Decrease In Glycation Research Articles

Effect of Citrullus colocynthis Extract on Glycated Hemoglobin Formation (In Vitro).

Diabetes mellitus (DM) is typically a disorder of carbohydrate, fat, and protein metabolism. It develops due to a lack of or loss associated with insulin and/or resistance to insulin. Regarding complications of chemical substance use, drugs with few complications and high-reliability tannins are needed. This study aimed to determine the effect and mechanism of action of Citrullus colocynthis extract on the formation of glycated hemoglobin (HbA1c). A solution containing hemoglobin and glucose was incubated for 1, 2, 3, 4, 30, and 60 days by adding Citrullus colocynthis extract or glutathione. Quantitative measurement of HbA1c was performed using ion-exchange chromatography. Data were analyzed using ANOVA and two-way repeated measures test. A p<0.05 was considered statistically significant. The Citrullus colocynthis extract in hyperglycemic conditions and with increasing time reduced the formation of HbA1c and thus inhibited the production of glycated proteins. By increasing the time and after initiation of reaction of extract concentrations (0, 0.1, 0.3, 0.5, and 1 g/dL), presently, there was a significant decrease in the formation of HbA1C compared to those in the control group (p<0.05). The decrease in glycation has been dose dependent. Therefore, Citrullus colocynthis could directly reduce the formation of HbA1c.

Role of the specifically targeted lysine residues in the glycation dependent loss of chaperone activity of αA- and αB-crystallins

Earlier studies have shown significant loss of chaperone activity in alpha-crystallin from diabetic lenses. In vitro glycation studies have suggested that glycation of alpha-crystallin could be the major cause of chaperone activity loss. The following lysine (K) residues in alpha-crystallin have been identified as the major glycation sites: K11, K78, and K166 in alpha A-crystallin and K90, K92, and K166 in alpha B-crystallin. The present study was aimed to assess the contribution of each of the above glycation site in the overall glycation and loss of chaperone activity by mutating them to threonine followed by in vitro glycation with fructose. Level of glycated protein (GP) was determined by phenylboronate affinity chromatography, advanced glycation end products (AGEs) by direct ELISA using anti-AGE polyclonal antibody, and chaperone activity by using alcohol dehydrogenase as the target protein. K11T, K78, and K166T mutants of alpha A showed 33, 17, and 27% decrease in GP and 32, 18, and 21% decrease in AGEs, respectively, as compared to alpha A-wt. Likewise, K90T, K92T, K90T/K92T, and K166T mutants of alpha B showed 18, 21, 29, and 12% decrease in GP and 22, 24, 32, and 16% decrease in AGEs, respectively. Chaperone activity also showed concomitant increase with decreasing glycation and AGEs formation. alpha A-K11T and alpha B-K90T/K92T mutants showed the largest decrease in glycation and increase in chaperone activity.

Free alanine, aspartic acid, or glutamic acid reduce the glycation of human lens proteins.

The amino acids lysine and glycine are reported to react with glucose at physiological pH and temperature and undergo non-enzymic glycation. Three other amino acids present in relatively larger amounts in the lens i.e. alanine, aspartic acid and glutamic acid were also found to undergo non-enzymic glycation as found by incorporation of uniformly labelled (U-[14C]) glucose into the amino acids. The glucose incorporation was 1.6 to 2.5% for alanine, 35 to 50% for aspartic acid and 2.3 to 3.3% for glutamic acid. Each amino acid of varying concentrations lowered the extent of in vitro glycation of lens proteins significantly in glucose-treated homogenates of normal lens from humans. The decrease in glycation for alanine was between 32 and 69%, that for aspartate was between 18 and 74%, and for glutamate was between 52 to 74%. Decreased glycation was greater for higher concentrations of glucose. Scavenging of intracellular glucose and decreasing the extent of glycation of lens proteins could be the mechanism of action by which the amino acids alanine, aspartic acid and glutamic acid could exercise a beneficial effect on cataract and diabetic retinopathy.

Decrease in Glycation of Lens Proteins by Lysine and Glycine by Scavenging of Glucose and Possible Mitigation of Cataractogenesis

The amino acid lysine has been reported to delay cataractogenesis by 'some unknown mechanism'. Lysine and glycine were found to react with glucose at physiological pH and temperature and undergo non-enzymic glycation. The formation of glycated lysine was shown by paper and thin-layer chromatography, HPLC and using an authentic sample of ε-fructosyl lysine. Confirmation was made by studies on incorporation of U-[ 14C]glucose into lysine and glycine. The extent of glycation of lysine was 15·5% in 96 hr and rose to 20% in 20 days. Lysine and glycine alone of varying concentrations lowered the extent of glycation of lens proteins significantly in glucose-treated homogenates of normal lens from humans and goats. Scavenging of intracellular glucose and thereby protecting the lens proteins from excessive glycation appears to be the mechanism of action by which lysine and glycine could exercise beneficial effect on cataract.

Decrease in skin collagen glycation with improved glycemic control in patients with insulin-dependent diabetes mellitus.

Glycation, oxidation, and nonenzymatic browning of protein have all been implicated in the development of diabetic complications. The initial product of glycation of protein, fructoselysine (FL), undergoes further reactions, yielding a complex mixture of browning products, including the fluorescent lysine-arginine cross-link, pentosidine. Alternatively, FL may be cleaved oxidatively to form N(epsilon)-(carboxymethyl)lysine (CML), while glycated hydroxylysine, an amino-acid unique to collagen, may yield N(epsilon)-(carboxymethyl)hydroxylysine (CMhL). We have measured FL, pentosidine, fluorescence (excitation = 328 nm, emission = 378 nm), CML, and CMhL in insoluble skin collagen from 14 insulin-dependent diabetic patients before and after a 4-mo period of intensive therapy to improve glycemic control. Mean home blood glucose fell from 8.7 +/- 2.5 (mean +/- 1 SD) to 6.8 +/- 1.4 mM (P less than 0.005), and mean glycated hemoglobin (HbA1) from 11.6 +/- 2.3% to 8.3 +/- 1.1% (P less than 0.001). These changes were accompanied by a significant decrease in glycation of skin collagen, from 13.2 +/- 4.3 to 10.6 +/- 2.3 mmol FL/mol lysine (P less than 0.002). However, levels of browning and oxidation products (pentosidine, CML, and CMhL) and fluorescence were unchanged. These results show that the glycation of long-lived proteins can be decreased by improved glycemic control, but suggest that once cumulative damage to collagen by browning and oxidation reactions has occurred, it may not be readily reversed. Thus, in diabetic patients, institution and maintenance of good glycemic control at any time could potentially limit the extent of subsequent long-term damage to proteins by glycation and oxidation reactions.

Effect of phosphate on the kinetics and specificity of glycation of protein.

The glycation (nonenzymatic glycosylation) of several proteins was studied in various buffers in order to assess the effects of buffering ions on the kinetics and specificity of glycation of protein. Incubation of RNase with glucose in phosphate buffer resulted in inactivation of the enzyme because of preferential modification of lysine residues in or near the active site. In contrast, in the cationic buffers, 3-(N-morpholino)propane-sulfonic acid and 3-(N-tris(hydroxymethyl)methyl-amino)-2-hydroxypropanesulfonic acid, the kinetics of glycation of RNase were decreased 2- to 3-fold, there was a decrease in glycation of active site versus peripheral lysines, and the enzyme was resistant to inactivation by glucose. The extent of Schiff base formation on RNAse was comparable in the three buffers, suggesting that phosphate, bound in the active site of RNase, catalyzed the Amadori rearrangement at active site lysines, leading to the enhanced rate of inactivation of the enzyme. Phosphate catalysis of glycation was concentration-dependent and could be mimicked by arsenate. Phosphate also stimulated the rate of glycation of other proteins, such as lysozyme, cytochrome c, albumin, and hemoglobin. As with RNase, phosphate affected the specificity of glycation of hemoglobin, resulting in increased glycation of amino-terminal valine versus intrachain lysine residues. 2,3-Diphosphoglycerate exerted similar effects on the glycation of hemoglobin, suggesting that inorganic and organic phosphates may play an important role in determining the kinetics and specificity of glycation of hemoglobin in the red cell. Overall, these studies establish that buffering ions or ligands can exert significant effects on the kinetics and specificity of glycation of proteins.