concluded that significant changes do occur in the AGE-like fluorophores of human lenses during the period of 40–50 years.
doi:10.1186/1471-2415-6-10
PMCID: PMC1450316  PMID: 16519820
14.  Immunochemical detection of glycated lens crystallins and their circulating autoantibodies in human serum during aging 
Molecular Vision  2008;14:2056-2066.
Purpose
The aim of this investigation was to exploit lens-specific glycated crystallins as an immunogen to detect human glycated crystallins and their circulating autoantibodies in human serum during aging in relation to the development of cataract.
Methods
Polyclonal antibodies were produced against human total lens proteins (40–80 years) in rabbits. The specificity of the antibodies produced were determined by antibody capture assay using purified human lens crystallins (high molecular weight fraction [HMW]+α, HMW+α-glycated, β, β-glycated, γ, and γ-glycated) as antigens. The cross-reactivity of these lens specific antibodies against rat β-, β-glycated, γ-, and γ-glycated lens crystallins was also analyzed. A non-competitive enzyme linked immunosorbent assay (ELISA) methodology was developed for the detection of circulating lens crystallins in human sera using HMW+α, HMW+α-glycated, β-, and β-glycated crystallins from humans and γ- and γ-glycated crystallins from rats as immobilized antigens. Circulating autoantibodies were also detected in human sera by antibody capture assay. The methodology was validated by evaluating 60 human serum samples collected from cataract patients and 30 human serum samples from apparently normal subjects belonging to the same age group.
Results
The polyclonal antibodies raised against human total lens proteins showed 90% and 65% cross-reactivity with rat γ- and β-crystallins, respectively, by ELISA. Further, these polyclonal antibodies were capable of detecting both native and in vitro synthesized glycated crystallins. Their IC50 values were observed to be (i) human total lens proteins (55 ng), (ii) human HMW+α (16.45 ng), (iii) human HMW+α-glycated (273 ng), (iv) human β- (37.82 ng), (v) human β-glycated (260 ng), (vi) rat γ- (105.34 ng), and (vii) rat γ-glycated (313 ng). The immunochemical analysis of human serum indicated a significant change (p<0.001) in the levels of circulating β-glycated and γ-glycated crystallins in the age group of 40–80 years with respect to their control groups. However, there was no statistically significant change in the levels of HMW+α-glycated crystallins in the age group of 40–80 years as compared to their age-matched controls. Notably, the levels of serum γ-glycated crystallins were found to be threefold higher than that of HMW+α-glycated and β-glycated crystallins in the age group of 70–80 years. Circulating autoantibodies to HMW+α-glycated, β-glycated, and γ-glycated crystallins were detected in the serum of both apparently normal and cataract patients in the age group of 40–80 years by antibody capture assay. The levels of these autoantibodies were significantly higher at every time point compared to their respective controls. Autoantibodies to γ-glycated crystallins were found to be twofold and 3.2 fold higher as compared to the levels of autoantibodies to β-glycated and HMW+α-glycated crystallins, respectively. Western blot and immunohistochemical analysis substantiated the observations made in non-competitive ELISA.
Conclusions
During the course of aging, leakage of lens crystallins (HMW+α, HMW+α-glycated, β, β-glycated, γ, and γ-glycated) elicit an immune response resulting in the formation of autoantibodies in cataract patients (40–80 years) as compared to age matched controls. This is the first experimental report where polyclonal antibodies raised against lens-specific glycated crystallins were capable of detecting the early leakage of glycated crystallins in human subjects. This immunochemical approach has implications in the early detection of senile cataract.
PMCID: PMC2584771  PMID: 19023447
15.  Antiglycation Effects of Carnosine and Other Compounds on the Long-Term Survival of Escherichia coli▿  
Applied and Environmental Microbiology  2010;76(24):7925-7930.
Glycation, or nonenzymatic glycosylation, is a chemical reaction between reactive carbonyl-containing compounds and biomolecules containing free amino groups. Carbonyl-containing compounds include reducing sugars such as glucose or fructose, carbohydrate-derived compounds such as methylglyoxal and glyoxal, and nonsugars such as polyunsaturated fatty acids. The latter group includes molecules such as proteins, DNA, and amino lipids. Glycation-induced damage to these biomolecules has been shown to be a contributing factor in human disorders such as Alzheimer's disease, atherosclerosis, and cataracts and in diabetic complications. Glycation also affects Escherichia coli under standard laboratory conditions, leading to a decline in bacterial population density and long-term survival. Here we have shown that as E. coli aged in batch culture, the amount of carboxymethyl lysine, an advanced glycation end product, accumulated over time and that this accumulation was affected by the addition of glucose to the culture medium. The addition of excess glucose or methylglyoxal to the culture medium resulted in a dose-dependent loss of cell viability. We have also demonstrated that glyoxylase enzyme GloA plays a role in cell survival during glycation stress. In addition, we have provided evidence that carnosine, folic acid, and aminoguanidine inhibit glycation in prokaryotes. These agents may also prove to be beneficial to eukaryotes since the chemical processes of glycation are similar in these two domains of life.
doi:10.1128/AEM.01369-10
PMCID: PMC3008226  PMID: 20952637
16.  Role of lipid peroxidation in the pathogenesis of myopic and senile cataract. 
AIMS/BACKGROUND: Increased production of free radicals, consumption of antioxidant, and oxidation of unsaturated lipids have been observed recently in cataractous lenses and active participation of the retina in human cataractogenesis has been proposed. To verify this hypothesis, the total (GSH) and oxidised (GSSG) glutathione concentrations were assayed in the lens and the malondialdehyde (MDA) levels assayed in the vitreous and in the lens of normal controls and patients with senile or myopic cataract. METHODS: The study was conducted on 34 lenses (nucleus and epinucleus) (nine clear lenses, 14 lenses with idiopathic senile cataract, and 11 lenses affected by severe myopic cataract) and vitreous of 19 (seven non-myopic, seven myopic, and five control) subjects. Glutathione determination was performed following the method of Reed, while malondialdehyde was assayed using a modification of the method of Dahle. RESULTS: Cataractous lenses showed a decreased content of GSH and increased concentration of GSSG compared with clear lenses. A higher oxidative consumption of GSH was found in myopic cataracts compared with senile ones. Also, increased levels of MDA were observed both in cataractous lenses and in the vitreous of myopic patients compared with the control and the senile ones. CONCLUSION: The observed alterations strongly suggest that retinal lipid peroxidation might play a key role in human cataractogenesis, especially in the myopic type.
PMCID: PMC505624  PMID: 8942384
17.  Effect of carnosine, aminoguanidine, and aspirin drops on the prevention of cataracts in diabetic rats 
Molecular Vision  2008;14:2282-2291.
Purpose
To investigate the effect of carnosine (CA), aminoguanidine (AG), and aspirin (ASA) drops, all inhibitors of glycation, on the development of diabetic cataract in rat.
Methods
Rats were made diabetic with streptozotocin, and based on the level of plasma glucose, they were assigned as non-diabetic rats (<14 mmol/l plasma glucose) and diabetic rats (>14 mmol/l plasma glucose). Animals in the treated groups received CA, AG, and ASA as drops to the left eyes starting from the day of streptozotocin injection. Progression of lens opacification was recorded using the slit lamp at regular time intervals. All the rats were killed after the week 13, and the levels of advanced glycation end products (AGE), glutathione reductase (GR), catalase (CAT), and glutathione (GSH) were determined.
Results
Lens opacification progressed in a biphasic manner in the diabetic rats, an initial slow increase during the first eight weeks of diabetes followed by a steep increase in the next five weeks. Carnosine treatment delayed the progression of cataracts in diabetic rats, and the delay was statistically significant on the fourth week of diabetes (p<0.05, when compared with untreated moderately diabetic rats). A decrease in the antioxidant enzymes of CAT and the level of GSH was found in the lens of the untreated diabetic rats at 13 weeks after injection. Some protection was provided in the treated eyes. The level of glycation in the untreated diabetic rats was significantly higher than that in the normal rats (p<0.001). After treatment with CA, AG, and ASA, those diabetic rats had a lower level of glycated lens protein compared to the untreated diabetic rats (p<0.001).
Conclusions
These results thus suggest that the effect of CA, AG, and ASA is indeed inhibition of the formation of AGEs. However, the effect of CA, AG, and ASA is overwhelmed by the excessive accumulation of AGEs in the severely diabetic rats. CA compared with AG and ASA treatment can delay the progression of lens opacification in the diabetic rats only during the earlier stages. It also protects against the inactivation of enzymes.
PMCID: PMC2600521  PMID: 19081783
18.  Topical application of L-arginine blocks advanced glycation by ascorbic acid in the lens of hSVCT2 transgenic mice 
Molecular Vision  2011;17:2221-2227.
Purpose
Previous experiments from our laboratory showed that the oral intake of selected guanidino compounds could block the formation of crystallin-bound advanced ascorbylation products. Here we tested whether these were also active when applied as eye drops.
Methods
Two month old hSVCT2 transgenic mice (n=10) were treated twice daily with one drop of 0.1% L-arginine, γ-guanidinobutyric acid (GBA), penicillamine (PA) or N-acetylcysteine (NAC) in one eye and vehicle only in the other eye. After seven months, lens crystallins were isolated, dialyzed, and proteolytically digested to determine the protein-bound fluorescence at 335/385 and 370/440 nm excitation/emission and the advanced glycation/ascorbylation endproducts carboxymethyl-lysine (CML), carboxyethyl-lysine (CEL), glucosepane, glyoxal, and methylglyoxal hydroimidazolones G-H1 and MG-H1. The topical uptake of L-arginine and NAC was also evaluated in vitro and in vivo in rabbit lens.
Results
In hSVCT2 mice, L-arginine decreased 335/385 and 370/440 nm fluorescence by 40% (p<0.001), CML, CEL, and glucosepane crystallin crosslinks by 35% (p<0.05), 30% (p<0.05), and 37% (p<0.05), respectively, without affecting MG-H1 and G-H1. NAC decreased 335/385 nm fluorescence by 50% (p<0.001) but, like PA and GBA, had no effect on other modifications. L-Arginine uptake into rabbit eyes treated topically reached identical lenticular plateau levels (~400 nmol/g wet weight) at 0.5% and 2.0% but levels remained three times higher at 5 h at 2% versus 0.5% concentration, respectively. In vitro studies showed a 100 fold higher L-arginine level than NAC levels, implicating high affinity uptake of the former.
Conclusions
L-Arginine when applied both orally and topically is a potent and broad suppressor of advanced ascorbylation in the lens. Its uptake in rabbit lens upon topical application suggests transcorneal uptake into the human lens should be feasible for testing its potential anticataract properties in clinical trials.
PMCID: PMC3164690  PMID: 21897744
19.  Determination of Dideoxyosone Precursors of AGEs in Human Lens Proteins 
Dideoxyosones (DDOs) are intermediates in the synthesis of advanced glycation end products (AGEs), such as pentosidine and glucosepane. Although the formation of pentosidine and glucosepane in the human lens has been firmly established, the formation of DDOs has not been demonstrated. The aim of this study was to develop a reliable method to detect DDOs in lens proteins. A specific DDO trapping agent, biotinyl-diaminobenzene (3,4-diamino-N-(3-{[5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl]aminopropyl) benzamide) (BDAB) was added during in vitro protein glycation or during protein extraction from human lenses. In vitro glycated human lens protein showed strong reaction in monomeric and polymeric crosslinked proteins by western blot and ELISA. Glycation of BSA in the presence of BDAB resulted in covalent binding of BDAB to the protein and inhibited pentosidine formation. Mass spectrometric analysis of lysozyme glycated in the presence of BDAB showed the presence of quinoxalines at lysine residues at positions K1, K33, K96, and K116. The ELISA results indicated that cataractous lens proteins contain significantly higher levels of DDO than non-cataractous lenses (101.9±67.8 AU/mg protein vs. 31.7±19.5 AU/mg protein, p<0.0001). This study provides first direct evidence of DDO presence in human tissue proteins and establishes that AGE crosslink synthesis in the human lens occurs via DDO intermediates.
doi:10.1016/j.abb.2011.07.012
PMCID: PMC3172345  PMID: 21820400
Glycation; advanced glycation endproducts; dideoxyosones; human lens proteins; cataract
20.  Advanced glycation end products and the absence of premature atherosclerosis in glycogen storage disease Ia 
Summary
Introduction
Despite their unfavourable cardiovascular risk profile, patients with glycogen storage disease type Ia (GSD Ia) do not develop premature atherosclerosis. We hypothesized that this paradox might be related to a decreased formation of advanced glycation end products (AGEs) resulting from lifetime low plasma glucose levels and decreased oxidative stress.
Methods
In 8 GSD Ia patients (age 20–24 years) and 30 matched controls we measured carotid intima-media thickness (IMT), skin autofluorescence (AF; a non-invasive index for AGEs), and specific AGEs (pentosidine, N-(carboxymethyl)lysine (CML), N-(carboxyethyl)lysine (CEL)) and collagen linked fluorescence (CLF, measured at excitation/emission wavelength combinations of 328/378 and 370/440 nm) in skin samples.
Results
Carotid IMT was significantly lower in GSD Ia patients. Skin AF did not differ between patients and controls. The skin samples showed higher CEL levels in the patient group (p=0.008), but similar levels of pentosidine, CML, and CLF. In the total group, skin AF correlated with CML (r=0.39, p=0.031), CLF 328/378 nm (r=0.53; p=0.002) and CLF 370/440 nm (r=0.60; p=0.001). In the control group, AF also correlated with the maximum carotid IMT (r=0.6; p=0.004).
Conclusion
Although our data confirm that GSD Ia patients present with a reduced burden of atherosclerosis, this phenomenon cannot be explained by differences in AGE accumulation as measured in the skin.
doi:10.1007/s10545-007-0507-0
PMCID: PMC2799632  PMID: 17570077
21.  Glycine therapy inhibits the progression of cataract in streptozotocin-induced diabetic rats 
Molecular Vision  2012;18:439-448.
Purpose
The purpose of this paper was to investigate the effect of the oral administration of L-glycine (Gly) on the development of diabetic cataract induced by streptozotocin (STZ) in rats.
Methods
Two groups of male Wistar rats were intraperitoneally injected with a single dose of STZ (65 mg/kg bodyweight). Then, one group of diabetic rats and a control group were administered with 1% of Gly in drinking water for three months, ad libitum. Cataract development was monitored biweekly through ophthalmoscope inspection and was classified into four stages. At the end of 12 weeks, the animals were sacrificed and some biochemical parameters were determined in their lenses. The parameters include advanced glycation end products (AGEs), glycated proteins, total and soluble protein, glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), aldose reductase (AR), and sorbitol dehydrogenase (SDH). Some parameters were also determined in the serum and blood of the rats.
Results
Diabetic cataract gradually progressed in the STZ-administered group with no other treatment. Consequently, up to the end of the experiment, 2/3 of the animals in this group reached to the last stage of the cataract (mature cataract). The progress of this process was much slower in the diabetic group that was treated with Gly. At the end of the study, the visual cataract score was significantly lower in the diabetic group treated with Gly compared to those administered with STZ. Some lens parameters, including glycated proteins, AGEs, SOD, and AR activities, were increased while some others, including soluble and total protein, GSH level, and CAT activity, were decreased due to diabetes induction. After Gly treatment, all the above-named parameters had reverse changes except for the CAT activity. The SDH activity in the lenses had no changes due to diabetes or treatment. In addition, this treatment significantly decreased the amount of serum glucose (Glc), serum AGEs, and glycated hemoglobin (HbA1c) in the diabetic rats. Gly also increased the ferric reducing antioxidant power (FRAP) in the serum of diabetic rats. However, the decreased bodyweight of animals due to diabetes induction was not compensated by Gly administration. It is important to note that Gly had no effect on normal rat parameters.
Conclusions
The results indicated that the oral administration of Gly significantly delayed the onset and the progression of diabetic cataract in rats. These effects were due to its antiglycating action and to a lesser extent, due to the inhibition of oxidative stress and polyol pathway.
PMCID: PMC3283203  PMID: 22355255
22.  Nonenzymatic glycation of human lens crystallin. Effect of aging and diabetes mellitus. 
Journal of Clinical Investigation  1984;74(5):1742-1749.
We have examined the nonenzymatic glycation of human lens crystallin, an extremely long-lived protein, from 16 normal human ocular lenses 0.2-99 yr of age, and from 11 diabetic lenses 52-82-yr-old. The glucitol-lysine (Glc-Lys) content of soluble and insoluble crystallin was determined after reduction with H-borohydride followed by acid hydrolysis, boronic acid affinity chromatography, and high pressure cation exchange chromatography. Normal lens crystallin, soluble and insoluble, had 0.028 +/- 0.011 nanomoles Glc-Lys per nanomole crystallin monomer. Soluble and insoluble crystallins had equivalent levels of glycation. The content of Glc-Lys in normal lens crystallin increased with age in a linear fashion. Thus, the nonenzymatic glycation of nondiabetic lens crystallin may be regarded as a biological clock. The diabetic lens crystallin samples (n = 11) had a higher content of Glc-Lys (0.070 +/- 0.034 nmol/nmol monomer). Over an age range comparable to that of the control samples, the diabetic crystallin samples contained about twice as much Glc-Lys. The Glc-Lys content of the diabetic lens crystallin samples did not increase with lens age.
PMCID: PMC425353  PMID: 6438156
23.  Antiglycating potential of Zingiber officinalis and delay of diabetic cataract in rats 
Molecular Vision  2010;16:1525-1537.
Purpose
Advanced glycation end products (AGE) are associated in the development of several pathophysiologies including diabetic cataract. Earlier we have reported that some common dietary agents have antiglycating activity and ginger (Zingiber officinalis) was one of the few prominent agents that effectively prevented AGE formation in vitro. In this study we investigated the potential of ginger to prevent diabetic cataract in rats.
Methods
Diabetes was induced in Wistar-NIN rats by intraperitoneal injection of streptozotocin (35 mg/kg bodyweight) and the control rats received vehicle alone. While a set of diabetic animals received AIN-93 diet, another set received either 0.5 or 3% ginger in their diet for a period of two months. Cataract progression was monitored by slit-lamp biomicroscope. At the end of two months, the animals were sacrificed to evaluate non-enzymatic glycation and osmotic stress in the eye lens.
Results
Slit-lamp examination revealed that feeding of ginger not only delayed the onset but also the progression of cataract in rats. Molecular analyses indicated that feeding of ginger significantly inhibited the formation of various AGE products including carboxymethyl lysine in the eye lens. In addition, ginger also countered hyperglycemia-induced osmotic stress in the lens.
Conclusions
The results indicated that ginger was effective against the development of diabetic cataract in rats mainly through its antiglycating potential and to a lesser extent by inhibition of the polyol pathway. Thus, ingredients of dietary sources, such as ginger, may be explored for the prevention or delay of diabetic complications.
PMCID: PMC2925903  PMID: 20806076
24.  Lens Aging: Effects of Crystallins 
Biochimica et biophysica acta  2009;1790(10):1095-1108.
The primary function of the eye lens is to focus light on the retina. The major proteins in the lens—a, b, and g-crystallins—are constantly subjected to age-related changes such as oxidation, deamidation, truncation, glycation, and methylation. Such age-related modifications are cumulative and affect crystallin structure and function. With time, the modified crystallins aggregate, causing the lens to increasingly scatter light on the retina instead of focusing light on it and causing the lens to lose its transparency gradually and become opaque. Age-related lens opacity, or cataract, is the major cause of blindness worldwide. We review deamidation, and glycation that occur in the lenses during aging keeping in mind the structural and functional changes that these modifications bring about in the proteins. In addition, we review proteolysis and discuss recent observations on how crystallin fragments generated in vivo, through their anti-chaperone activity may cause crystallin aggregation in aging lenses. We also review hyperbaric oxygen treatment induced guinea pig and ‘humanized’ ascorbate transporting mouse models as suitable options for studies on age-related changes in lens proteins.
doi:10.1016/j.bbagen.2009.05.008
PMCID: PMC2743770  PMID: 19463898
lens crystallins; aging; lens opacity; chaperones; deamidation; glycation; oxidation; peptides
25.  Clinical Study of Advanced Glycation End Products in Egyptian Diabetic Obese and Non-Obese Patients 
Advanced glycation end products (AGEs) are complex, heterogenous molecules generated by glycation and oxidation of proteins in vivo, which are thought to markedly increase in diabetic patients. One of the recently identified AGEs is carboxy methyl lysine (CML), which is the main ligand of receptors for advanced glycation end products (RAGE). The present study aimed to assess the effect of obesity on such pathways in presence and absence of Type 2 diabetes mellitus. CML, soluble receptors for advanced glycation end products (sRAGE), HbA1C, lipid profile, liver function tests and kidney function tests were determined in 29 diabetic obese, 29 diabetic non-obese, 15 non-diabetic obese and 15 non-diabetic non-obese subjects. The study compared obese and non-obese subjects in presence and absence of type 2 diabetes. The results showed a significant increase in CML and a significant decrease in sRAGE in each of the diabetic obese group when compared with the diabetic non-obese group and the non-diabetic obese group when compared with the non-diabetic non-obese group. A significant positive correlation was found between CML and markers of obesity (body mass index and waist/hip ratio). These results suggest that obesity can increase CML independent of diabetes and support the reports that CML could be generated from both sugars and lipids. The present study suggests that treatment using glycation inhibitors like aminoguanidine or recombinant sRAGE will not only retard the diabetic complications, but may also have a prophylactic effect.
PMCID: PMC3614834  PMID: 23675236
advanced glycation end products; carboxymethyllysine; receptors for advanced glycation end products; obesity and type 2 diabetes mellitus

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