Glycation Of Lens Proteins Research Articles

Identification of genes contributing to attenuation of rat model of galactose-induced cataract by pyruvate.

Cataracts are a disease that reduces vision due to opacity formation of the lens. Diabetic cataracts occur at young age and progress relatively quickly, so the development of effective treatment has been awaited. Several studies have shown that pyruvate inhibits oxidative stress and glycation of lens proteins, which contribute to onset of diabetic cataracts. However, detailed molecular mechanisms have not been revealed. In this study, we attempted to reduce galactose-induced opacity by pyruvate with rat ex vivo model. Rat lenses were extracted and cultured in galactose-containing medium to induce lens opacity. After opacity had developed, continued culturing with pyruvate in the medium resulted in a reduction of lens opacity. Subsequently, we conducted microarray analysis to investigate the genes that contribute to the therapeutic effect. We performed quantitative expression measurements using RT-qPCR for extracted genes that were upregulated in cataract-induced lenses and downregulated in pyruvate-treated lenses, resulting in the identification of 34 candidate genes. Functional analysis using the STRING database suggests that metallothionein-related factors (Mt1a, Mt1m, and Mt2A) and epithelial-mesenchymal transition-related factors (Acta2, Anxa1, Cd81, Mki67, Timp1, and Tyms) contribute to the therapeutic effect of cataracts.

Topical Application of Deglycating Enzymes as an Alternative Non-Invasive Treatment for Presbyopia.

Presbyopia is an age-related vision disorder that is a global public health problem. Up to 85% of people aged ≥40 years develop presbyopia. In 2015, 1.8 billion people globally had presbyopia. Of those with significant near vision disabilities due to uncorrected presbyopia, 94% live in developing countries. Presbyopia is undercorrected in many countries, with reading glasses available for only 6-45% of patients living in developing countries. The high prevalence of uncorrected presbyopia in these parts of the world is due to the lack of adequate diagnosis and affordable treatment. The formation of advanced glycation end products (AGEs) is a non-enzymatic process known as the Maillard reaction. The accumulation of AGEs in the lens contributes to lens aging (leading to presbyopia and cataract formation). Non-enzymatic lens protein glycation induces the gradual accumulation of AGEs in aging lenses. AGE-reducing compounds may be effective at preventing and treating AGE-related processes. Fructosyl-amino acid oxidase (FAOD) is active on both fructosyl lysine and fructosyl valine. As the crosslinks encountered in presbyopia are mainly non-disulfide bridges, and based on the positive results of deglycating enzymes in cataracts (another disease caused by glycation of lens proteins), we studied the ex vivo effects of topical FAOD treatment on the power of human lenses as a new potential non-invasive treatment for presbyopia. This study demonstrated that topical FAOD treatment resulted in an increase in lens power, which is approximately equivalent to the correction obtained by most reading glasses. The best results were obtained for the newer lenses. Simultaneously, a decrease in lens opacity was observed, which improved lens quality. We also demonstrated that topical FAOD treatment results in a breakdown of AGEs, as evidenced by gel permeation chromatography and a marked reduction in autofluorescence. This study demonstrated the therapeutic potential of topical FAOD treatment in presbyopia.

Hesperetin treatment attenuates glycation of lens proteins and advanced‑glycation end products generation.

Advanced glycation end products (AGEs) in lens proteins increase with aging, thus inducing cataracts and/or presbyopia. Hesperetin (Hst), which is an abundant plant flavanone largely derived from citrus species, and its derivatives attenuate cataracts and presbyopia invivo and invitro; however, no reports have described its effects on AGE formation in lens proteins. The present study demonstrated that AGEs in lens proteins increase with age in mice. Additionally, it showed that Hst can prevent AGEs and N(ε)‑carboxymethyl‑lysine generation and modification of lens proteins using invitro in human lens epithelial cell lines and exvivo in mouse lens organ cultures. Furthermore, treatment with Hst prevented lens hardening and decreased chaperone activity in lens proteins. These results suggested that Hst and its derivatives are good candidates for the prevention of presbyopia and cataracts.

Inhibition of lens protein glycation and aldose reductase activity using Aqueous extract of Morinda Citrifolia and Ocimum Sanctum in Glucose induced diabetic cataract

Diabetes mellitus is a chronic disorder characterized by persistent elevated blood glucose level in the body which leads to various secondary complications like cataract. The understanding of mechanisms by which glucose exerts its toxicity is of utmost importance for rational pharmacological interventions to treat diabetic cataract. Plants have always been an exemplary source of drugs and many of the currently available drugs have been derived directly or indirectly from them. The aim of the present study was to explore local antiglycation and anticataract potential of M. citrifolia and O. sanctum in goat lenses using glucose induced cataract model. A total of 120 goat lenses were divided into four groups of 30 each viz. Normal Control, Experimental diabetic cataract, Experimental diabetic cataract + Extract of Morinda citrifolia (0.25%), Experimental diabetic cataract Extract of Ocimum sanctum (0.25%). Extent of protein glycation and activity of aldose reductase were measured in lens homogenates. Experimental cataractous lenses showed increased levels of both glycation of protein as well as aldose reductase activity. However, lenses treated with M. citrifolia as well as O. sanctum showed significant decreased levels of both glycation of protein and aldose reductase activity.

Identification of potential phytochemical lead against diabetic cataract: An insilico approach

Cataract is one of the leading causes for blindness among worldwide devoid of effective treatment. Glycation of γ-crystallin under hyperglycemic condition leads to diabetic cataract formation. It is important to design a drug which could inhibit lens protein glycation and prevent the diabetic cataract. The use of plant based natural compounds could have potential bioavailability and fewer toxicity. The current investigation aim is to determine the efficient phytochemicals for inhibition of γ-crystallin glycation. In this study the phytochemicals with anticataract property were virtually screened against γ-crystallin. The potent phytochemicals were filtered based on their interaction site and binding affinity. These phytochemicals were further scrutinized and evaluated with pharmacokinetics, druggability and Pharmacophore feature analysis. Among the phytochemicals, Alizarin (polyketide), Pseudobaptigenin (flavonoid) and Ellagic acid (tannin) are three phytochemicals which had preferred druglike behavior and binding affinity for the inhibition of glycation against γ-crystallin protein. The features of these three phytochemicals could be used as prototype for synthesizing new molecules against diabetic cataract.

Lens protein glycation in diabetic and senile cataract patients

Introduction: Cataract is an abnormality in lens, characterized by decreased transparency and increased cloudiness and may progress to severe visual impairment if left untreated. Glycation of lens protein crystallins is considered as the major mechanism for the formation of cataract. The study was undertaken with the aim to estimate glycated protein in lens of diabetic cataract patients and to compare it with senile cataracts. Materials and Methods: A total of 60 lenses were collected. Of which, 30 were from diabetic cataract subjects and rest 30 were from subjects of senile cataract. Glycated protein was measured in the lenses of all participants. Results: The level of lens glycated protein was found to be significantly increased in subjects of type 2 diabetes mellitus compared to senile cataract patients. Conclusion: In conclusion, the results of the present study indicate that glycation of lens protein may play an important role in the development of cataract with greater extent in diabetic patients compared to senile group. Keywords: Cataract, Crystallins, Diabetes mellitus, Glycation.

Dietary flavonoids inhibit the glycation of lens proteins: implications in the management of diabetic cataract

The intervention of functional foods as complementary therapeutic approach for the amelioration of diabetes and sugar induced cataractogenesis is more appreciated over the present day chemotherapy agents owing to their nontoxic and increased bioavailability concerns. Dietary flavonoids, a class of bioactive phytochemicals is known to have wide range of biological activities against variety of human ailments. In the present study, we demonstrate anti-cataract effect of eight dietary flavonoids in sugar induced lens organ culture study. We present data on processes like inhibition of glycation-induced lens cloudiness, lens protein aggregation, glycation reaction and advanced glycation end products formation that can act as biochemical markers for this disease. The selected flavonoids were also tested for their aldose reductase (AR) inhibition (experimental and in silico). The molecular dynamics simulation results shed light on mechanistic details of flavonoid induced AR inhibition. The outcome of the present study clearly focuses the significance of kaempferol, taxifolin and quercetin as potential candidates for controlling diabetic cataract.

Zinc Supplementation Ameliorates Diabetic Cataract Through Modulation of Crystallin Proteins and Polyol Pathway in Experimental Rats.

Non-enzymatic glycation of lens proteins and elevated polyol pathway in the eye lens have been the characteristic features of a diabetic condition. We have previously reported the benefits of zinc supplementation in reducing hyperglycemia and associated metabolic abnormalities and oxidative stress in diabetic rats. The current study explored whether zinc supplementation protects against cataractogenesis through modulation of glycation of lens proteins, elevated polyol pathway, oxidative stress, and proportion of different heat shock proteins in the eye lens of diabetic rats. Streptozotocin-induced diabetic rats were fed with a zinc-enriched diet (5 and 10 times of normal) for 6weeks. Supplemental zinc alleviated the progression and maturation of diabetes-induced cataract. Zinc was also effective in preventing the reduced content of total and imbalanced proportion of soluble proteins in the lens. Supplemental zinc also alleviated cross-linked glycation and concomitant expression of the receptor of glycated products and oxidative stress indicators in the eye lens. Zinc supplementation further induced the concentration of heat shock protein in the eye lens of diabetic rats, specifically α-crystallin. Zinc supplementation counteracted the elevated activity and expression of polyol pathway enzymes and molecules in the lens. The results of this animal study endorsed the advantage of zinc supplementation in exerting the antiglycating influence and downregulating polyol pathway enzymes to defer cataractogenesis in diabetic rats.

Functional food supplements to ameliorate the secondary complications in high fructose fed diabetic rats.

Functional foods are the most natural and safest source of health ingredients, providing health benefits beyond basic nutrition, and hence can be used as supplements for the prevention of secondary complications in diabetes. Persistent diabetes may cause glycation of various tissue proteins such as of those in lens, kidney, blood, and brain, which may further lead to the development of pathological conditions such as cataract and cardiovascular diseases. This study on adult rats was designed to assess if the functional food supplements A and B (proprietary blends of antioxidant rich plant materials) can reduce secondary complications such as cataract, dyslipidemia, and oxidative stress under severe diabetic conditions. After nine weeks of intervention of the supplements, it was found that the % HbA1c levels in the formulation group B significantly (p < 0.05) lowered (10.9%) followed by those in group A (11.1%) as compared to those in the diabetic fructose control (DFC) group (15.1%); moreover, plasma insulin levels were significantly (p < 0.01) improved in the formulation B group (9.8 mU L-1) as compared to those in the DFC group (8.5 mU L-1). The significantly higher level of plasma TEAC in group B (27.5 mg dL-1) (p < 0.02) and group A (26.6 mg dL-1) (p < 0.05) indicates an improved plasma antioxidants status as compared to that in DFC group (21.7 mg dL-1). Both the formulation groups A and B showed a decrease in AGEs and tryptophan fluorescence, which suggests amelioration of the glycation of lens proteins as compared to that in the DFC group. The present results indicate that the formulations A and B exhibit antiglycating and antioxidant potentials by inhibiting the high fructose-induced glycation in diabetic rats; hence, they may have therapeutic value as functional foods in the effective management of secondary complications associated with severe diabetic conditions.

Featured Article: Inhibition of diabetic cataract by glucose tolerance factor extracted from yeast.

Diabetes leads to many complications; among them is the development of cataract. Hyperglycemia brings to increased polyol concentration in the lens, to glycation of lens proteins, and to elevated level of ROS (Reactive Oxygen Species) causing oxidative stress. The glucose tolerance factor (GTF) was found by several groups to decrease hyperglycemia and oxidative stress both in diabetic animals and humans. The aim of our study was to explore the damages induced by high glucose to the eye lens and to assess the protective effects of GTF both in vivo and in vitro The in vivo study included control healthy rats, streptozotocin (STZ) diabetic untreated rats, and STZ diabetic rats orally treated with 15 doses of GTF. The diabetic untreated rats developed cataracts, whereas the development of cataract was totally or partially prevented in GTF treated animals. In vitro studies were done on bovine lenses incubated for 14 days. Half of the lenses were incubated in normal glucose conditions, and half in high glucose conditions (450 mg%). To one group of the normal or high glucose condition GTF was added. The optical quality of all the lenses was measured daily by an automated scanning laser system. The control lenses, whether with or without GTF addition, did not show any reduction in their quality. High glucose conditions induced optical damage to the lenses. Addition of GTF to high glucose conditions prevented this damage. High glucose conditions affected the activity of aldose reductase and sodium potassium ATPase in lens epithelial cell. Addition of GTF decreased the destructive changes induced by high glucose conditions. The amount of soluble cortical lens proteins was decreased and structural changes were detected in lenses incubated in high glucose medium. These changes could be prevented when GTF was added to high glucose medium. Our findings demonstrate the anticataractogenic potential of GTF.

Effects of L-Carnitine and Cinnamon Extract Treatment on Lens Crystallins of Rats Fed High Fructose Diet

Problem statement: Rats fed high dietary fructose are documented to f orm an acquired model of insulin resistance; the present study aims to investigate possible changes in lens crystallin s of rats fed high fructose diet and the effects of admi nistration of each exogenous L-Carnitine (CA) and Cinnamon Extract (CE) on protein glycation, oxidati ve stress and redox homeostasis in this rat model. Approach: A total number of 60 male Wister rats of body weig ht 120-160 g were divided into 4 groups of 15 rats each. Group 1 received control di et, while groups 2, 3 and 4: rats received high fructose diet (60g/100 g diet). After 2 weeks from fructose feeding, animals of group 3 were treated with L-carnitine (300 mg g -1 body weight/day i.p.), while animals of group 4 we re treated with cinnamon extract (0.5 mL/rat/day orally). At the en d of experimental period (30 days), serum levels of glucose and insulin were determined. Lenses of each animal were dissected; molecular weights of crystalline, oxidative stress markers, early glycat ion of lens proteins and carbonyl group were assaye d. Results: A significant decline in antioxidants and increase in lipid peroxidation products, protein oxidation and protein glycation were observed in lens samples obtained from fructose-fed rats. Administration of each CA and CE to fructose-fed rats significantly attenu ated oxidative damage and protein glycation and ret urned levels of antioxidants to near those in control gro up. Chromatographic analysis of lens crystalline of rats fed high fructose diet showed diffused peaks, indicatin g crystalline aggregation. Conclusion: The results of the present study indicate that dietary fructose di sturbs lens integrity and administration of L-carni tine or cinnamon extract may safeguard the lens by minimizing the protein aggregation, preventing glycation and oxidative stress in animals fed high fructose diet. L-carnitine has more potent effects than CE in this animal model.

Associations between nutrition and cataract.

Blindness due to opacification of the lens, or cataract, afflicts 50 million persons worldwide. In the United States over 541,000 cataract extractions are done annually at a cost of over $3.8 billion. Conservative estimates indicate that the prevalences of cataracts in Americans aged 65-75 and 75-85 years are 18% and 46%, respectively. Cataracts are even more prevalent in some other populations. It is estimated that the need for cataract extractions would be diminished by half if onset of cataract could be delayed by only ten years. Hypotheses regarding the etiology of cataract include oxidative perturbations of protein metabolism, diverse pathologic conditions, and perhaps glycation of lens proteins. Epidemiologic data indicate that elevated plasma levels of specific nutrients (i.e., carotenoids, ascorbate, tocopherol, and taurine) are associated with diminished incidence of certain types of cataract. Biochemical evidence suggests that each of these compounds can delay photooxidative damage to lens proteins. Roles in lens metabolism for selenium and tryptophan have been suggested. Elucidation of mechanisms by which caloric restriction delays cataract development is a promising area of current research.

Tryptophan Metabolites from Young Human Lenses and the Photooxidation of Ascorbic Acid by UVA Light

To determine whether there are UVA light-responsive sensitizers in young human lenses capable of initiating the oxidation of ascorbic acid in the absence of oxygen. Lens homogenates were fractionated, and low-molecular-weight (LMW) components were separated from the proteins by filtration through a 3000-MWt cutoff filter. Aliquots of each fraction were assayed for sensitizer activity by UVA irradiation (337-nm cutoff filter) with 0.1 mM ascorbic acid, measuring ascorbate oxidation by loss of absorbance at 265 nm. Two major peaks were isolated from a human lens water-soluble (WS)-LMW fraction on a reversed-phase column and were identified by mass spectrometry. All human lens fractions oxidized ascorbate when irradiated by UVA light. Most of the sensitizer activity in young human lenses was in the LMW fractions. An action spectrum for the WS-LMW fraction from human lens showed activity throughout the UVA region. Assays with and without oxygen showed little or no difference in ascorbate oxidized, arguing for a direct transfer of an electron in a so-called type 1 reaction. A human lens WS-LMW fraction contained two major peaks of activity. The greater peak was identified as 3-hydroxykynurenine glucoside (3OHKG) by mass spectrometry and its absorption spectrum, whereas the lesser peak was identified as 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid glucoside (AHBG). The activities were 1.1 and 2.8 nmol of ascorbate oxidized in 30 minutes/nmol 3OHKG and AHBG, respectively. The filter compounds present in human lenses can absorb UVA light and cause the oxidation of ascorbic acid in the presence and absence of oxygen, possibly initiating the glycation of lens proteins.

Delay of diabetic cataract in rats by the antiglycating potential of cumin through modulation of α-crystallin chaperone activity

α-Crystallin, a molecular chaperone of the eye lens, plays an important role in maintaining the transparency of the lens by preventing the aggregation/inactivation of several proteins and enzymes in addition to its structural role. α-Crystallin is a long-lived protein and is susceptible to several posttranslational modifications during aging, more so in certain clinical conditions such as diabetes. Nonenzymatic glycation of lens proteins and decline in the chaperone-like function of α-crystallin have been reported in diabetic conditions. Therefore, inhibitors of nonenzymatic protein glycation appear to be a potential target to preserve the chaperone activity of α-crystallin and to combat cataract under hyperglycemic conditions. In this study, we investigated the antiglycating potential of cumin in vitro and its ability to modulate the chaperone-like activity of α-crystallin vis-à-vis the progression of diabetic cataract in vivo. Aqueous extract of cumin was tested for its antiglycating ability against fructose-induced glycation of goat lens total soluble protein (TSP), α-crystallin from goat lens and a nonlenticular protein bovine serum albumin (BSA). The antiglycating potential of cumin was also investigated by feeding streptozotocin (STZ)-induced diabetic rats with diet containing 0.5% cumin powder. The aqueous extract of cumin prevented in vitro glycation of TSP, α-crystallin and BSA. Slit lamp examination revealed that supplementation of cumin delayed progression and maturation of STZ-induced cataract in rats. Cumin was effective in preventing glycation of TSP and α-crystallin in diabetic lens. Interestingly, feeding of cumin to diabetic rats not only prevented loss of chaperone activity but also attenuated the structural changes of α-crystallin in lens. These results indicated that cumin has antiglycating properties that may be attributed to the modulation of chaperone activity of α-crystallin, thus delaying cataract in STZ-induced diabetic rats.

CHANGES IN REDOX RATIO AND PROTEIN GLYCATION IN PRECATARACTOUS LENS FROM FRUCTOSE‐FED RATS: EFFECTS OF EXOGENOUS l‐CARNITINE

1. Rats fed high dietary fructose are documented to form an acquired model of insulin resistance. The present study measured the effects of administration of L-carnitine (CA) on lens protein glycation, oxidative stress and redox homeostasis in rats fed a high-fructose diet. 2. Animals were divided into four groups: (i) an untreated control group (fed starch diet); (ii) an untreated fructose-group (fed a high-fructose diet); (iii) a CA-treated (300 mg/kg per day), fructose-fed group; and (iv) a CA-treated, starch-fed group. After 60 days treatment, lenses were dissected and multiple oxidative stress markers, glycation of proteins and the ratio of oxidized to reduced glutathione (GSSG/GSH) were determined. 3. A significant decline in enzyme and non-enzyme anti-oxidants and an increase in lipid peroxidation products, protein oxidation, protein glycation, GSSG/GSH ratio and aldehyde formation were observed in lens samples obtained from fructose-fed rats. Administration of CA to fructose-fed rats significantly attenuated oxidative damage and protein glycation and returned levels of anti-oxidants to near those seen in the control group. 4. The results of the present study indicate that dietary fructose disturbs lens integrity and exogenous CA may safeguard the lens by preventing glycation and oxidative stress.

Lens fluorescence and accommodative amplitude in pre-presbyopic and presbyopic subjects

Accommodative amplitude (AA; the difference, measured in diopters, between the near and far points of vision) declines steadily with age such that, by midlife, most individuals are unable to focus clearly on near objects and, thus, are said to be presbyopic. Conversely, intrinsic lens fluorescence (LF) increases steadily with age. Previous studies have suggested that AA and LF are negatively correlated, independent of age. Were this to be the case, it might suggest that the biochemical modifications underlying increased tissue fluorescence (for example, glycation of lens proteins) contribute to presbyopia. We used quantitative techniques to re-evaluate the relationship between AA and LF in 161 healthy volunteers aged between 25 and 70. Our data confirmed that AA decreases with age, becoming essentially zero by age 55, and LF increases with age. However, in marked contrast to previous reports, statistical analysis failed to detect any correlation between LF and AA independent of age. Thus, the biochemical processes responsible for increased LF observed in the aged lens are unlikely to contribute directly to presbyopia.

A comparative study of lens protein glycation in various forms of cataract

The aim of this study was to estimate and compare the levels of glycated protein in the lens extracted from patients with cataract of various etiologies. A total of 72 cataract lens were collected. The levels of total proteins, glucose and glycated protein in the lens were studied. Plasma protein and fasting glucose levels were also estimated. The amount of glycation of lens was significantly higher in case of hypermature senile cataract (p<0.01) when compared with other types of cataracts. The levels of lens glucose between the various types of cataracts did not differ significantly. These results indicate that the level of lens glucose alone is not the only determining factor of lens protein glycation.

The effect of UVA light on the anaerobic oxidation of ascorbic acid and the glycation of lens proteins.

To determine whether UVA-excited human lens chromophores can cause the oxidation of ascorbic acid in the absence of oxygen, and whether these oxidation products are capable of glycating lens proteins. The oxidation of ascorbic acid, mediated by UVA irradiation in the presence of aged human lens proteins, was measured in the absence of oxygen by the decrease in absorbance at 265 nm in vitro. An action spectrum from 320 to 400 nm was determined for both ascorbate oxidation and the photobleaching of the lens yellow pigments at lambda = 350 nm. The UVA-mediated oxidation products of [U-(14)C]ascorbate were quantified by HPLC. Glycation was assayed by the UVA-dependent incorporation of [U-(14)C]ascorbate into lens proteins with a water-insoluble (WI) fraction in vitro, with incubated whole human lenses, and with a WI fraction after a 5- to 7-day exposure to ambient sunlight. An enzymatic digest of [U-(14)C]ascorbate-labeled proteins was fractionated over HPLC columns and compared with the 330-nm absorbance profile of a proteolytic digest of aged human lens proteins. Aged human lens WI proteins absorbed UVA light (86 J/h per square centimeter) and oxidized 33 to 45 nanomoles of ascorbate over 1 hour in the absence of oxygen. No ascorbate oxidation was detected, however, in the dark control. An action spectrum showed that ascorbate oxidation occurred throughout the UVA region, with lambda(max) at 350 nm, which was similar to the action spectrum obtained for the photobleaching of the lens chromophores. Anaerobic UVA irradiation of aged human lens proteins for 2 hours with [U-(14)C]ascorbate resulted in a 40% loss of ascorbate with the accumulation of dehydroascorbic acid, diketogulonic acid, and oxalate. After subsequent incubation for 24 hours, the ascorbate oxidation products disappeared, with a corresponding incorporation of radioactivity into lens proteins. Chromatography of enzymatic digests of the labeled proteins produced peaks that coeluted with several of the 330-nm absorbing peaks in an aged human lens protein digest. Irradiation of whole human lenses for 2 hours caused a 33% loss of total lens ascorbate. UVA irradiation of aged human lenses for 2 hours resulted in the incorporation of ascorbate into lens proteins during the ensuing 24 hours in the dark. Exposure of aged human lens WI proteins to reflected ambient sunlight (1.1 J/h per square centimeter) for 5 to 7 days in the absence of oxygen also produced an increased incorporation of [(14)C]ascorbate into protein when compared with dark control samples. These data argue that UVA light can cause an oxidation of ascorbic acid in the absence of oxygen, due to the activation of the sensitizers present in aged human lens WI proteins. The oxidation products formed were the same as those seen in the presence of oxygen, and were rapidly incorporated into protein, apparently by Maillard-type chemistry. These data argue that ascorbate glycation can occur under the low oxygen levels thought to exist in the human lens nucleus in vivo.

Non-enzymatic glycation of lens proteins and haemoglobin-inhibition by pyruvate: an in-vivo study.

Previous studies have demonstrated that pyruvate can prevent protein glycation and oxidative stress under in-vitro conditions. The aim of this study was to examine the in-vivo effectiveness of this metabolite against glycation of lens crystallins and haemoglobin in galactosemic rats. Sprague-Dawley rats were maintained on a 30% or 50% galactose-containing diet in the absence or presence of 2% or 5% pyruvate in food and water, respectively. The animals were killed subsequently and the extent of glycation of lens crystallins and haemoglobin was determined using an affinity column chromatograpic technique. Maintenance of rats on the high galactose diet resulted in a significant increase in glycation of both the proteins. The increase was faster and more substantial in the animals maintained on the 50% galactose diet than that in the animals fed a 30% galactose diet. The increase in the latter was also very significant. Supplementation with pyruvate inhibited the process. The inhibition is attributable to a competitive binding of pyruvate to the protein NH2 groups as well as to the antioxidant effect of the compound. The studies therefore suggest that this and other alpha-keto-acids may be physiologically useful in minimizing glycation and oxidative stress induced tissue pathology by the hyperglycaemic conditions, such as diabetes and galactosemia. The results are also considered pharmacologically significant.

Temporal association between lens protein glycation and cataract development in diabetic rats.

In an attempt to shed more light on the relation between the glycation process and structural protein alterations, we followed the formation of glycated products in the lenses of hyperglycaemic Wistar rats during a period of 5 months following alloxan diabetes inducement. The study groups included non-diabetic (control), untreated diabetic rats (D), and diabetic rats receiving insulin alone or in combination with phlorizin, an inhibitor of renal tubular glucose transport. Lenses were removed at 4 and 20 weeks, and advanced glycation products in alkalisoluble lens proteins were determined by their characteristic spectrofluorescence (emission at 385 nm with excitation of 335 nm). In 20-week untreated diabetic rats as compared to control rats, a significant increase was observed in the fluorescence level (3.25 +/- 1.02 vs 1.61 +/- 0.17 FU/mg, p < 0.001), while in 4-week animals the increase was from 1.26 +/- 0.11 FU/mg in controls to 1.80 +/- 0.25 FU/mg in diabetics (P < 0.001). Daily treatment of 20-week diabetic rats with insulin alone (2.46 +/- 0.48 FU/mg) or in combination with phlorizin (2.30 +/- 0.26 FU/mg) did not significantly influence lens fluorescence level. The amount of glucosebound ketoamine linkage was estimated after acid hydrolysis as released 5-hydroxymethylfurfural (HMF). In 20-week controls, it was slightly higher than in 4-week controls (0.57 +/- 0.31 vs 0.41 +/- 0.20 nmol HMF/mg, respectively). The diabetic group showed a significant increase, however. In 4-week diabetics, a level of 1.07 +/- 0.36 nmol HMF/mg was found, while in 20-week animals the glycated protein amount rose to 2.46 +/- 0.79 nmol HMF/mg. In addition to the increases in glycated content with continuing diabetic hyperglycaemia, significant changes in protein composition of alkali-soluble lenses developed. The SDS-PAGE pattern showed an appearance of protein polymers of heterogeneous size (C 4 weeks 3.0 +/- 1.1% vs C 20 weeks 17.9 +/- 2.9%, D 4 weeks 7.3 +/- 2.1% vs D 20 weeks 19.8 +/- 3.6%) and the proteins of high molecular weight (HMW) failed to penetrate into the gel. Only a small amount of these HMW proteins was present in controls (C 20 weeks 2.5 +/- 1.2%) and short-term diabetes (D 4 weeks 0.8 +/- 0.2%), whereas in long-term untreated diabetes there was a dramatic increase (D 20 weeks 30.5 +/- 3.2%) with a corresponding decrease in other peaks. All diabetic animals from this group had macroscopically detectable cataractous lenses. Treatment with insulin or insulin/phlorizin followed the HMW protein level of the untreated animals (28.2 +/- 4.0% or 27.08 +/- 3.3% vs 30.52 +/- 3.32%).