Detection of nitrotyrosine in the diabetic plasma: evidence of oxidative stress (original) (raw)
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Biomedicine, 2022
Introduction and Aim: 3-Nitrotyrosine (NT) has been recognized as a marker of oxidative stress in diabetes mellitus. NT has also been studied in diverse metabolic conditions. The aim of our study was oriented towards the role of NT as a predictor of oxidative stress mediated impending nephropathy in diabetes mellitus and that with reference to albuminuria. Materials and Methods: A total of 150 type 2 diabetics in the age group 35-50 years were enrolled as three groups, comprising 50 each, based on albuminuria. 50 healthy age and gender matched subjects constituted the control group. Serum NT and Insulin were assessed by ELISA. HbA1c was quantitated by immunoturbidimetric method and microalbumin was assessed by turbilatex method. Routine biochemistry was enabled through ERBA EM-200 fully automated analyzer. Stringent quality control was affected. The study was begun following approval accorded by the competent committees. Results: NT levels were positively correlated with albumin-creatinine ratio and insulin resistance. NT could be used as a predictor of impending vascular complications in diabetic nephropathy. Conclusion: NT levels could act as a predictor of oxidative stress mediated diabetic nephropathy in the light of albuminuria.
Role of Hyperglycemia in Nitrotyrosine Postprandial Generation
2016
OBJECTIVE — Recently, much attention has been paid to the possibility that postprandial hyperglycemia may be a cardiovascular risk factor in diabetes. Oxidative stress has been involved in the pathogenesis of diabetic complications, and increased plasma levels of nitrotyrosine, a product of peroxynitrite action, have been found in the plasma of diabetic subjects. The aim of the present study was to evaluate whether postprandial hyperglycemia is accompanied by ni-trotyrosine generation and, if so, to explore a possible direct role of hyperglycemia in such a phenomenon. RESEARCH DESIGN AND METHODS — A total of 23 type 2 diabetic patients and 15 matched normal healthy subjects were recruited for this study. Two different tests were per-formed in diabetic patients: a standard meal preceded by regular insulin (0.15 units/kg body wt) or insulin aspart (0.15 units/kg body wt) to achieve different levels of postprandial hyperglyce-mia. The meal test was also performed in healthy control sub...
Antioxidants
Diabetes mellitus (DM) is a strong risk factor for the development of cardiovascular diseases (CVDs), which are the most important cause of morbidity and mortality in the population of patients living with DM. DM is associated with lipid metabolism disorders characterized by a decrease in the high-density lipoprotein blood concentration, an increase in the triglyceride blood concentration, and the presence of modified lipoproteins not routinely measured in clinical practice. Nitrated lipoproteins are produced by the nitration of the tyrosyl residues of apolipoproteins by myeloperoxidase. There is some evidence from the research conducted showing that nitrated lipoproteins may play a role in the development of cardiovascular dysfunction, but this issue requires further investigation. It was found that the nitration of HDL particles was associated with a decrease in caspase-3 and paraoxonase-1 activity, as well as a decrease in the activity of cholesterol transport via ABCA1, which re...
IUBMB Life, 2007
In the systemic circulation, LDL occurs in the form of a weakly nitrated LDL-albumin complex (LAC). The question here is whether LAC (or HDL) is able to denitrate the albumin-bound 3-NO 2 -tyrosine (3NT). Nitrated albumin was incubated in the presence of lipoprotein fraction (LPF) to be tested, with or without Ca 21 . After precipitation and centrifugation, supernatants (SNs) and protein pellets (PP) were collected. HCl proteolysis was carried out with deuterated 3NT as an internal standard, and amino acids were derivatized for GC-MS analysis, whereas SNs were used for NO 2 2 /NO 3 2 -fluorimetric assays. A loss of 3NT, higher with albumin-low LDL than with albumin-rich LDL or HDL, was found in PP only in the presence of Ca 21 . c-Tocopherol loading of LPF inhibited 3NT loss. 3NT loss was found for the first time to be stoichiometrically equivalent to NO 3 2 , proving that the 3NT loss must be ascribed to a 3NT-denitrating nitratase activity. 3NT loss and NO 3 2 production that clearly cannot be attributed to PON-1 were impaired by D-penicillamine and phenylacetate, inhibitor, and substrate of PON-1, respectively, leading to speculate on the active site. Finally, nitratase activity and albumin contribute to beneficially convert peroxynitrite (ONOO 2 ) into nonbioactive NO 3 2 . But, in inflammatory conditions, xanthine oxidoreductase is expressed leading to detrimentally reduce O 2 and NO 3 2 into O 2 2 and NO that may interact, reconstituting the ONOO 2 pool. The real consequence of nitratase activity and the physiological significance of nitration/denitration processes remain to be explored.
Diabetes, 2000
The role of nitric oxide (NO) and free radicals in the development of microvascular disease in type 1 diabetes remains unclear. We have measured NO and isoprostane (a stable marker of in vivo lipid peroxidation) production in 13 type 1 diabetic subjects with normal urinary albumin excretion and 13 healthy volunteers. Whole-body NO synthesis was quantified by measuring the urinary excretion of 15 N-nitrate after the intravenous administration of L-[ 15 N] 2 -arginine. The urinary excretion of the major urinary metabolite of 15-F 2t -isoprostane (8-iso-prostaglandin-F 2␣ ), 2,3-dinor-5,6-dihydro-F 2t -IsoP, was quantified as a marker of in vivo lipid peroxidation. Whole-body NO synthesis was significantly higher in diabetic subjects compared with control subjects (342 vs. 216 nmol 15 N-nitrate/mmol creatinine [95% CI of the difference 45-207], P = 0.005). This increase was not explained by a difference in renal function between the 2 groups. There was no difference in 2,3-dinor-5,6-dihydro-F 2t -IsoP excretion between diabetic subjects and control subjects (44.8 ± 7.8 vs. 41.4 ± 10.0 ng/mmol creatinine, mean ± 95% CI). However, there was an inverse correlation between NO synthesis and free radical activity in subjects with diabetes (r = -0.62, P = 0.012) that was not observed in control subjects (r = 0.37, P = 0.107). We conclude that whole-body NO synthesis is higher in type 1 diabetic subjects with normal urinary albumin excretion than in control subjects. The inverse correlation between isoprostane production and NO synthesis in diabetic subjects is consistent with the hypothesis that NO is being inactivated by reactive oxygen species. NMMA, N G -monomethyl-L-arginine; MANOVA, multivariate analysis of variance; NO, nitric oxide; O 2 -, superoxide; ONOO -, peroxynitrite; UAER, urinary albumin excretion rate.
Hepatic nitrosative stress in experimental diabetes
Journal of Diabetes and its Complications, 2012
The effects of the inhibition of nitrosative stress by aminoguanidine in an experimental model of diabetes mellitus (DM) were investigated. Methods: Twenty-one male Wistar rats were divided into three groups: control (CO), diabetic (DM), and diabetic treated with aminoguanidine (DM + AG). Aminoguanidine (aminoguanidine hemisulfate salt, Sigma Chemical Co., St. Louis, MO, USA) was used at a dose of 50 mg/kg (i.p.) during the last 30 days of the experiment. The expression levels of liver lipoperoxidation (TBARS-nmol/mg protein), inducible oxide nitric synthase (iNOS), nitrotyrosine and the NFκB nuclear transcription factor p65 were examined using western blot analysis. Results: The DM group demonstrated an increase in lipoperoxidation and in the expression of iNOS, nitrotyrosine and p65. Aminoguanidine reduced hepatic lipid peroxidation and protein expression levels of iNOS, nitrotyrosine and p65. Conclusion: Aminoguanidine treatment reduces liver oxidative and nitrosative stress in diabetic animals. In addition, aminoguanidine reduced the expression of p65 in the liver.
Free Radical Biology and Medicine, 2018
The generation of 3-nitrotyrosine, within proteins, is a post-translational modification resulting from oxidative or nitrative stress. It has been suggested that this modification could be used as a biomarker for inflammatory diseases. Despite the superiority of mass spectrometry-based determinations of nitrotyrosine, in a high-throughput clinical setting the measurement of nitrotyrosine by an enzyme-linked immunosorbent assay (ELISA) is likely to be more cost-effective. ELISAs offer an alternative means to detect nitrotyrosine, but many commercially available ELISAs are insufficiently sensitive to detect nitrotyrosine in healthy human serum. Here, we report the development, validation and clinical application of a novel electrochemiluminescence-based ELISA for nitrotyrosine which provides superior sensitivity (e.g. a 50-fold increase in sensitivity compared with one of the tested commercial colorimetric ELISAs). This nitrotyrosine ELISA has the following characteristics: a lower limit of quantitation of 0.04 nM nitrated albumin equivalents; intra-and inter-assay coefficients of variation of 6.5% and 11.3%, respectively; a mean recovery of 106 ± 3% and a mean linearity of 0.998 ± 0.001. Far higher nitration levels were measured in normal human blood cell populations when compared to plasma. Mass spectrometry was used to validate the new ELISA method. The analysis of the same set of chemically modified albumin samples using the ELISA method and mass spectrometry showed good agreement for the relative levels of nitration present in each sample. The assay was applied to serum samples from patients undergoing elective surgery which induces the human inflammatory response. 3 Matched samples were collected before and one day after surgery. An increase in nitration was detected following surgery (median (IQR): 0.59 (0.00-1.34) and 0.97 (0.00-1.70) nitrotyrosine (fmol of nitrated albumin equivalents/mg protein) for pre-and postsurgery respectively. The reported assay is suitable for nitrotyrosine determination in patient serum samples, and may also be applicable as a means to determine oxidative stress in primary and cultured cell populations.
Acta diabetologica, 2009
The aim of this study was to evaluate the activity of semicarbazide-sensitive amine oxidase (SSAO) and the total nitrite and nitrate (NO( x )) concentrations in serum from type 2 diabetic patients and control subjects in order to evaluate if they could be used as novel diabetic markers. We studied 38 type 2 diabetic patients and 35 control subjects. Serum samples from those subjects were evaluated by radiochemical methods for SSAO activity using (14)C-benzylamine. Serum NO( x ) concentrations were obtained as an index of nitric oxide production by the Griess reaction. Serum SSAO activity was higher in type 2 diabetic patients than in control group and serum SSAO in type 2 diabetic correlated with age, serum creatinine and total cholesterol. Serum NO( x ) levels in type 2 diabetic patients were also significantly higher than those in the control group. Serum NO( x ) levels in control group correlated with serum SSAO activity. In conclusion, the increase in the SSAO activity and NO( x...