Phototransformations of advanced glycation end products in the human eye lens due to ultraviolet A light irradiation.

Previous studies from this laboratory have shown that ultraviolet A (UVA) light can bleach the yellow advanced glycation end products (AGEs) of aged and cataractous human lenses. The AGEs OP-lysine and argpyrimidine are two UVA-absorbing posttranslational modifications that are abundant in the eye lens. The purpose of this study was to outline the changes in these two AGEs due to UVA irradiation. The changes of OP-lysine, OP-phenethylamine (a phenethylamine analogue of OP-lysine), and argpyrimidine due to irradiation with UVA light in the presence or absence of air and ascorbic acid were followed by different spectral methods. Aged human lenses were similarly irradiated in artificial aqueous humor. The amounts of OP-lysine in the irradiated lenses and in the corresponding dark controls were determined by HPLC. Both OP-lysine and argpyrimidine decreased 20% when irradiated with UVA light in the absence of ascorbic acid. Under the same conditions, OP-lysine was bleached 80% in the presence of ascorbic acid during irradiation experiments. In contrast, argpyrimidine UVA light bleaching was not affected by the presence of ascorbic acid. Interestingly the major product of OP-phenethylamine after UVA irradiation in the presence of ascorbic acid was phenethylamine, which indicates that the entire heterocycle of this AGE was cleaved and the initial amino group was restored. Some AGEs in the human eye lens can be transformed by UVA light.

Mass spectrometric monitoring of albumin in uremia.

BACKGROUND: Advanced glycation end products (AGEs) are a novel class of uremic toxins. In plasma, they are present in proteins and also in low molecular mass peptides. AGE-modified peptides are thought to bind and modify plasma proteins. Monitoring of the consequent increase in molecular mass of serum albumin may be used in surveillance of the clinical management of uremia. METHODS: We investigated molecular mass changes of human serum albumin (HSA) glycated by glucose and methylglyoxal in vitro and of subjects with moderate renal impairment, end-stage renal disease (ESRD), ESRD on hemodialysis, and normal healthy controls by matrix-assisted laser desorption ionization mass spectrometry. RESULTS: Fatty acid-free HSA had a molecular mass of 66,446 +/- 114 D. Mean (+/-SD) molecular mass increases were HSA minimally glycated by glucose 399 +/- 88 D (N = 5, P < 0.001), HSA highly glycated by glucose 6780 +/- 122 D (N = 5, P < 0.001), HSA minimally glycated by methylglyoxal 73 +/- 121 D (N = 5, P > 0.05), and HSA without fatty acid removal 535 +/- 90 D (N = 5, P < 0.001). For HSA of human subjects, mean (+/- SD) molecular mass increases were normal healthy controls 243 +/- 97 D (N = 5), moderate renal impairment 350 +/- 83 D (P > 0.05 with respect to controls, N = 5), ESRD 498 +/- 128 (P < 0.02 with respect to controls, N = 3), and ESRD on hemodialysis 438 +/- 85 D (P < 0.02 with respect to controls, N = 5). The mean molecular mass of albumin of all groups was increased significantly with respect to that of fatty acid free albumin (P < 0.001). CONCLUSIONS: Only ESRD was associated with a significant increase in the molecular mass of HSA in vivo. Since this mass increase was very low and much lower than reported for AGE-modified peptides, it may reflect AGE formation on HSA by alpha-oxoaldehydes that accumulate in uremia, rather than modification of albumin by AGE-modified peptides. The molecular mass of HSA in vivo was indicative of a minimal and not a high extent of glycation.

Kinetics and mechanism of the reaction of aminoguanidine with the alpha-oxoaldehydes glyoxal, methylglyoxal, and 3-deoxyglucosone under physiological conditions.

Aminoguanidine (AG), a prototype agent for the preventive therapy of diabetic complications, reacts with the physiological alpha-oxoaldehydes glyoxal, methylglyoxal, and 3-deoxyglucosone (3-DG) to form 3-amino-1,2,4-triazine derivatives (T) and prevent glycation by these agents in vitro and in vivo. The reaction kinetics of these alpha-oxoaldehydes with AG under physiological conditions pH 7.4 and 37 degrees was investigated. The rate of reaction of AG with glyoxal was first order with respect to both reactants; the rate constant k(AG,G) was 0.892 +/- 0.037 M(-1) sec(-1). The kinetics of the reaction of AG with 3-DG were more complex: the rate equation was d[T](o)/dt (initial rate of T formation) = [3-DG](k(AG,3-DG)[AG] + k(3-DG)), where k(AG,3-DG) = (3. 23 +/- 0.25) x 10(-3) M(-1) sec(-1) and k(3-DG) = (1.73 +/- 0.08) x 10(-5) sec(-1). The kinetics of the reaction of AG with methylglyoxal were consistent with the reaction of both unhydrated (MG) and monohydrate (MG-H(2)O) forms. The rate equation was d[T](o)/dt = ¿k(1)k(AG,MG)/(k(-1) + k(AG,MG)[AG]) + k(AG, MG-H(2)O)¿[MG-H(2)O][AG], where the rate constant for the reaction of AG with MG, k(AG,MG), was 178 +/- 15 M(-1) sec(-1) and for the reaction of AG with MG-H(2)O, k(AG,MG-H(2)O), was 0.102 +/- 0.001 M(-1) sec(-1); k(1) and k(-1) are the forward and reverse rate constants for methylglyoxal dehydration MG-H(2)O right harpoon over left harpoon MG. The kinetics of these reactions were not influenced by ionic strength, but the reaction of AG with glyoxal and with methylglyoxal under MG-H(2)O dehydration rate-limited conditions increased with increasing phosphate buffer concentration. Kinetic modelling indicated that the rapid reaction of AG with the MG perturbed the MG/MG-H(2)O equilibrium, and the ratio of the isomeric triazine products varied with initial reactant concentration. AG is kinetically competent to scavenge the alpha-oxoaldehydes studied and decrease related advanced glycated endproduct (AGE) formation in vivo. This effect is limited, however, by the rapid renal elimination of AG. Decreased AGE formation is implicated in the prevention of microvascular complications of diabetes by AG.

Methylglyoxal-modified arginine residues--a signal for receptor-mediated endocytosis and degradation of proteins by monocytic THP-1 cells.

Non-enzymatic glycosylation or glycation of proteins to form advanced glycation endproducts (AGE) has been proposed as a process which provides a signal for the degradation of proteins. Despite this, the AGE which act a recognition factor for receptor-mediated endocytosis and degradation of glycated proteins by monocytes and macrophages has not been identified. Methylglyoxal, a reactive alpha-oxoaldehyde and physiological metabolite, reacted irreversibly with arginine residues in proteins to form Ndelta-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine and Ndelta-(5-methyl-4-imidazolon-2-yl)ornithine residues. Human serum albumin minimally-modified with methylglyoxal (MG(min)-HSA) was bound by cell surface receptors of human monocytic THP-1 cells in vitro at 4 degrees C: the binding constant K(d) value was 377 +/- 35 nM and the number of receptors per cell was 5.9 +/- 0.2 X 10(5) (n = 12). N alpha-Acetyl-Ndelta-(5-hydro-5-methyl-4-imidazolon-2-yl)orni thine displaced MG(min)-HSA from THP-1 cells, suggesting that the Ndelta-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine residue was the receptor recognition factor. At 37 degrees C, MG(min)-HSA was internalised by THP-1 cells and degraded. Similar binding and degradation of human serum albumin modified by glucose-derived AGE was found but only when highly modified. MG(min)-HSA, therefore, is the first example of a protein minimally-modified by AGE-like compounds that binds specifically to monocyte receptors. The irreversible modification of proteins by methylglyoxal is a potent signal for the degradation of proteins by monocytic cells in which the arginine derivative, Ndelta-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine, is the receptor recognition factor. This factor is not present in glucose-modified proteins.

Interaction of glycyl-phenylalanine with carbohydrates as a model of N-terminal glycation of proteins.

Glycation is thought to be a factor in the development of cataract in elderly and diabetic patients. In this report, we describe our initial investigations on the reaction of the dipeptide glycyl-phenylalanine (GF) with different carbonyl compounds as a model of protein glycation. The results obtained demonstrate that the interaction of GF with carbohydrates has features in common with glycation of lens membrane proteins and lens crystallins.

Biological effects of a novel intestinal peptide--inhibiting enterocytogenin on cultured 3T3 mouse fibroblasts and L5178Y mouse lymphoma cells.

The effects of a new intestinal peptide, inhibiting enterocytogenin (IEG) derived from pig intestinal mucosa were studied in vitro on 3T3 mouse fibroblasts and L5178Y mouse lymphoma cell line. IEG caused considerable growth inhibition together with specific morphological changes, necrotic effects as well as formation of monolayers at the highest concentration applied (1000 micrograms/ml). A biologically active fraction (IEG-BAF) derived by further purification of IEG by gel-filtration, proved to possess most of the described activity. The concentrations of IEG and IEG-BAF inhibiting the growth of L5178Y lymphoma cells by 50% (IC50 values) were calculated to be 759 micrograms/ml and 192 micrograms/ml, respectively. IEG-BAF has a molecular mass of 4450 +/- 180 Da and is most probably a peptidylnucleotidate as revealed by spectral analysis.