2-4-dinitrophenylhydrazine and pimagedine

Advanced glycation end product (AGE) formation is related to hyperglycemia in diabetes but not in uremia, because plasma AGE levels do not differ between diabetic and nondiabetic hemodialysis patients. The mechanism of this phenomenon remains elusive. Previously, it was suggested that elevation of AGE levels in uremia might result from the accumulation of unknown AGE precursors. The present study evaluates the in vitro generation of pentosidine, a well identified AGE structure. Plasma samples from healthy subjects and nondiabetic hemodialysis patients were incubated under air for several weeks. Pentosidine levels were determined at intervals by HPLC assay. Pentosidine rose to a much larger extent in uremic than in control plasma. Pentosidine yield, i.e., the change in pentosidine level between 0 and 4 wk divided by 28 d, averaged 0.172 nmol/ml per d in uremic versus 0.072 nmol/ml per d in control plasma (P < 0.01). The difference in pentosidine yield between uremic and control plasma was maintained in samples ultrafiltrated through a filter with a 5000-Da cutoff value and fortified with human serum albumin (0.099 versus 0.064 nmol/ml per d; P < 0.05). Pentosidine yield was higher in pre- than in postdialysis plasma samples (0.223 versus 0.153 nmol/ml per d; P < 0.05). These results suggest that a large fraction of the pentosidine precursors accumulated in uremic plasma have a lower than 5000 Da molecular weight. Addition of aminoguanidine and OPB-9195, which inhibit the Maillard reaction, lowered pentosidine yield in both uremic and control plasma. When ultrafiltrated plasma was exposed to 2,4-dinitrophenylhydrazine, the yield of hydrazones, formed by interaction with carbonyl groups, was markedly higher in uremic than in control plasma. These observations strongly suggest that the pentosidine precursors accumulated in uremic plasma are carbonyl compounds. These precursors are unrelated to glucose or ascorbic acid, whose concentration is either normal or lowered in uremic plasma. They are also unrelated to 3-deoxyglucosone, a glucose-derived dicarbonyl compound whose level is raised in uremic plasma: Its addition to normal plasma fails to increase pentosidine yield. This study reports an elevated level of reactive carbonyl compounds ("carbonyl stress") in uremic plasma. Most have a lower than 5000 Da molecular weight and are thus partly removed by hemodialysis. Their effect on pentosidine generation can be inhibited by aminoguanidine or OPB-9195. Carbonyl

Topics: Aged; Aldehydes; Arginine; Carboxylic Acids; Deoxyglucose; Depression, Chemical; Esters; Glycosylation; Guanidines; Humans; Ketones; Lysine; Maillard Reaction; Male; Middle Aged; Molecular Weight; Phenylhydrazines; Renal Dialysis; Thiadiazoles; Thiazolidines; Ultrafiltration; Uremia

Several mechanisms have been postulated for the formation of advanced glycation endproducts (AGEs) from glycated proteins; they all feature protein-bound carbonyl intermediates. Using 2,4-dinitrophenylhydrazine (DNPH), we have detected these intermediates on bovine serum albumin, lysozyme and beta-lactoglobulin after in vitro glycation by glucose or fructose. Carbonyls were formed in parallel with AGE-fluorophores, via oxidative Maillard reactions. Neither Amadori nor Heyns products contributed to the DNPH reaction. Fluorophore and carbonyl yields were much enhanced in lipid-associated proteins, but both groups could also be detected in lipid-free proteins. When pre-glycated proteins were incubated in the absence of free sugar, carbonyl groups were rapidly lost in a first-order reaction, while fluorescence continued to develop beyond the 21 days of incubation. Another unexpected finding was that not all carbonyl groups were blocked by aminoguanidine, although there was complete inhibition of reactions leading to AGE-fluorescence. It is suggested that carbonyls acting as fluorophore precursors react readily with aminoguanidine, while others are resistant to this hydrazine, possibly because they are involved in ring closure. Factors influencing the relative rates of acyclisation and hydrazone formation are discussed, together with possible implications for antiglycation therapy.

Topics: Fluorescence; Fructose; Glucose; Glycation End Products, Advanced; Guanidines; Lactoglobulins; Muramidase; Pentetic Acid; Phenylhydrazines; Serum Albumin, Bovine

Nonreductive modification of proteins with glyceraldehyde forming 2-oxo-3-hydroxypropylated protein is mechanistically analogous to nonenzymic glycation reactions. The latent cross-linking potential of glyceraldehyde as a consequence of the reactivity of the carbonyl function of 2-oxo-3-hydroxypropyl groups of nonreductively modified protein has been now investigated. Reaction of RNase A (0.5 mM) with glyceraldehyde (20 mM) at pH 7.4 and 37 degrees C for 4 h resulted in the intermolecular cross-linking of the protein, with the concomitant development of a yellow chromophore with two new absorption bands having maxima around 305 and 375 nm. The product exhibited a fluorescence that had excitation and emission maxima around 365 and 450 nm, respectively. The presence of NaCNBH3 during the reaction, which selectively reduces the Schiff base adducts of aldotriose to form 2,3-dihydroxypropyl groups on proteins, inhibited both the cross-linking reaction and the development of the absorption and fluorescence characteristics. The hydroxymethyl group of the aldotriose is not an essential moiety since the cross-linking potential of glyceraldehyde is comparable to that of glyceraldehyde 3-phosphate. The formation of cross-links appears to involve the carbonyl function of the keto amines resulting in the formation of Schiff base adducts (ketimine linkages) as the initial event. Consistent with this, incubation of 2-oxo-3-hydroxypropylated RNAse A with [14C]glycine ethyl ester resulted in the incorporation of the reagent into the protein. The cross-linking reaction was inhibited when the reaction of RNase A with glyceraldehyde was carried out in the presence of amino compounds, such as glycine ethyl ester, ethanolamine, glucosamine, and aminoguanidine.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amines; Borohydrides; Cross-Linking Reagents; Glyceraldehyde; Guanidines; Ketones; Oxidation-Reduction; Phenylhydrazines; Proteins; Ribonuclease, Pancreatic; Spectrometry, Fluorescence