Aminoguanidine and the effects of modified LDL on cultured retinal capillary cells.

PURPOSE: Compared with normal low density lipoprotein (N-LDL), LDL minimally modified in vitro by glycation, minimal oxidation, or glycoxidation (G-, MO-, GO-LDL) decreases survival of cultured retinal capillary endothelial cells and pericytes. Similar modifications occurring in vivo in diabetes may contribute to retinopathy. The goal of this study was to determine whether low concentrations of aminoguanidine might prevent cytotoxic modification of LDL and/or protect retinal capillary cells from previously modified LDL. METHODS: Minimal in vitro modification of LDL (3 days, 37 degrees C) was achieved with glucose (0, 50 mM), under antioxidant conditions (for N-LDL, G-LDL), or under mild oxidant conditions (for MO-, GO-LDL) in the presence/absence of aminoguanidine (0, 1, 10, 100 microM). Glucose and aminoguanidine were then removed by dialysis. Confluent bovine retinal capillary endothelial cells (n = 13) and pericytes (n = 14) were exposed to LDL (100 mg/l) for 3 days, with and without aminoguanidine (100 microM) in media. Cell counts were determined by hemocytometer. RESULTS: A decrease in cell counts after exposure to modified compared with N-LDL was confirmed (P < 0.001) but was significantly mitigated if LDL had been modified in the presence of aminoguanidine (P < 0.001). Aminoguanidine was as effective at 1 microM as at the higher concentrations. Aminoguanidine (100 microM) present in culture media conferred no additional protection, and showed slight evidence of toxicity. Aminoguanidine present during LDL modification had no effect on measured glycation or oxidation products, or on LDL oxidizability. CONCLUSIONS: Very low concentrations of aminoguanidine mitigate toxicity of LDL exposed to stresses that simulate the diabetic environment. This action may contribute to the beneficial effects of aminoguanidine observed in experimental diabetic retinopathy.

New biomarkers of Maillard reaction damage to proteins.

The amount of advanced glycation end-products (AGE) in tissue proteins increases in diabetes mellitus, and the concentration of a subclass of AGEs, known as glycoxidation products, also increases with chronological age in proteins. The rate of accumulation of glycoxidation products is accelerated in diabetes and age-adjusted concentrations of two glycoxidation products, N epsilon-(carboxymethyl)lysine (CML) and pentosidine, correlate with the severity of complication in diabetic patients. Although AGEs and glycoxidation products are implicated in the development of diabetic complications, these compounds are present at only trace concentrations in tissue proteins and account for only a fraction of the chemical modifications in AGE proteins prepared in vitro. The future of the AGE hypothesis depends on the chemical characterization of a significant fraction of the total AGEs in tissue proteins, a quantitative assessment of their effects on protein structure and function, and an assessment of their role as mediators of biological responses. In this manuscript we describe recent work leading to characterization of new AGEs and glycoxidation products. These compounds include: (1) the imidazolone adduct formed by reaction of 3-deoxyglucosone with arginine residues in protein; (2) N epsilon-(carboxyethyl)lysine, an analogue of CML formed on reaction of methylglyoxal with lysine; (3) glyoxal-lysine dimer; and (4) methyl-glyoxal-lysine dimer, which are imidazolium crosslinks formed by reaction of glyoxal or methylglyoxal with lysine residues in protein. The presence of 3-deoxyglucosone, methylglyoxal and glyoxal in vivo and the formation of the above AGEs in model carbonyl-amine reaction systems suggests that these AGEs are also formed in vivo and contribute to tissue damage resulting from the Maillard reaction.

L-Arginine inhibits in vitro nonenzymatic glycation and advanced glycosylated end product formation of human serum albumin.

L-Arginine (Arg) has a structure similar to that of aminoguanidine (AG) and may inhibit glycation and advanced glycosylated end product (AGE) formation. Human serum albumin (HSA) (100mg/ml) was incubated for 2 weeks with glucose (200mM) at 37°C or with glucose and equimolar concentrations of Arg, N-α-acetyl Arg, or AG with or without 25mM diethylenetriaminepentaacetic acid (DTPA). In the absence of DTPA, electrospray ionization mass spectrometry showed a 70% reduction of covalently bound glucose in the presence of Arg and a 30% reduction with AG. Digestibility by trypsin of HSA incubated with glucose and Arg was similar to that of HSA incubated alone. This suggests less covalent modification of HSA in the presence of Arg as compared with the absence of Arg. When incubations contained DTPA, autoradiography showed less(14)C labeling of HSA subunits in the presence of Arg and AG. When theα-amino group of Arg was blocked with an acetyl group, labeling was similar to that of HSA incubated with glucose, suggesting involvement of theα-amino group in the inhibition. Fluorescence of HSA at ex370 and em440 was reduced with Arg, but AG was more effective than Arg. These results suggest that Arg, like AG, can inhibit glycation and AGE formation.

N epsilon-(carboxymethyl)lysine is a dominant advanced glycation end product (AGE) antigen in tissue proteins.

Advanced glycation end products (AGEs) and glycoxidation products are formed during Maillard or browning reactions between sugars and proteins and are implicated in the pathophysiology of aging and the complications of diabetes. To determine the structure of AGEs, antibodies were prepared to protein browned by incubation with glucose and used in ELISA assays to measure AGEs formed in model reactions between bovine serum albumin (BSA) or N alpha-acetyllysine and glucose, fructose, or glyoxal. AGEs were formed from glucose and fructose only under oxidative conditions, but from glyoxal under both oxidative and antioxidative conditions. Gel permeation chromatographic analysis indicated that a similar AGE was formed in reactions of N alpha-acetyllysine with glucose, fructose, and glyoxal and that this AGE co-eluted with authentic N alpha-acetyl-N epsilon-(carboxymethyl)lysine. Amino acid analysis of AGE proteins revealed a significant content of N epsilon-(carboxymethyl)lysine (CML). In ELISA assays using polyclonal antibodies against AGE proteins, CML-BSA (approximately 25 mol of CML/mol of BSA), prepared by chemical modification of BSA, was a potent inhibitor of the recognition of AGE proteins and of AGEs in human lens proteins. We conclude that AGEs are largely glycoxidation products and that CML is a major AGE recognized in tissue proteins by polyclonal antibodies to AGE proteins.

Mechanism of autoxidative glycosylation: identification of glyoxal and arabinose as intermediates in the autoxidative modification of proteins by glucose.

Glycation and oxidation reactions contribute to protein modification in aging and diabetes. Formation of dicarbonyl sugars during autoxidation of glucose is the hypothetical first step in the autoxidative glycosylation and subsequent browning of proteins by glucose [Wolff, S. P., & Dean, R. T. (1987) Biochem. J. 245, 243-250]. In order to identify the dicarbonyl sugar(s) formed during autoxidation of glucose under physiological conditions, glucose was incubated in phosphate buffer (pH 7.4) at 37 degrees C under air (oxidative conditions) or nitrogen with transition metal chelators (antioxidative conditions). Dicarbonyl compounds were analyzed spectrophotometrically and by HPLC after reaction with Girard-T reagent. Carbohydrates were analyzed by gas chromatography-mass spectrometry. Both dicarbonyl sugar and arabinose concentrations increased with time and glucose concentration in incubations conducted under oxidative conditions; only trace amounts of these products were detected in glucose incubated under antioxidative conditions. HPLC analysis of adducts formed with Girard-T reagent indicated that glyoxal was the only alpha-dicarbonyl sugar formed on autoxidation of glucose. Glyoxal and arabinose accounted for > or = 50% of the glucose lost during a 21 day incubation. Neither glucosone nor its degradation product, ribulose, was detectable. Reaction of glyoxal with RNase yielded the glycoxidation product, N epsilon-(carboxymethyl)lysine, while arabinose is a source of pentosidine. Our results implicate glyoxal and arabinose as intermediates in the browning and crosslinking of proteins by glucose under oxidative conditions. They also provide a mechanism by which antioxidants and dicarbonyl trapping reagents, such as aminoguanidine, limit glycoxidation reactions and support further evaluation of these types of compounds for inhibition of chemical modification and crosslinking of proteins during aging and diabetes.

3-Deoxyfructose concentrations are increased in human plasma and urine in diabetes.

3-Deoxyglucosone (3-DG) is a reactive dicarbonyl sugar thought to be a key intermediate in the nonenzymatic polymerization and browning of proteins by glucose. 3-DG may be formed in vivo from fructose, fructose 3-phosphate, or Amadori adducts to protein, such as N epsilon-fructoselysine (FL), all of which are known to be elevated in body fluids or tissues in diabetes. Modification of proteins by 3-DG formed in vivo is thought to be limited by enzymatic reduction of 3-DG to less reactive species, such as 3-deoxyfructose (3-DF). In this study, we have measured 3-DF, as a metabolic fingerprint of 3-DG, in plasma and urine from a group of diabetic patients and control subjects. Plasma and urinary 3-DF concentrations were significantly increased in the diabetic compared with the control population (0.853 +/- 0.189 vs. 0.494 +/- 0.072 microM, P < 0.001, and 69.9 +/- 44.2 vs. 38.7 +/- 16.1 nmol/mg creatinine, P < 0.001, respectively). Plasma and urinary 3-DF concentrations correlated strongly with one another, with HbA1c (P < 0.005 in all cases), and with urinary FL (P < 0.02 and P = 0.005, respectively). The overall increase in 3-DF concentrations in plasma and urine in diabetes and their correlation with other indexes of glycemic control suggest that increased amounts of 3-DG are formed in the body during hyperglycemia in diabetes and then metabolized to 3-DF. These observations are consistent with a role for increased formation of the dicarbonyl sugar 3-DG in the accelerated browning of tissue proteins in diabetes.

Glycation, glycoxidation, and cross-linking of collagen by glucose. Kinetics, mechanisms, and inhibition of late stages of the Maillard reaction.

The Maillard or browning reaction between sugar and protein contributes to the increased chemical modification and cross-linking of long-lived tissue proteins in diabetes. To evaluate the role of glycation and oxidation in these reactions, we have studied the effects of oxidative and antioxidative conditions and various types of inhibitors on the reaction of glucose with rat tail tendon collagen in phosphate buffer at physiological pH and temperature. The chemical modifications of collagen that were measured included fructoselysine, the glycoxidation products N epsilon-(carboxymethyl)lysine and pentosidine and fluorescence. Collagen cross-linking was evaluated by analysis of cyanogen bromide peptides using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by changes in collagen solubilization on treatment with pepsin or sodium dodecylsulfate. Although glycation was unaffected, formation of glycoxidation products and cross-linking of collagen were inhibited by antioxidative conditions. The kinetics of formation of glycoxidation products proceeded with a short lag phase and were independent of the amount of Amadori adduct on the protein, suggesting that autoxidative degradation of glucose was a major contributor to glycoxidation and cross-linking reactions. Chelators, sulfhydryl compounds, antioxidants, and aminoguanidine also inhibited formation of glycoxidation products, generation of fluorescence, and cross-linking of collagen without significant effect on the extent of glycation of the protein. We conclude that autoxidation of glucose or Amadori compounds on protein plays a major role in the formation of glycoxidation products and cross-liking of collagen by glucose in vitro and that chelators, sulfhydryl compounds, antioxidants, and aminoguanidine act as uncouplers of glycation from subsequent glycoxidation and cross-linking reactions.

Role of oxygen in cross-linking and chemical modification of collagen by glucose.

The role of oxygen in chemical modification and cross-linking of rat tail collagen by glucose was studied at physiological pH and temperature in vitro. Cross-linking of collagen under air depended on glucose concentration, but was inhibited under antioxidative conditions (nitrogen atmosphere with transition metal chelators). The cross-linking reaction under air depended on phosphate buffer concentration, but this effect was eliminated by addition of chelators, identifying trace metal ions in the buffer as catalysts of oxidative cross-linking reaction. Antioxidative conditions had no effect on glycation, that is, formation of fructose lysine, but inhibited formation of the glycoxidation products N epsilon-(carboxymethyl)lysine and pentosidine as well as the development of fluorescence in glycated collagen. Glycation itself decreased during continued incubation of the collagen without glucose; however, cross-linking and concentrations of glycoxidation products and fluorescence in collagen were not reversible under either oxidative or antioxidative conditions. These observations are consistent with recent studies in vivo on the reversibility of collagen glycation, the irreversibility of formation of glycoxidation products and fluorescence, and the strong correlations between glycoxidation products and fluorescence in collagen (1). These results indicate that oxidation reactions play a critical role in the extended chemical modification and cross-linking of collagen by glucose and suggest that measurement of glycoxidation products should be useful for assessing cumulative chemical modification of collagen by glucose in vivo.

Detection of 3-deoxyfructose and 3-deoxyglucosone in human urine and plasma: evidence for intermediate stages of the Maillard reaction in vivo.

3-Deoxyglucose (3-deoxy-D-erythro-hexos-2-ulose) (3-DG) is a reactive dicarbonyl intermediate involved in the polymerization and browning of proteins by glucose in vitro. Damage to protein by formation of 3-DG in vivo is thought to be limited by enzymes which convert 3-DG to less reactive species, such as 3-deoxyfructose (3-DF). We have developed a sensitive and specific assay for measuring 3-DG and 3-DF in human urine and plasma. In this assay, 3-DG and 3-DF are reduced to 3-deoxy-hexitols (3-DH), using either NaBH4 or NaBD4, and then analyzed by selected ion monitoring gas chromatography-mass spectrometry. Based on comparative analysis of samples reduced with NaBD4 versus NaBD4, 3-DH in urine was derived exclusively (greater than 99%) from 3-DF, while 3-DG accounted for approximately 15% of 3-DH in plasma. The concentrations of 3-DH in fasting human urine and plasma were 5.3 +/- 1.5 micrograms/mg creatinine (n = 18) and 7.2 +/- 1.7 micrograms/dl (n = 18), respectively. The concentrations of 3-DG and 3-DF in plasma (n = 7) were 1.0 +/- 0.2 and 6.7 +/- 1.6 micrograms/dl, respectively. These results suggest that several milligrams of 3-DG are formed in the body per day and detoxified by reduction to 3-DF and support the role of 3-DG as an intermediate in the browning of protein via the Maillard reaction in vivo.

Effect of diabetes and aging on carboxymethyllysine levels in human urine.

Carboxymethyllysine (CML) has been identified as a modified amino acid that accumulates with age in human lens proteins and collagen. CML may be formed by oxidation of fructoselysine (FL), the Amadori adduct formed on nonenzymatic glycosylation of lysine residues in protein, or by reaction of ascorbate with protein under autoxidizing conditions. We proposed that measurements of tissue and urinary CML may be useful as indices of oxidative stress or damage to proteins in vivo. To determine the extent to which oxidation of nonenzymatically glycosylated proteins contributes to urinary CML, we measured the urinary concentrations of FL and CML in diabetic (n = 26) and control (n = 28) patients. The urinary concentration of FL correlated strongly with HbA1 measurements and was significantly higher in diabetic compared with control samples (9.2 +/- 6.5 and 4.0 +/- 2.8 micrograms/mg creatinine, respectively; P less than 0.0001). There was also a strong correlation between the concentrations of CML and FL in both diabetic and control urine (r = 0.67, P less than 0.0001) but only a weakly significant increase in the CML concentration in diabetic compared with control urine (1.2 +/- 0.5 and 1.0 +/- 0.3 micrograms/mg creatinine, respectively; P = 0.05). The molar ratio of CML to FL was significantly lower in diabetic compared with control patients (0.25 +/- 0.12 and 0.43 +/- 0.16, respectively; P less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)