Identification of Glyoxal and Arabinose as Intermediates in the Autoxidative Modification of Proteins by Glucose

Abstract

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.

In Vitro Kinetic Studies of Formation of Antigenic Advanced Glycation End Products (AGEs)

“…Such fluorescent changes can be produced by dicarbonyl or glycoxidation products that arise from free sugar, from the initial Schiff bases, and from Amadori and other intermediates (50 -52). Carboxymethyllysine (CmL), a notable AGE, can also arise from a variety of glycoxidation intermediates besides the Amadori product (35,36,53). The acid-stable fluorescent AGE pentosidine (54 -56) can be utilized in principle, but the chemical work-up, protein hydrolysis, and high performance liquid chromatography separation required for each time point makes its use inconvenient for large sets of kinetics.…”

“…Instead, aminoguanidine, through its guanidinium functionality, was found to inhibit another AGE formation pathway by scavenging reactive dicarbonyl intermediates (Scheme 1) that arise from glycoxidation during glycation (65)(66)(67)(68). Dicarbonyls can include glyoxal and glycoaldehyde that arise from free sugar or from Schiff bases (via the Namiki pathway) and "glucosones" (deoxydiketoses or deoxyaldoketoses) which can arise from Amadori intermediates (35,36,38,53). Hirsch et al (66) found very rapid irreversible formation of 5-and 6-substituted triazines from reaction of aminoguanidine with model dicarbonyls, while Chen and Cerami (68) have reported that reaction of a model Amadori compound with aminoguanidine only leads to formation of triazine and bis-hydrazone products of dicarbonyl fragments derived from the Amadori compound.…”

Renal clearance of glycolaldehyde- and methylglyoxal-modified proteins in mice is mediated by mesangial cells through a class A scavenger receptor (SR-A)

“…AGE proteins are characterised physicochemically by their fluorescent, brown color and intramolecular and intermolecular crosslinking [1], and biologically by their recognition by specific AGE receptors [2]. Recent studies reported that several aldehydes such as glycolaldehyde (GA), glyoxal, methylglyoxal (MG), and 3-deoxyglucosone are generated during the Maillard reaction from glucose, a Schiff base, or Amadori products [3,4]. A much stronger chemical reactivity than that of glucose indicates the important role of these aldehydes in the in vivo generation of AGE structures [5][6][7].…”

Unexpected elevation of pentosidine formation in collagen incubated with glucose by low concentrations of the AGE-inhibitor aminoguanidine

“…Aminoguanidine prevents pentosidine formation at high concentrations above 18 mmol/l through two possible mechanisms: dicarbonyl trapping and chelating activity [4,7]. At concentrations lower than 18 mmol/l, the unexpected increase in pentosidine formation may result from a reaction of aminoguanidine with (i) auto-oxidation products of glucose (arabinose or glyoxal [10]), and (ii) with two accessible lysines and/or hydroxylysines at a suitable distance to form pentosidine. In the presence of oxygen and a few free metal ions, this could take place as long as aminoguanidine chelating activity is not complete.…”