2-4-dinitrophenylhydrazine and Uremia

Oxidative stress has been defined as a loss of balance between free radical production and the antioxidant systems. There have been many reports of increased production of oxidants and decreased levels of antioxidants in chronic renal failure (CRF) patients. An increase in oxidative stress may contribute to the development of oxidative protein damage in CRF. Our aim was to reveal oxidative modifications of plasma proteins by measuring 2,4-dinitrophenylhydrazine reactive carbonyl derivates (PCO), protein thiol (P-SH) and reduced glutathione (GSH) in predialytic uraemic and haemodialysed (HD) patients before and after dialysis. We included 20 predialytic uraemic patients, 20 HD patients and 20 healthy volunteers in our study. PCO concentration in predialytic uraemic patients increased compared with the concentration of the control group and this increase was more profound in HD patients. P-SH concentrations were significantly decreased in haemodialytic patients compared with those of controls. GSH level was higher in HD patients (both before and after dialysis). Increased PCO and decreased P-SH concentrations in all patient groups in comparison to the control subjects indicate increased protein oxidation. Our data in ESRD patients propose plasma protein carbonyl derivates and thiol concentrations as novel specific markers for oxidative protein damage.

Topics: Aged; Blood Proteins; Case-Control Studies; Female; Glutathione; Humans; Kidney Failure, Chronic; Male; Middle Aged; Oxidation-Reduction; Oxidative Stress; Phenylhydrazines; Renal Dialysis; Sulfhydryl Compounds; Uremia

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