Plasma Nitrotyrosine Research Articles

Development and application of novel ELISA-based analytical tools for assessing nitroxidative distress biomarkers in ischemic stroke: implications for improved diagnosis and clinical management

ABSTRACT Ischemic cerebrovascular accident (iCVA) is a public health issue, whose subjacent events involve the development of nitroxidative distress. Identifying biomarkers that assist in the diagnosis of this disease has clinically relevant implications. The aim of this study was to develop an analytic tool for measuring nitroxidative distress biomarkers, intended for application in clinical practice to enhance patient healthcare. Three enzyme linked immunosorbent assays (ELISA) were developed, with different detection objectives. One of them, in a sandwich format, quantifies the amount of fibrinogen in human plasma, an important glycoprotein involved in the blood coagulation process, contributing to thrombus formation and thereby participating in the mechanism of ischemic stroke. Another ELISA, also in a sandwich format, detects the presence of nitrotyrosine residues in fibrinogen from human plasma, a nitroxidative posttranslational modification resulting from the attack of peroxynitrite by-products on tyrosine residues present in proteins. The third one, in inhibition format, determines human plasma nitrotyrosine total content and was used to analyze human plasma samples from control and iCVA patients. Those two groups of plasma samples were analyzed using inhibition ELISA, revealing statistically significant differences in their nitrotyrosine content and molar ratios of nitrotyrosine to fibrinogen, which were higher in the iCVA group. This study provides evidence that nitroxidative distress occurs in ischemic stroke, as indicated by the detection of the biomarker nitrotyrosine. This finding supports other studies that also identified nitrotyrosine in ischemic stroke, through several different methods. This specific ELISA method is applicable for the rapid analysis of clinical samples, making it a potential clinical tool for assessing iCVA patients.

Age-Related COVID-19 Influence on Male Fertility.

The impact of coronavirus on the reproductive health of men attracts the special attention of many researchers. While studies suggest changes in sperm parameters and the possibility of testicular inflammation, further studies are needed to elucidate any potential age-related changes in these findings, which is the purpose of the present study. The semen quality parameters, cytokine concentration, and markers of the pro- and antioxidant system were assessed in 60 men five to seven months after the coronavirus infection and in 77 controls (without a history of coronavirus infection). Additionally, participants were divided into two age groups: less than 35 years and 35 years or older. Notably increased round cell count in ejaculate and reduced sperm hyaluronan binding ability were observed among post-infection patients younger than 35 years. In the same group, a decline in seminal plasma zinc levels and nitrotyrosine in the cell fraction was found. In men over 35 years of age, Coronavirus Disease 2019 (COVID-19) led to increased sperm DNA fragmentation, a decrease in the total antioxidant capacity, and an elevation in the levels of interleukin-1β and interleukin-10. The concentration of interleukin-1β decreased over time following recovery in all affected patients. The data obtained suggest the potential adverse impact of the coronavirus infection on male reproductive health; however, these effects appear to be age-dependent.

Chronic SIRT1 supplementation in diabetic mice improves endothelial function by suppressing oxidative stress.

Enhancing SIRT1 activity exerts beneficial cardiovascular effects. In diabetes, plasma SIRT1 levels are reduced. We aimed to investigate the therapeutic potential of chronic recombinant murine SIRT1 (rmSIRT1) supplementation to alleviate endothelial and vascular dysfunction in diabetic mice (db/db). Left internal mammary arteries obtained from patients undergoing coronary artery bypass grafting with or without a diagnosis of diabetes were assayed for SIRT1 protein levels. Twelve-week-old male db/db mice and db/+ controls were treated with vehicle or rmSIRT1 intraperitoneally for 4 weeks, after which carotid artery pulse wave velocity (PWV) and energy expenditure/activity were assessed by ultrasound and metabolic cages, respectively. Aorta, carotid, and mesenteric arteries were isolated to determine endothelial and vascular function using the myograph system.Arteries obtained from diabetic patients had significantly lower levels of SIRT1 relative to non-diabetics. In line, aortic SIRT1 levels were reduced in db/db mice compared to db/+ mice, while rmSIRT1 supplementation restored SIRT1 levels. Mice receiving rmSIRT1 supplementation displayed increased physical activity and improved vascular compliance as reflected by reduced PWV and attenuated collagen deposition. Aorta of rmSIRT1-treated mice exhibited increased endothelial nitric oxide (eNOS) activity, while endothelium-dependent contractions of their carotid arteries were significantly decreased, with mesenteric resistance arteries showing preserved hyperpolarization. Ex vivo incubation with reactive oxygen species (ROS) scavenger Tiron and NADPH oxidase inhibitor apocynin revealed that rmSIRT1 leads to preserved vascular function by suppressing NADPH oxidase (NOX)-related ROS synthesis. Chronic rmSIRT1 treatment resulted in reduced expression of both NOX1 and NOX4, in line with a reduction in aortic protein carbonylation and plasma nitrotyrosine levels. In diabetic conditions, arterial SIRT1 levels are significantly reduced. Chronic rmSIRT1 supplementation improves endothelial function and vascular compliance by enhancing eNOS activity and suppressing NOX-related oxidative stress. Thus, SIRT1 supplementation may represent novel therapeutic strategy to prevent diabetic vascular disease.

Biomarkers of oxidative stress and liver function in early lactation and their relationship with the reproductive efficiency of multiparous grazing dairy cows in Argentina. A retrospective study.

This study aimed to analyze the possible relationship between days to conception and different oxidative stress (OS) biomarkers and liver functional parameters in multiparous dairy cows. Besides, a fast reliable method for the accurate measurement of malondialdehyde (MDA) by liquid chromatography-tandem mass spectrometry was developed in several matrices. During lactation, the days to conception of 28 cows were determined for a retrospective study. According to this parameter, cows were divided into two groups: high and low days to conception (HDC and LDC, respectively). Blood, urine and liver biopsies were sampled 21days before the expected calving date, and 7 and 21days after calving. The method developed for MDA was validated according to international requirements. The lower limit of quantification was 0.25µmol/L for plasma and urine and 10.00µmol/L for liver tissue. No differences between groups were observed in the systemic concentration of non-esterified fatty acids, β-hydroxybutyric acid and liver triacylglycerol content (P > 0.05). Cholesterol concentration was higher in the LDC than in the HDC group (P < 0.05). Plasma 3-nitrotyrosine (3-NT) concentration was lower in the LDC than in the HDC group on day 21 post-calving (P < 0.05). Superoxide dismutase activity was higher in the LDC than in the HDC group (P < 0.05). Particularly, in the liver, 3-NT and MDA concentrations were lower in the LDC than in the HDC group (P < 0.05). These results allow inferring that the amelioration of OS biomarkers in plasma and liver could be related to a better reproductive performance of dairy cows.

P66Shc is increased in peripheral blood mononuclear cells of the patients with obstructive sleep apnea.

Objective: Obstructive sleep apnea (OSA) is characterized by nocturnal intermittent hypoxemia and linked to oxidative stress. Evidence demonstrated that p66Shc plays a key role in regulating oxidative stress. This study aimed to investigate the expression of p66Shc in peripheral blood mononuclear cells (PBMCs) of patients with OSA and the association with polysomnographic parameters. Methods: Fifty-four OSA subjects and 19 no OSA controls were enrolled in this study. All the subjects underwent standard polysomnography. P66Shc mRNA and protein levels in the PBMCs were detected by quantitative real-time polymerase chain reaction and western blotting. Plasma 3-nitrotyrosine (3-NT), oxidized low density lipoprotein (oxLDL), and advanced oxidation protein products (AOPP) were measured by ELISA method. Results: P66Shc mRNA and protein levels in PBMCs were significantly higher in OSA patients than in controls. P66Shc mRNA was positively correlated with plasma 3-NT, oxLDL, AOPP, hypopnea index (AHI), oxygen desaturation index (ODI), percentage of total sleep time with oxygen saturation (SaO2) below 90% (CT90), epworth sleepiness scale (ESS) and lymphocytes; negatively correlated with lowest SaO2 (LSaO2) and mean SaO2 (MSaO2). Further multivariate linear regression analysis showed that p66Shc mRNA levels were independently associated with AHI, MSaO2 and CT90. Conclusions: Oxidative stress regulator p66Shc may play a role in the pathophysiology of OSA and might serve as a potential biomarker for this disease.

Biomarkers of oxidative stress in urine and plasma of operators at six Singapore printing centers and their association with several metrics of printer-emitted nanoparticle exposures

Inhalation of nanoparticles emitted from toner-based printing equipment (TPE), such as laser printers and photocopiers, also known as PEPs, has been associated with systemic inflammation, hypertension, cardiovascular disease, respiratory disorders, and genotoxicity. Global serum metabolomics analysis in 19 healthy TPE operators found 52 dysregulated biomolecules involved in upregulation of inflammation, immune, and antioxidant responses and downregulation of cellular energetics and cell proliferation. Here, we build on the metabolomics study by investigating the association of a panel of nine urinary OS biomarkers reflecting DNA/RNA damage (8OHdG, 8OHG, and 5OHMeU), protein/amino acid oxidation (o-tyrosine, 3-chlorotyrosine, and 3-nitrotyrosine), and lipid oxidation (8-isoprostane, 4-hydroxy nonenal, and malondialdehyde [MDA]), as well as plasma total MDA and total protein carbonyl (TPC), with several nanoparticle exposure metrics in the same 19 healthy TPE operators. Plasma total MDA, urinary 5OHMeU, 3-chlorotyrosine, and 3-nitrotyrosine were positively, whereas o-tyrosine inversely and statistically significantly associated with PEPs exposure in multivariate models, after adjusting for age and urinary creatinine. Urinary 8OHdG, 8OHG, 5OHMeU, and total MDA in urine and plasma had group mean values higher than expected in healthy controls without PEPs exposure and comparable to those of workers experiencing low to moderate levels of oxidative stress (OS). The highest exposure group had OS biomarker values, most notably 8OHdG, 8OHG, and total MDA, that compared to workers exposed to welding fumes and titanium dioxide. Particle number concentration was the most sensitive and robust exposure metric. A combination of nanoparticle number concentration and OS potential of fresh aerosols is recommended for larger scale future studies.

Comparison of Five Oxidative Stress Biomarkers in Vegans and Omnivores from Germany and Finland.

When the amount of reactive oxygen species produced by human metabolism cannot be balanced by antioxidants, this phenomenon is commonly referred to as oxidative stress. It is hypothesised that diets with high amounts of plant food products may have a beneficial impact on oxidative stress status. However, few studies have examined whether a vegan diet is associated with lower oxidative stress compared to an omnivorous diet. The present cross-sectional study aimed to compare the levels of five oxidative stress biomarkers in vegans and omnivores. Data of 36 vegans and 36 omnivores from Germany and of 21 vegans and 18 omnivores from Finland were analysed. HPLC coupled with mass spectrometry or fluorescence detection and ELISA methods were used to measure the oxidative stress biomarkers malondialdehyde (MDA), protein carbonyls and 3-nitrotyrosine in plasma and 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 8-iso-prostaglandin F2α (8-iso-PGF2α) in 24 h urine. Analyses of variance and covariance, considering potential confounders, were used. Vegans and omnivores showed no differences in MDA and protein carbonyl concentrations. In Finnish but not in German vegans, the concentrations of 3-nitrotyrosine were lower compared to those in omnivores (p = 0.047). In Germany, vegans showed lower excretion levels of 8-iso-PGF2α than omnivores (p = 0.002) and with a trend also of 8-OHdG (p = 0.05). The sensitivity analysis suggests lower 8-iso-PGF2α excretion levels in women compared to men, independently of the dietary group. The present study contributes to expanding our knowledge of the relationship between diet and oxidative stress and showed that 3-nitrotyrosine, 8-OHdG and 8-iso-PGF2α tended to be lower in vegans. Furthermore, studies are recommended to validate the present findings.

Nitric Oxide Therapy Attenuates Heart Failure with Preserved Ejection Fraction

IntroductionNitric oxide (NO) based therapeutics have proven to exert cardioprotective actions in the setting of heart failure with reduced ejection fraction (HFrEF). However, NO therapy has failed to exert beneficial effects in clinical studies of heart failure with preserved ejection fraction (HFpEF).We investigated (1) the effects of the NO donor, sodium nitrite, as a monotherapy, (2) hydralazine monotherapy, a known vasodilator and antioxidant, and (3) combination therapy with both drugs in a murine model of HFpEF. We assessed left ventricular (LV) function, oxidative/nitrosative stress, exercise tolerance, and cardiac fibrosis.Materials and MethodsMale, C57/BL6N (n=15 per group) were placed on a Western high fat diet (60% kCal from fat) and treated with L‐NG‐Nitro arginine methyl ester (L‐NAME, 0.5 g/L/day) in the drinking water beginning at 10 weeks of age. At 15 weeks of age, mice were randomly assigned to four separate groups for five additional weeks: HFpEF control, sodium nitrite in drinking water (75 mg/L), hydralazine (2 mg/kg/day, i.p., b.i.d.), or the combination of sodium nitrite and hydralazine.Plasma was collected to measure circulating 8‐isoprostane and 3‐nitrotyrosine levels, biomarkers of oxidative and nitrosative stress respectively. In addition, cardiac tissue was harvested at 20 weeks for Masson’s Trichrome staining and to measure cardiac nitrite levels. Echocardiography and exercise tolerance testing was performed at 10, 15, and 20 weeks of age.ResultsThere was a significant increase in circulating nitrite levels (p &lt; 0.05) in the sodium nitrite + hydralazine group compared to the control and either monotherapy groups. There was no significant difference in the cardiac tissue nitrite levels between the combination therapy and the control group. Plasma 8‐isoprostane and 3‐nitrotyrosine levels were significantly (p &lt; 0.05) reduced in the sodium nitrite + hydralazine group compared to the HFpEF control group, indicating a reduction in oxidative and nitrosative stress. Although there was no significant difference in the extent of interstitial cardiac fibrosis between groups, there was a significant decrease (p &lt; 0.05) in myocardial perivascular fibrosis in the sodium nitrite + hydralazine group compared to control. Left ventricular end‐diastolic pressure (LVEDP) was significantly reduced in all treatment groups, and LVEDP was reduced to the greatest extent in the nitrite + hydralazine group vs. HFpEF control. Furthermore, we observed a significant (p &lt; 0.01) improvement in LV diastolic function (i.e., E/e’) in the sodium nitrite and hydralazine group that was superior to either of the treatments alone. There was no significant difference in the exercise distance or duration among the various groups.ConclusionThese data demonstrate that the combination of a powerful antioxidant with NO donor therapy significantly enhances the beneficial effects NO therapy in the setting of severe HFpEF.

Endothelial Pannexin 3 – B Cell Lymphoma‐6 Interactions Protect Against Oxidative Stress

Pannexin channel isoforms (Panx1‐3) are thought to release nucleotides into the extracellular milieu and have been shown to effect vascular hemodynamics. For this reason, we examined their mRNA and protein expression in hypertensive humans and genetically‐inbred hypertensive mice. In both mouse and humans, we found a significant reduction in Panx3 expression in resistance artery endothelium. Thus, we hypothesized Panx3 may be a regulator of vascular function. In en face endothelial preparations from 3rd order mesenteric arteries, we localized Panx3 to the Golgi Apparatus as opposed to Panx1 which localized to the plasma membrane. Next, we generated an inducible, endothelial cell Panx3 knockout mouse (Panx3ECKO). Radiotelemetry revealed a renin‐independent spontaneous hypertension, with unremarkable immune infiltration in the kidney. There was no change in cytoplasmic or released ATP. To understand how Panx3 may regulate blood pressure, we examined whether Panx3 interacted with B Cell Lymphoma 6 (BCL6), a potential binding partner. En face proximity ligation assays demonstrated an interaction between Panx3 and BCL6 in the Golgi. Panx3ECKO mice exhibited significantly decreased BCL6 protein, hinting that Panx3 may stabilize BCL6 by binding at the BCL6 ubiquitin sites. In silico “threading” of the Panx3 sequence onto the cryo‐EM structure of Panx1 confirmed this site of interaction. BCL6 is a NFκB repressor, thus its degradation in Panx3ECKO mice caused an increase in NFκB activity with IκBα and p100 significantly upregulated. A novel mimetic peptide designed to block Panx3‐BCL6 interactions was administered into C57Bl/6J mice, which recapitulated these results. In addition, Panx3ECKO mice had increased endothelial NOX4 (but not NOX1 or NOX2), likely due to increased NFκB activity—this correlated with a significant increase in plasma H2O2, nitrotyrosine (NT3), and 4‐hydroxynonenal (4‐HNE). In line with this observation, 3rd order mesenteric arteries from Panx3ECKO mice constricted (not dilated) to acetylcholine, which was rescued with the H2O2‐scavenger catalase (1000U/mL), suggesting that vascular oxidative stress drives hypertension in Panx3ECKO mice. Interestingly, Panx3ECKO mice also exhibit a significant increase in IL‐4 receptors on endothelium, and increased IL‐4 cytokines in bone marrow lysates. Because IL‐4 can drive BCL6 expression in other cell types, we suggest a possible homeostatic immune‐endothelial signaling axis. These data elucidate a novel Golgi‐localized oxidative signaling pathway in endothelium with a potential immune‐derived negative feedback loop.

Hyperglycemia and Loss of Redox Homeostasis in COVID-19 Patients.

The infection with SARS-CoV-2 impairs the glucose–insulin axis and this contributes to oxidative (OS) and nitrosative (NSS) stress. Here, we evaluated changes in glucose metabolism that could promote the loss of redox homeostasis in COVID-19 patients. This was comparative cohort and analytical study that compared COVID-19 patients and healthy subjects. The study population consisted of 61 COVID-19 patients with and without comorbidities and 25 healthy subjects (HS). In all subjects the plasma glucose, insulin, 8-isoprostane, Vitamin D, H2S and 3-nitrotyrosine were determined by ELISA. The nitrites (NO2−), lipid-peroxidation (LPO), total-antioxidant-capacity (TAC), thiols, glutathione (GSH) and selenium (Se) were determined by spectrophotometry. The glucose, insulin and HOMA-IR (p < 0.001), 8-isoprostanes, 3-nitrotyrosine (p < 0.001) and LPO were increased (p = 0.02) while Vitamin D (p = 0.01), H2S, thiols, TAC, GSH and Se (p < 0.001) decreased in COVID-19 patients in comparison to HS. The SARS-CoV-2 infection resulted in alterations in the glucose–insulin axis that led to hyperglycemia, hyperinsulinemia and IR in patients with and without comorbidities. These alterations increase OS and NSS reflected in increases or decreases in some oxidative markers in plasma with major impact or fatal consequences in patients that course with metabolic syndrome. Moreover, subjects without comorbidities could have long-term alterations in the redox homeostasis after infection.

Application of an optimized and validated LC–MS/MS method for the quantification of free 3-nitrotyrosine in plasma, urine and liver tissue of lactating dairy cows

In dairy cows, the high energy demand and oxygen requirements during the transition period could increase oxidative stress (OS), impairing the challenging metabolic adaptation needed during this period. Also, OS and health could be affected by some environmental conditions such as heat stress. In this sense, 3-nytrotyrosine (3-NT) has been described as a biomarker of OS. Therefore, the aim of this study was to develop and validate a fast, reliable method for the accurate determination of free 3-NT in blood, urine and liver samples by liquid chromatography–tandem mass spectrometry (LC-MS/MS). To this end, during the transition period, 3-NT concentrations were analyzed in samples from dairy cows grouped according to different environmental conditions: cows that gave birth during winter (winter group, WG; n = 18) and cows that gave birth during spring (spring group, SG; n = 18). Additionally, other plasma parameters such as β-hydroxybutyrate acid (BHBA) and glucose concentrations and liver triacylglycerol (TAG) content and aspartate transaminase activity were exhaustively studied. Samples were collected at 21 (±3) days before the expected calving date, and at 7 (±3) and 21 (±3) days after calving. The analysis of the samples involved a purification step by solid-phase extraction. The method developed was validated in terms of linearity, specificity, precision, accuracy, recovery, matrix effect, dilution and analyte stability. The lower limit of quantification of the method was 1.02 ng/mL for plasma, 0.24 mg/L for urine and 0.05 mg/kg for liver tissue. Coefficients of variation for the concentrations tested were < 15% and accuracy was within 85–115%. 3-NT concentration was higher in cows of the SG than in those of the WG in the three matrices evaluated. BHBA and glucose concentrations were higher and lower, respectively, in cows of the SG. BHBA concentration was particularly higher in cows of the SG on day 21 postpartum. Liver TAG content and aspartate transaminase activity were also higher in cows of the SG on the postpartum days. These results suggest that the methodology developed and validated by LC-MS/MS for the quantification of 3-NT in different fluids and liver tissue could be a very useful parameter to evaluate OS in dairy cows during the transition period.

Assessing non-protein nitrogen sources in commercial dry dog foods.

Protein is a macronutrient required by dogs for growth and maintenance metabolism. However, a portion of the crude protein listed on pet foods may actually arise from non-digestible organic nitrogen or potentially toxic inorganic non-protein nitrogen sources. Neither non-protein source is retained or used by the animal. However, these compounds may result in adverse effects such as methemoglobin formation and increased oxidative stress or potentially beneficial effects such as improved vascular distensibility and decreased inflammation. To analyze nitrogen retention and screen for non-protein nitrogen, four commercial, dry kibble dog foods and one laboratory-made diet were evaluated and then fed to beagles during two separate feeding trials. During the first trial, dogs were randomly assigned each diet (n = 4 dogs/diet) and fed chromium oxide-coated diets for 48 h, followed by total urine and marked fecal collection, as well as plasma collection for total nitrogen, nitrate, ammonia, and urea determination. The amount of nitrogen retained (93%–96%) did not differ among commercial diets. Protein total tract apparent digestibility (TTAD) ranged from 69% to 84%, with the high protein diets significantly higher than the laboratory-made and mid-ranged diets (1-way ANOVA: P < 0.05). The high protein diet also contained the highest concentration of nitrate with subsequent elevations in plasma nitrotyrosine levels (indicator of oxidative stress). During the second trial, eight dogs (n = 8) were fed the same diets for 6 d, after which echocardiography was completed with blood, urine, and feces collected. For health end-points, methemoblobin, plasma nitrotyrosine, and C-reactive protein (CRP; indicator of inflammation) levels were measured. Methemoglobin levels were significantly lower in the high protein diet (P > 0.05), possible due to the stimulation of methemoglobin reductase while nitrotyrosine was unchanged and CRP was undetectable. Furthermore, there was a positive relationship between crude protein, crude fat (simple linear regression: P = 0.02, r2 > 0.6), price (P = 0.08, r2 > 0.6), and caloric density (P = 0.11, r2 > 0.6). There were no significant cardiovascular differences among any of the diets (P > 0.05). Ultimately, this study shows that in commercial diets, price does reflect protein content but that feeding dogs high protein diets for a long period of time may provide an excess in calories without a change in cardiovascular function or detectable increases in inflammation.

Cross-Talk Between Nitrosative Stress, Inflammation and Hypoxia-Inducible Factor in Patients with Adrenal Masses.

BackgroundAdrenal masses are the most common of all human tumors. The role of nitrosative stress and inflammation in cancer development has already been demonstrated. However, it is not known whether they are involved in the pathogenesis of adrenal tumors. The aim of the study was to investigate a cross-talk between nitrosative stress, inflammation and hypoxia-inducible factor (HIF-1α) in 75 patients with different types of adrenal masses (non-functional incidentaloma, pheochromocytoma and Cushing’s/Conn’s adenoma).MethodsThe plasma concentrations of total nitric oxide (NO), S-nitrosothiols, peroxynitrite nitrotyrosine and the activity of serum myeloperoxidase (MPO) were measured spectrophotometrically, whereas concentrations of interleukin 1 beta (IL-1β), tumor necrosis factor α (TNF-α) and hypoxia-inducible factor 1 alpha (HIF-1α) were measured using commercial ELISA kits. The control group consisted of 50 healthy people matched by age and sex to the study group. The number of subjects was determined a priori based on our previous experiment (power of the test = 0.9; α = 0.05).ResultsWe found significantly higher nitrosative stress (↑nitric oxide, ↑peroxynitrite, ↑S-nitrosothiols and ↑nitrotyrosine) in the plasma of patients with adrenal tumors, which was accompanied by increased inflammatory (↑myeloperoxidase, ↑interleukin 1 beta and ↑tumor necrosis factor α) and hypoxia (HIF-1α) biomarkers. Peroxynitrite and nitrotyrosine were positively correlated with aldosterone level. Nitrosative stress was also associated with inflammation and HIF-1α. Interestingly, plasma nitrotyrosine and serum MPO differentiated patients with adrenal tumor from healthy individuals with high sensitivity and specificity. Moreover, using multivariate regression analysis, we showed that ONOO− and IL-1β depended on cortisol level, while ONOO−, nitrotyrosine and HIF-1α were associated with aldosterone. Unfortunately, none of the assessed biomarkers differentiated between tumor types studied, suggesting that the severity of nitrosative damage and inflammation are similar in patients with incidentaloma, pheochromocytoma, and Cushing’s or Conn’s adenoma.ConclusionAdrenal tumors are associated with increased protein nitration/S-nitrosylation and inflammation.

The effect of different dietary ratios of lysine, arginine and methionine on protein nitration and oxidation reactions in turkey tissues and DNA

An assumption was made in the study that the optimal inclusion levels and ratios of lysine (Lys), arginine (Arg) and methionine (Met) in diets with Lys content consistent with National Research Council (NRC) recommendations (1994) contribute to stimulate the antioxidant defense system and prevent disorders resulting from the oxidation and nitration of biologically important molecules. The experiment was carried out on 864 one-day-old Hybrid Converter turkeys divided into six experimental groups (8 replicates per group and 18 birds per replicate) receiving different levels of Arg and Met. Chickens from group Arg90Met30 received 90% Arg and 30% Met relative to Lys; Arg90Met45 – 90% Arg and 45% Met relative to Lys; Arg100Met30 – 100% Arg and 30% Met relative to Lys; Arg100Met45 – 100% Arg and 45% Met relative to Lys; Arg110Met30 – 110% Arg and 30% Met relative to Lys and Arg110Met45 – 110% Arg level and 45% Met level relative to the content of dietary Lys. In comparison with turkeys fed diets with moderate Arg content (100% of Lys content), a decrease in dietary Arg level (90% of Lys content) led to a decrease in plasma 3-nitrotyrosine (3-NT) concentration (163.6 vs. 141.0), whereas an increase in dietary Arg level (110% of Lys content) led to an increase in plasma 3-NT concentration (163.6 vs. 202.6). In comparison with turkeys fed diets with moderate Arg content (100% of Lys content), the lowest dietary Arg level (90% of Lys content) decreased superoxide dismutase (SOD) activity in the intestinal wall (19.68 vs. 17.41) and in the liver (11.51 vs. 7.94), increased SOD activity in the blood (507.6 vs. 961.4) and in breast muscles (6.26 vs. 7.43) and increased the concentration of malondiadehyde in breast muscles (1.10 vs. 1.50). An increase in dietary Met content from 30 to 45% of Lys content caused a decrease in plasma protein carbonyl concentration (4.33 vs. 3.8) and catalase activity in breast muscles (54.70 vs. 49.66), and an increase in SOD activity in the liver (8.90 vs. 10.41). The highest dietary Arg level (110% of Lys content) did not induce the oxidation of lipids, proteins or DNA, but it increased the risk of protein nitration. The lowest dietary Arg level (90% of Lys content) deteriorated the antioxidant status of turkeys. Regardless of dietary Arg levels, an increase in Met content from 30 to 45% of Lys content stimulated the antioxidant defense system of turkeys.

Endothelial dysfunction and body mass index: is there a role for plasma peroxynitrite?

BackgroundEndothelial function is dependent on the balance between vasoconstrictive and vasodilatory substances. The endothelium ability to produce nitric oxide is one of the most crucial mechanisms in regulating vascular tone. An increase in inducible nitric oxide synthase contributes to endothelial dysfunction in overweight persons, while oxidative stress contributes to the conversion of nitric oxide to peroxynitrite (measured as nitrotyrosine in vivo) in underweight persons. The objective of this study was to elucidate the interaction of body composition and oxidative stress on vascular function and peroxynitrite. This was done through an experimental design with three weight groups (underweight, normal weight and overweight), with four treatment arms in each. Plasma nitrotyrosine levels were measured 15–20 h post lipopolysaccharide (LPS) treatment, as were aortic ring tension changes. Acetylcholine (ACh) and sodium nitroprusside (SNP) challenges were used to observe endothelial-dependent and endothelial-independent vascular relaxation after pre-constriction of aortic rings with phenylephrine.ResultsNitrotyrosine levels in saline-treated rats were similar among the weight groups. There was a significant increase in nitrotyrosine levels between saline-treated rats and those treated with the highest lipopolysaccharide doses in each of the weight groups. In response to ACh challenge, Rmax (percentage reduction in aortic tension) was lowest in overweight rats (112%). In response to SNP, there was an insignificantly lower Rmax in the underweight rats (106%) compared to the normal weight rats (112%). Overweight rats had a significant decrease in Rmax (83%) in response to SNP, signifying involvement of a more chronic process in tension reduction changes. A lower Rmax accompanied an increase in peroxynitrite after acetylcholine challenge in all weight groups.ConclusionsEndothelial dysfunction, observed as an impairment in the ability to reduce tension, is associated with increased plasma peroxynitrite levels across the spectrum of body mass. In higher-BMI rats, an additional role is played by vascular smooth muscle in the causation of endothelial dysfunction.

Investigation of DNA damage and protein damage caused by oxidative stress in canine visceral leishmaniasis

Leishmaniasis, considered by the World Health Organization as one of the most important zoonotic diseases, causes death when it is not treated in its self-healing skin form. The aim of this study was to investigate DNA damage and oxidative protein damage that occurs in dogs infected with Leishmania infantum. The study group consisted of 25 dogs, including 10 clinically healthy dogs aged 2 to 6 years and 15 dogs infected with Leishmania infantum diagnosed by means of the rk39 dipstick test and the immunofluorescence antibody test (IFAT). The effects of oxidative stress on protein were evaluated in the dogs infected with Leishmania infantum by determining plasma malondialdehyde, protein carbonyl groups, nitrotyrosine, and total antioxidant capacity in blood. DNA damage, on the other hand, was determined by the COMET method. Plasma protein carbonyl content (PCO) and nitrotyrosine (NT) levels, which are considered as indicators of protein damage, were found to be higher in the dogs infected with Leishmania infantum compared to the control group, but the difference in both values was not statistically significant (p &gt; 0.05). Plasma malondialdehyde (MDA), an indicator of lipid peroxidation, was significantly higher in the dogs infected with Leishmania infantum than in the control group. TAC levels, however, were lower in the leishmanial dogs (p &lt; 0.05). According to the results of the COMET assay, lymphocyte cells were damaged in the leishmanial dogs, and both tail intensity (TI) and tail moment (TM) values were higher in those dogs than they were in the control group (p &lt; 0.05, p &lt; 0.01). The parasites caused oxidative stress through protein and DNA damage in the host and decreased the antioxidant capacity concentration that prevents destructive effects.

Consumption of Anacardium Occidentale L. (Cashew Nuts) Inhibits Oxidative Stress through Modulation of the Nrf2/HO-1 and NF-kB Pathways.

Ischemia/reperfusion injury is a severe disorder associated with a high mortality. Several antioxidant and pharmacological properties of cashew nuts (Anacardium occidentale L.) and its metabolites from different countries have recently been described. It is a medicinal plant with important therapeutic effects. This study aimed to verify the effect of an oral administration of cashew nuts in a rat model of ischemia/reperfusion (I/R). Adult male rats were subjected to intestinal I/R injury by clamping the superior mesenteric artery for 30 min and then allowing animals to 1 h of reperfusion. Rats subjected to I/R of the gut showed a significant increase in different biochemical markers. In particular, we evaluated lipid peroxidation, tissue myeloperoxidase activity, protein carbonyl content, reactive oxygen species generation and decreased antioxidant enzyme activities. Western blot analysis showed the activation of the NRF2 and NF-kB pathways. Increased immunoreactivity to nitrotyrosine, PARP, P-selectin, and ICAM-1 was observed in the ileum of rats subjected to I/R. Administration of cashew nuts (100 mg/kg) significantly reduced the mortality rate, the fall in arterial blood pressure, and oxidative stress and restored the antioxidant enzyme activities by a mechanism involving both NRF2 and NF-kB pathways. Cashew nuts treatments reduced cytokines plasma levels, nitrotyrosine, and PARP expression as well as adhesion molecules expressions. Additionally, cashew nuts decreased the intestinal barrier dysfunction and mucosal damage, the translocation of toxins and bacteria, which leads to systemic inflammation and associated organs injuries in particular of liver and kidney. Our study demonstrates that cashew nuts administration exerts antioxidant and pharmacological protective effects in superior mesenteric artery occlusion–reperfusion shock.

Influence of bariatric surgery induced weight loss on oxidative DNA damage

Obesity is associated with elevated cancer risk, which may be represented by elevated genomic damage. Oxidative stress plays a key role in obesity related detrimental health consequences including DNA oxidation damage. The elevated cancer risk in obesity may be a consequence. Weight loss has been shown to reduce genomic damage, but the role of oxidative stress in that has not been clarified.The aim of this study is therefore to investigate the influence of bariatric surgery induced weight loss on DNA oxidation damage in morbidly obese subjects. For this aim, we used cryopreserved peripheral blood mononuclear cells in the FPG comet assay. Advanced protein oxidation products and 3-nitrotyrosine were measured as oxidative and nitrative protein stress markers. Furthermore, expression of oxidative stress related proteins HSP70 and Nrf2 as well as mitochondrial enzyme citrate synthase and NADPH oxidase subunit p22 phox were analysed.Our findings revealed significantly reduced DNA strand breaks, but DNA base oxidation was not reduced. We observed significant reduction in plasma AOPPs and 3-nitrotyrosine, which indicated an improvement in oxidative/nitrative stress. However, expression of HSP70 and Nrf2 were not altered after weight loss. In addition, expression of citrate synthase and p22 phox were also unaltered.Overall, bariatric surgery induced significant reduction in excess body weight and improved the patients’ health status, including reduced DNA strand breaks and slightly improved antioxidant status in some of the investigated endpoints, while cellular ROS formation and DNA oxidation damage stayed unaltered. This complex situation may be due to combined beneficial effects of weight loss and burdening of the body with fat breakdown products. In the future, collecting samples two years after surgery, when patients have been in a weight plateau for some time, might be a promising approach.

Acute Exenatide Therapy Attenuates Postprandial Vasodilation in Humans with Prediabetes: A Randomized Controlled Trial.

Background: The state of prediabetes comprises atherosclerotic changes leading to decreased vascular function in humans. This study examined the effects on incretin mimetics on vascular physiology in the prediabetic postprandial state. Methods: Fifteen obese adults with prediabetes participated in a randomized, crossover, double-blinded trial comparing the postprandial effects of exenatide, saxagliptin, and placebo on peripheral vasodilation. All studies utilized a standardized high-fat meal. Resting and peak forearm blood flow (FBF) were measured via strain gauge venous occlusion plethysmography, and makers of vascular dysfunction were measured in plasma. Results: Exenatide attenuated resting FBF at 3 hr (P = 0.003) and 6 hr (P = 0.056) postmeal, compared to placebo. Nonsignificant reductions in resting FBF were observed between saxagliptin and placebo at the same time points. No group differences were observed for peak FBF, plasma nitrotyrosine, and plasma 8-iso-prostaglandin F2alpha. A transient increase in plasma triglyceride was abated in the exenatide group, when compared to saxagliptin and placebo groups. Only exenatide group showed no significant upsurge in plasma insulin. Plasma-free fatty acids significantly declined in all three groups, although less markedly for exenatide. Postmeal glucose increased at 2 hr with placebo and saxagliptin, but simultaneously decreased with exenatide. Conclusions: Acute treatment with exenatide blunted the postprandial vasodilatory effect of a high-fat meal in prediabetes. Exenatide's acute effects derived primarily from multiple endothelium-independent processes. Trial Registration Number: NCT02104739.

The Rectus Sheath Block (RSB) Analgesia Following Laparotomy Could Affect Malonidialdehyde (MDA) Concentrations in Benign Disease and Cancer

The aim of the study was to compare the MDA (malonidialdehyde) plasma concentrations versus CAT (catalase)/NT (nitrotyrosine) plasma concentrations, patient satisfaction and pain score at rest/pressure to the wound area in laparotomy patients with rectus sheath block (RSB) analgesia. Initially, 56 patients were randomized to four groups; control group (n=12), single-dose (n=16), repeated-dose (n=12) and continuous infusion (n=16) RSB analgesia groups. The plasma concentrations of CAT, NT and MDA markers were measured just before, immediately after and 24 h after operation. The RSB analgesia enhanced significantly patient satisfaction (p=0.001). The plasma MDA decreased immediately after operation (POP1) and the postoperative decrease between the preoperative and the POP1 values in the MDA marker were statistically significant (p<0.001). In linear mixed model, the time effect in both the single group and in the benign group in plasma NT biomarker was statistically significant (p=0.001, p=0.013, respectively). The median plasma MDA concentrations (ng/ml) following surgery were significantly lower in patients with cancer versus patients with benign disease (589 vs. 852, p=0.021). Jitterplots of the individual plasma NT versus plasma MDA showed that there was significant correlation in benign and cancer patients (r=0.347, p<0.001). Plasma MDA decreased significantly after operation in all patients and cancer patients had significantly lower MDA concentrations following surgery than patients with benign disease.