Nikolaos Karachalias

Quantitative screening of advanced glycation endproducts in cellular and extracellular proteins by tandem mass spectrometry.

Glycation of proteins forms fructosamines and advanced glycation endproducts. Glycation adducts may be risk markers and risk factors of disease development. We measured the concentrations of the early glycation adduct fructosyl-lysine and 12 advanced glycation endproducts by liquid chromatography with tandem mass spectrometric detection. Underivatized analytes were detected free in physiological fluids and in enzymic hydrolysates of cellular and extracellular proteins. Hydroimidazolones were the most important glycation biomarkers quantitatively; monolysyl adducts (N(epsilon)-carboxymethyl-lysine and N(epsilon)-1-carboxyethyl-lysine) were found in moderate amounts, and bis(lysyl)imidazolium cross-links and pentosidine in lowest amounts. Quantitative screening showed high levels of advanced glycation endproducts in cellular protein and moderate levels in protein of blood plasma. Glycation adduct accumulation in tissues depended on the particular adduct and tissue type. Low levels of free advanced glycation endproducts were found in blood plasma and levels were 10-100-fold higher in urine. Advanced glycation endproduct residues were increased in blood plasma and at sites of vascular complications development in experimental diabetes; renal glomeruli, retina and peripheral nerve. In clinical uraemia, the concentrations of plasma protein advanced glycation endproduct residues increased 1-7-fold and free adduct concentrations increased up to 50-fold. Comprehensive screening of glycation adducts revealed the relative and quantitative importance of alpha-oxoaldehyde-derived advanced glycation endproducts in physiological modification of proteins-particularly hydroimidazolones, the efficient renal clearance of free adducts, and the marked increases of glycation adducts in diabetes and uraemia-particularly free advanced glycation endproducts in uraemia. Increased levels of these advanced glycation endproducts were associated with vascular complications in diabetes and uraemia.

High-dose thiamine therapy counters dyslipidemia and advanced glycation of plasma protein in streptozotocin-induced diabetic rats.

Abstract: The streptozotocin‐induced (STZ) diabetic rat experimental model of diabetes on insulin maintenance therapy exhibits dyslipidemia, mild thiamine deficiency, and increased plasma protein advanced glycation end products (AGEs). The reversal of thiamine deficiency by high‐dose thiamine and S‐benzoylthiamine monophosphate (benfotiamine) prevented the development of incipient nephropathy. Recently, we reported that high‐dose thiamine (but not benfotiamine) countered diabetic dyslipidemia. To understand further the differences between the effects of thiamine and benfotiamine therapy, we quantified the levels of the AGEs in plasma protein. We found hydroimidazolone AGE residues derived from glyoxal and methylglyoxal, G‐H1 and MG‐H1, were increased 115% and 68% in STZ diabetic rats, with respect to normal controls, and were normalized by both thiamine and benfotiamine; whereas N‐carboxymethyl‐lysine (CML) and N‐carboxyethyl‐lysine (CEL) residues were increased 74% and 118% in STZ diabetic rats and were normalized by thiamine only. The lack of effect of benfotiamine on plasma CML and CEL residue concentrations suggests there may be important precursors of plasma protein CML and CEL residues other than glyoxal and methylglyoxal. These are probably lipid‐derived aldehydes.

Estimation of α-oxoaldehydes formed from the degradation of glycolytic intermediates and glucose fragmentation in blood plasma of human subjects with uraemia

Abstract The concentrations of α-oxoaldehyde glycating agents in the blood plasma of human subjects with uraemia were investigated. The concentrations of glyoxal, methylglyoxal and ribosone were increased markedly with impairment of renal function.

Reaction kinetics of the scavenging of α-oxoaldehydes by aminoguanidine under physiological conditions

Abstract The rate of reaction of aminoguanidine with α-oxoaldehydes derived from glucose fragmentation and glycolytic intermediates was investigated. Aminoguanidine reacted most rapidly with methylglyoxal, glyoxal and hydroxypyruvaldehyde.