Jonas T. C. Sertorio

Relationship between systemic nitric oxide metabolites and cyclic GMP in healthy male volunteers

Aim:  Nitric oxide (NO) is an endogenous mediator of many physiological processes, many of which are mediated by cyclic guanosine 3′,5′‐monophosphate (cGMP). Much effort has been made to validate clinical markers of NO production or bioavailability. While the measurement of plasma nitrate, nitrite, and cGMP concentrations have been suggested to reflect endogenous production of NO, there is no study showing whether there is correlation between these three markers. In the present study, we investigate whether there is correlation between the plasma concentrations of nitrate, nitrite, and cGMP in a relatively homogeneous group of 141 healthy subjects.

Blood transfusions increase circulating plasma free hemoglobin levels and plasma nitric oxide consumption: a prospective observational pilot study

IntroductionThe increasing number of reports on the relation between transfusion of stored red blood cells (RBCs) and adverse patient outcome has sparked an intense debate on the benefits and risks of blood transfusions. Meanwhile, the pathophysiological mechanisms underlying this postulated relation remain unclear. The development of hemolysis during storage might contribute to this mechanism by release of free hemoglobin (fHb), a potent nitric oxide (NO) scavenger, which may impair vasodilation and microcirculatory perfusion after transfusion. The objective of this prospective observational pilot study was to establish whether RBC transfusion results in increased circulating fHb levels and plasma NO consumption. In addition, the relation between increased fHb values and circulating haptoglobin, its natural scavenger, was studied.MethodsThirty patients electively received 1 stored packed RBC unit (n = 8) or 2 stored packed RBC units (n = 22). Blood samples were drawn to analyze plasma levels of fHb, haptoglobin, and NO consumption prior to transfusion, and 15, 30, 60 and 120 minutes and 24 hours after transfusion. Differences were compared using Pearsons chi-square test or Fishers exact test for dichotomous variables, or an independent-sample t test or Mann-Whitney U test for continuous data. Continuous, multiple-timepoint data were analyzed using repeated one-way analysis of variance or the Kruskall-Wallis test. Correlations were analyzed using Spearman or Pearson correlation.ResultsStorage duration correlated significantly with fHb concentrations and NO consumption within the storage medium (r = 0.51, P < 0.001 and r = 0.62, P = 0.002). fHb also significantly correlated with NO consumption directly (r = 0.61, P = 0.002). Transfusion of 2 RBC units significantly increased circulating fHb and NO consumption in the recipient (P < 0.001 and P < 0.05, respectively), in contrast to transfusion of 1 stored RBC unit. Storage duration of the blood products did not correlate with changes in fHb and NO consumption in the recipient. In contrast, pre-transfusion recipient plasma haptoglobin levels inversely influenced post-transfusion fHb concentrations.ConclusionThese data suggest that RBC transfusion can significantly increase post-transfusion plasma fHb levels and plasma NO consumption in the recipient. This finding may contribute to the potential pathophysiological mechanism underlying the much-discussed adverse relation between blood transfusions and patient outcome. This observation may be of particular importance for patients with substantial transfusion requirements.

Increased circulating cell-free hemoglobin levels reduce nitric oxide bioavailability in preeclampsia

Contrasting with increased nitric oxide (NO) formation during healthy pregnancy, reduced NO bioavailability plays a role in preeclampsia. However, no study has examined whether increased NO consumption by enhanced circulating levels of cell-free hemoglobin plays a role in preeclampsia. We studied 82 pregnant women (38 healthy pregnant and 44 with preeclampsia). To assess NO bioavailability, we measured plasma and whole blood nitrite concentrations using an ozone-based chemiluminescence assay. Plasma ceruloplasmin concentrations and plasma NO consumption (pNOc) were assessed and plasma hemoglobin (pHb) concentrations were measured with a commercial immunoassay. We found lower whole blood and plasma nitrite concentrations in preeclamptic patients (-48 and -39%, respectively; both P<0.05) compared with healthy pregnant women. Plasma samples from preeclamptic women consumed 63% more NO (P=0.003) and had 53% higher pHb and 10% higher ceruloplasmin levels than those found in healthy pregnant women (P<0.01). We found significant positive correlations between pHb and pNOc (r=0.61; P<0.0001), negative correlations between pNOc and whole blood or plasma nitrite concentrations (P=0.02; r=-0.32 and P=0.01; r=-0.34, respectively), and negative correlations between pHb and whole blood or plasma nitrite concentrations (P=0.03; r=-0.36 and P=0.01; r=-0.38, respectively). These findings suggest that increased pHb levels lead to increased NO consumption and lower NO bioavailability in preeclamptic compared with healthy pregnant women.

Hemolysis during cardiac surgery is associated with increased intravascular nitric oxide consumption and perioperative kidney and intestinal tissue damage

Introduction: Acute kidney injury (AKI) and intestinal injury negatively impact patient outcome after cardiac surgery. Enhanced nitric oxide (NO) consumption due to intraoperative intravascular hemolysis, may play an important role in this setting. This study investigated the impact of hemolysis on plasma NO consumption, AKI, and intestinal tissue damage, after cardiac surgery. Methods: Hemolysis (by plasma extracellular (free) hemoglobin; fHb), plasma NO-consumption, plasma fHb-binding capacity by haptoglobin (Hp), renal tubular injury (using urinary N-Acetyl-β-D-glucosaminidase; NAG), intestinal mucosal injury (through plasma intestinal fatty acid binding protein; IFABP), and AKI were studied in patients undergoing off-pump cardiac surgery (OPCAB, N = 7), on-pump coronary artery bypass grafting (CABG, N = 30), or combined CABG and valve surgery (CABG+Valve, N = 30). Results: FHb plasma levels and NO-consumption significantly increased, while plasma Hp concentrations significantly decreased in CABG and CABG+Valve patients (p < 0.0001) during surgery. The extent of hemolysis and NO-consumption correlated significantly (r2 = 0.75, p < 0.0001). Also, NAG and IFABP increased in both groups (p < 0.0001, and p < 0.001, respectively), and both were significantly associated with hemolysis (Rs = 0.70, p < 0.0001, and Rs = 0.26, p = 0.04, respectively) and NO-consumption (Rs = 0.55, p = 0.002, and Rs = 0.41, p = 0.03, respectively), also after multivariable logistic regression analysis. OPCAB patients did not show increased fHb, NO-consumption, NAG, or IFABP levels. Patients suffering from AKI (N = 9, 13.4%) displayed significantly higher fHb and NAG levels already during surgery compared to non-AKI patients. Conclusions: Hemolysis appears to be an important contributor to postoperative kidney injury and intestinal mucosal damage, potentially by limiting NO-bioavailability. This observation offers a novel diagnostic and therapeutic target to improve patient outcome after cardiothoracic surgery.

Environmental Exposure to Methylmercury is Associated with a Decrease in Nitric Oxide Production

Some studies have recently suggested that mercury (Hg)-exposed populations face increased risks of cardiovascular diseases, and experimental data indicate that such risks might be due to reductions in nitric oxide bioavailability. However, no previous study has examined whether Hg exposure affects plasma nitrite concentrations in humans as an indication of nitric oxide production. Here, we investigated whether there is an association between circulating nitrite and Hg concentrations in whole blood, plasma and hair from an exposed methylmercury (MeHg) population. Hair and blood samples were collected from 238 persons exposed to MeHg from fish consumption. Hg concentrations in plasma (PHg), whole blood (BHg) and hair Hg (HHg) were determined by inductively coupled plasma-mass spectrometry. Mean BHg content was 49.8 +/- 35.2 microg/l, mean PHg was 7.8 +/- 6.9 microg/l and HHg 14.6 +/- 10.6 microg/g. Mean plasma nitrite concentration was 253.2 +/- 105.5 nM. No association was found between plasma nitrite concentration and BHg or HHg concentrations in a univariate model. However, multiple regression models adjusted for gender, age and fish consumption showed a significant association between plasma nitrite and plasma Hg concentration (beta = -0.1, p < 0.001). Our findings constitute preliminary clinical evidence that exposure to MeHg may cause inhibitory effects on the production of endothelial nitric oxide.

Elevated Plasma Hemoglobin Levels Increase Nitric Oxide Consumption in Experimental and Clinical Acute Pulmonary Thromboembolism

Objectives:We examined whether experimental lung embolization with autologous blood clots or with the infusion of microspheres increase cell-free hemoglobin levels and nitric oxide consumption by plasma samples from anesthetized lambs. These parameters were also measured in patients with acute pulmonary thromboembolism at baseline conditions and after thrombolysis, and in healthy controls. Design:Controlled animal and clinical studies. Setting:University research laboratory and university hospital. Subjects:Sheep and humans. Interventions:Anesthetized lambs were embolized with intravenous injections of autologous blood clots or repeated injections of 300 &mgr;m microspheres. Control animals received saline. Blood samples were drawn from patients with acute pulmonary thromboembolism at baseline conditions and after thrombolytic therapy with streptokinase or alteplase. Measurements and Main Results:Hemodynamic measurements were performed and plasma cell-free hemoglobin concentrations were measured. A nitric oxide consumption assay was used to measure nitric oxide consumption by plasma samples. Embolization with blood clots or microspheres increased mean pulmonary artery pressure from ~15 to ~40 mm Hg in lambs. Both plasma hemoglobin concentrations and nitric oxide consumption increased in proportion to the hemodynamic alterations and correlated significantly. Patients with acute pulmonary thromboembolism had higher plasma hemoglobin concentrations and nitric oxide consumption than healthy controls. Thrombolysis with streptokinase or alteplase further increased both parameters, which peaked 1–3 days after thrombolysis. Conclusions:Our results show consistent evidence indicating a new mechanism involving increased hemoglobin decompartmentalization and augmented nitric oxide consumption, possibly contributing to the hemodynamic derangement of acute pulmonary thromboembolism.

Alterations in cyclic GMP levels in preeclampsia may reflect increased B-type natriuretic peptide levels and not impaired nitric oxide activity.

OBJECTIVES We compared nitrite, B-type natriuretic peptide (BNP), and cGMP levels in preeclamptic with those found in healthy pregnant. METHODS We studied 21 healthy pregnant and 27 preeclamptic. Plasma cGMP and BNP levels were determined by ELISA. Nitrite levels were determined by chemiluminescence. RESULTS Higher cGMP and BNP, and lower nitrite levels were found in preeclamptic versus healthy pregnant. CONCLUSIONS Altered cGMP levels reflect increased BNP levels and not impaired nitric oxide activity in preeclampsia.

Aminoguanidine produces beneficial haemodynamic effects in a canine model of acute pulmonary thromboembolism.

Aim:  Activating the nitric oxide (NO)‐cyclic guanosine 3′,5′‐monophosphate (cGMP) pathway improves haemodynamics following acute pulmonary thromboembolism (APT). However, the role of NO synthase (NOS) isoforms in the responses to APT has not been determined. We examined the effects of selective and non‐selective inducible NOS (iNOS) inhibition.

Proton pump inhibitors and nitric oxide mechanisms in type 2 diabetes.

To the Editor : Hove et al carried out an interesting randomised prospective placebo-controlled study to examine the effects of esomeprazole, a proton pump inhibitor (PPI) on insulin secretion, HbA1c and risk factors for cardiovascular disease in patients with type 2 diabetes [1]. In contrast with their initial hypothesis, they found that treatment with the PPI did not improve insulin secretion or glycaemic control in patients with type 2 diabetes. Curiously, they reported that daytime systolic blood pressure and 24 h diastolic blood pressure tended to increase in patients treated with esomeprazole. While the authors try to explain their apparently unexpected findings, there is now recent evidence strongly suggesting new mechanisms involving the potent vasodilator NO [2], which may help to clarify their findings. Bioconversion of nitrate to nitrite in the entero-salivary circulation is followed by nitrite reduction to NO by enzymatic and non-enzymatic pathways [2] and nitrite infusion in humans causes rapid vasodilation at physiological concentrations [3]. Nitrate reduction to nitrite requires the presence of anaerobic bacteria in the mouth and under the acidic conditions of the stomach nitrite can be converted to NO [2]. Interestingly, orally administered nitrate and nitrite have been associated with blood pressure lowering [4, 5] and PPIs attenuate nitrite-induced decreases in blood pressure [6, 7]. It is highly probable that esomeprazole may have increased blood pressure in the study by Hove et al by inhibiting the effects of nitrate and nitrite in the diet of their diabetic patients. Further supporting this idea, nitrate has been shown to reverse features of the metabolic syndrome, ameliorate glucose tolerance and reduce blood pressure in nitric oxide synthase-deficient mice [8] and PPIs may blunt the effects of nitrite on metabolic variables as well. In conclusion, the results reported by Hove et al are consistent with the idea that PPIs can contribute to cardiovascular pathogenesis by impairing the nitrate–nitrite–NO pathway [6, 7]. This mechanism may be particularly relevant in type 2 diabetic patients who are prescribed PPIs.

Homocysteine and nitrite levels are modulated by MTHFR 677C>T polymorphism in obese women treated with simvastatin.

Higher homocysteine (Hcy) levels are associated with cardiovascular risk. The aim of the present study was to evaluate the effect of simvastatin treatment on circulating Hcy levels in obese women without hypertension, diabetes or dyslipidaemia; and to determine whether the 677C>T polymorphism located in methylenetetrahydrofolate reductase (NAD(P)H) (MTHFR) gene modulates the effects of this treatment on Hcy and nitrite (as a biomarker of nitric oxide (NO) bioavailability). Twenty‐five obese women (body mass index ≥ 30 kg/m2) who had received 20 mg/day simvastatin for 6 weeks were enrolled in the study. Venous blood samples were collected to measure plasma biomarkers and gene polymorphisms. Simvastatin treatment significantly reduced total cholesterol, low‐density lipoprotein–cholesterol, thiobarbituric acid‐reactive substances, high‐sensitivity C‐reactive protein and Hcy, whereas nitrite levels were increased. The reduction in Hcy levels in carriers of the T allele was −20.3% compared with –9.4% in patients with the CC genotype. Importantly, before treatment, nitrite levels were significantly higher in patients with the CC genotype compared with T allele carriers, whereas after treatment these levels were similar between groups. Our findings demonstrate that obese women without comorbidities and carrying the T variant of the 677C>T polymorphism of MTHFR exhibit benefits with simvastatin treatment, mainly in terms of increased NO levels.