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To the Editor: In diabetes, protein modification by hyperglycaemia leads to Amadori products like HbA1c, which in turn can be oxidised to form advanced glycation end products (AGE), and particularly pentosidine [1]. Pentosidine crosslinks peptidic chains, alters physicochemical properties of proteins and contributes to the development of diabetic complications. Skin collagen pentosidine levels, adjusted for age, have been shown to correlate with the severity of complications in Type 1 diabetic patients [2]. More recently, when adjusted for age and diabetes duration, they were significantly associated with nephropathy and neuropathy [3]. Aminoguanidine (pimagedine) inhibits advanced glycation by preventing conversion of Amadori products into AGE [4]. It was tested successfully in experimental models of diabetes, reducing proteinuria and retinopathy, and improving nerve conduction velocity [5]. While screening various inhibitors of AGE formation in collagen incubated with glucose, we used aminoguanidine as reference. We observed that, at aminoguanidine concentrations higher than 18 mmol/l, aminoguanidine reduced pentosidine formation as expected, but increased it significantly at lower concentrations. This was confirmed in different experiments. Insoluble collagen from bovine Achilles tendon (9 mg/ml, Sigma, St Louis, Mo., USA) was incubated in 200 mmol/l sodium phosphate buffer, pH=7.4, with or without 250 mmol/l glucose, in the absence or presence of aminoguanidine hydrochloride (obtained from equimolar amounts of aminoguanidine bicarbonate [Sigma] and HCl in 400 mmol/l phosphate buffer) at concentrations ranging from 1.6 mmol/l to 89 mmol/l. This was done for 28 days at 37 °C with toluene (10 μl/1 ml). Each condition was repeated in quadruplicates. After incubation, collagen was washed, lyophilised and submitted to acid hydrolysis under vacuum. Pentosidine was measured by HPLC [1], hydroxyproline was determined colorimetrically [6] and the pentosidine : hydroxyproline molar ratio was calculated. Specific advanced glycation by pentosidine (SAGP) was evaluated as follows: (sample pentosidine:hydroxyproline – pentosidine:hydroxyproline of control without glucose) × 100/ (pentosidine:hydroxyproline of collagen + glucose – pentosidine:hydroxyproline of collagen without glucose). Results were presented as means ± SEM. Statistical comparisons were performed by analysis of variance. Four experiments were conducted: a preliminary experiment (y) and three further experiments a, b, c. In experiment c, EDTA (Sigma) and diethylene triamine pentaacetic acid (DTPA) (Acros Organics, Geel, Belgium) were tested at 10 and 36.8 mmol/l, to compare the activity of chelators with that of aminoguanidine, which is reported to possess chelating activity [7]. In experiment y, in the presence of glucose alone, the pentosidine hydroxyproline ratio was 31.6±1.7 pmol/μmol; 40 mmol/l aminoguanidine reduced SAGP to 24±4% of this collagen + glucose control (p<0.001), but 8 mmol/l aminoguanidine unexpectedly increased SAGP to 172±5% (p<0.001). This paradoxical observation was confirmed in experiments a and b, using extended aminoguanidine concentration scales. Results are shown in Figure 1. The maximum paradoxical increase in SAGP was observed with 8 mmol/l aminoguanidine. Increases were found at concentrations from 1.6 mmol/l to 18 mmol/l. However, at 40 mmol/l and 89 mmol/l aminoguanidine, the inhibitory effect of aminoguanidine was confirmed (33±3%, p<0.001, and 31±5%, p<0.001, at 40 mmol/l; 3±1%, p<0.001, and 2±1%, p<0.001 at 89 mmol/l; in experiments a and b respectively). In experiment c, we found a significant reduction of SAGP in the presence of EDTA (34±12%, p<0.001, and 25±3%, p<0.001, at 10 mmol/l and 36.8 mmol/l respectively) and a marked fall in the presence of DTPA (5±1%, p<0.001, and 6±3%, p<0.001). We presented a preliminary abstract of our observations at the International Symposium on Maillard Reaction in Kumamoto [8]. Since then, paradoxical increases in pentosidine formation after ribose glycation of bovine serum albumin have been observed using a novel pyridoxamine derivative and various AGE inhibitors [9]. Formation of AGE from glucose, but not from pentose, needs oxygen and trace metal ions [7]. Under our conditions of glycation by glucose, pentosidine is formed in the presence of air and trace amounts of metal ions. As DTPA traps these, pentosidine formation is almost completely inhibited. 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. Indeed its apparent binding constant for copper is approximately 2 mmol/l [7]. Increased pentosidine formation from glucose in albumin, in the presence of metal ions and 5 mmol/l aminoguanidine (+21%), was recently reported [9], with pentosidine formation inhibited by 25 mmol/l aminoguanidine. Our model of AGE formation in high glucose appears to mimic in vivo conditions in the extracellular matrix of diabetic patients and in some cells with high glucose, such as endothelial cells, exaggerating however the glucose level. Interestingly, aminoguanidine was tested in clinical trials, significantly decreasing circulating LDL levels by 28% after 1 Diabetologia (2004) 47:959–961

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