Nitrite and tempol combination promotes synergic effects and alleviates right ventricular wall stress during acute pulmonary thromboembolism.

INTRODUCTION: The mechanical obstruction and pulmonary vasoconstriction are major determinants of the sudden right ventricular (RV) afterload increases observed during acute pulmonary thromboembolism (APT). Vasodilators and antioxidants agents have been shown to mitigate pulmonary hypertension. We examined whether sodium nitrite and the antioxidant tempol combination could be advantageous in an APT sheep model. METHODS: APT was induced in anesthetized sheep by autologous blood clots (250 mg/kg) into the right atrium. Thirty minutes after APT induction, the animals received a continuous infusion of tempol (1.0 mg/kg/min), increasing sodium nitrite infusion (5, 15, and 50 µmol/kg), or a simultaneous combination of both drugs. Saline was used as a control treatment. Hemodynamic measurements were carried out every 15 min. Also, whole blood nitrite and serum 8-isoprostanes levels were measured. RESULTS: APT induced sustained pulmonary hypertension, increased dp/dtmax, and rate pressure product (RPP). Nitrite or tempol treatments attenuated these increases (P < 0.05). When both drugs were combined, we found a robust reduction in the RV RPP compared with the treatments alone (P < 0.05). The sole nitrite infusion increased blood nitrite concentrations by 35 ± 6 µM (P < 0.05), whereas the nitrite and tempol combination produced higher blood nitrite concentrations by approximately 54 ± 7 µM. Tempol or nitrite infusions, both alone or combined, blunted the increases in 8-isoprostane concentrations observed after APT. CONCLUSIONS: Nitrite and tempol combination protects against APT-induced RV wall stress. The association of both drugs may offer an advantage to treat RV failure during severe APT.

Sickle Cell Trait Increases Red Blood Cell Storage Hemolysis and Post-Transfusion Clearance in Mice.

BACKGROUND: Transfusion of blood at the limits of approved storage time is associated with lower red blood cell (RBC) post-transfusion recovery and hemolysis, which increases plasma cell-free hemoglobin and iron, proposed to induce endothelial dysfunction and impair host defense. There is noted variability among donors in the intrinsic rate of storage changes and RBC post-transfusion recovery, yet genetic determinants that modulate this process are unclear. METHODS: We explore RBC storage stability and post-transfusion recovery in murine models of allogeneic and xenogeneic transfusion using blood from humanized transgenic sickle cell hemizygous mice (Hbatm1PazHbbtm1TowTg(HBA-HBBs)41Paz/J) and human donors with a common genetic mutation sickle cell trait (HbAS). FINDINGS: Human and transgenic HbAS RBCs demonstrate accelerated storage time-dependent hemolysis and reduced post-transfusion recovery in mice. The rapid post-transfusion clearance of stored HbAS RBC is unrelated to macrophage-mediated uptake or intravascular hemolysis, but by enhanced sequestration in the spleen, kidney and liver. HbAS RBCs are intrinsically different from HbAA RBCs, with reduced membrane deformability as cells age in cold storage, leading to accelerated clearance of transfused HbAS RBCs by entrapment in organ microcirculation. INTERPRETATION: The common genetic variant HbAS enhances RBC storage dysfunction and raises provocative questions about the use of HbAS RBCs at the limits of approved storage.

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-ß-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 (r (2) = 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 (R s = 0.70, p < 0.0001, and R s = 0.26, p = 0.04, respectively) and NO-consumption (Rs = 0.55, p = 0.002, and R s = 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.

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/m(2) ) 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.

The antioxidant tempol decreases acute pulmonary thromboembolism-induced hemolysis and nitric oxide consumption.

INTRODUCTION: Acute pulmonary thromboembolism (APT) is a critical condition associated with acute pulmonary hypertension. Recent studies suggest that oxidative stress and hemolysis contribute to APT-induced pulmonary hypertension, possibly as a result of increased nitric oxide (NO) consumption. We hypothesized that the antioxidant tempol could attenuate APT-induced hemolysis, and therefore attenuate APT-induced increases in plasma NO consumption. MATERIALS AND METHODS: APT was induced in anesthetized sheep with autologous blood clots. The hemodynamic effects of tempol infused at 1.0mg/kg/min 30 min after APT were determined. Hemodynamic measurements were carried out every 15 min. To assess oxidative stress, serum 8-isoprostanes levels were measured by ELISA. Plasma cell-free hemoglobin concentrations and NO consumption by plasma samples were determined. An in vitro oxidative AAPH-induced hemolysis assay was used to further validate the in vivo effects of tempol. RESULTS: APT caused pulmonary hypertension, and increased pulmonary vascular resistance in proportion with the increases in 8-isoprostanes, plasma cell-free hemoglobin concentrations, and NO consumption by plasma (all P<0.05). Tempol attenuated the hemodynamic alterations by approximately 15-20% and blunted APT-induced increases in 8-isoprostanes, in cell-free hemoglobin concentrations, and the increases in NO consumption by plasma (P<0.05). Tempol dose-dependently attenuated AAPH-induced in vitro hemolysis (P<0.05). CONCLUSIONS: Our findings are consistent with the idea that antioxidant properties of tempol decrease APT-induced hemolysis and nitric oxide consumption, thus attenuating APT-induced pulmonary hypertension.

Acute cardiac and hemodynamic effects of sildenafil on resistant hypertension.

PURPOSE: Failure to control blood pressure (BP) despite the use of three or more drugs characterizes resistant hypertension (RHTN). Impaired endothelial function is associated with this condition and phosphodiesterase-5 inhibitors (PDE5i)-inhibiting cGMP breakdown-reduce BP in RHTN patients. We hypothesized that acute administration of PDE5i could ameliorate hemodynamic, endothelial parameters and left ventricular diastolic function (LVDF) in RHTN patients. Also, an exploratory analysis was performed to assess the influence of the T-786C endothelial NO synthase polymorphism on those responses. METHODS: Subjects (n = 26) underwent a 6-month clinical screening for RHTN diagnosis. Increasing doses of oral sildenafil were given at 30 min intervals (37.5, 50 and 100 mg) while continuous non-invasive hemodynamic measures were assessed. LVDF, flow mediated dilation (FMD), nitrite and cGMP levels were also determined. RESULTS: Mean arterial pressure and total peripheral resistance decreased in all patients (84.17 ± 21.04 to 75 ± 17.21 mmHg; 1149 ± 459.7 to 1037 ± 340 dyn.s/cm(-5), respectively). Likewise, sildenafil improved diastolic dysfunction parameters (Left atrial volume: 25 ± 5.8 to 20 ± 4.4; IVRT: 104 ± 19.33 to 88 ± 15.22; E/e' septal: 9.7 ± 3.8 to 7.9 ± 2.9; E/e' lateral: 7.7 ± 3.4 to 6.4 ± 3.2). No statistical changes were found in FMD, nitrite and cGMP with PDE5i. CONCLUSION: Our data suggest PDE5i acutely improves diastolic function and hemodynamic profile in RHTN subjects, despite unchanging endothelial dysfunction.

Antihypertensive effects of inducible nitric oxide synthase inhibition in experimental pre-eclampsia.

Upregulation of inducible nitric oxide synthase (iNOS) has been reported in both experimental and clinical hypertension. However, although pro-inflammatory cytokines that up-regulate iNOS contribute to pre-eclampsia, no previous study has tested the hypothesis that a selective iNOS inhibitor (1400 W) could exert antihypertensive effects associated with decreased iNOS expression and nitrosative stress in pre-eclampsia. This study examined the effects of 1400 W in the reduced uteroplacental perfusion pressure (RUPP) placental ischaemia animal model and in normal pregnant rats. Sham-operated and RUPP rats were treated with daily vehicle or 1 mg/kg/day N-[3-(Aminomethyl) benzyl] acetamidine (1400 W) subcutaneously for 5 days. Plasma 8-isoprostane levels, aortic reactive oxygen species (ROS) levels and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ROS production were evaluated by ELISA, dihydroethidium fluorescence microscopy and lucigenin chemiluminescence respectively. Inducible nitric oxide synthase expression was assessed by western blotting analysis and aortic nitrotyrosine was evaluated by immunohistochemistry. Mean arterial blood pressure increased by ~30 mmHg in RUPP rats, and 1400 W attenuated this increase by ~50% (P < 0.05). While RUPP increased plasma 8-isoprostane levels, aortic ROS levels, and NADPH-dependent ROS production (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). Moreover, while RUPP increased iNOS expression and aortic nitrotyrosine levels (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). These results clearly implicate iNOS in the hypertension associated with RUPP. Our findings may suggest that iNOS inhibitors could be clinically useful in the therapy of pre-eclampsia, especially in particular groups of patients genetically more prone to express higher levels of iNOS. This issue deserves further confirmation.

Maternal flow-mediated dilation and nitrite concentration during third trimester of pregnancy and postpartum period.

OBJECTIVES: To compare maternal flow-mediated dilation (FMD) of the brachial artery and nitrite concentration between third trimester of pregnancy (3rdT) and postpartum (PP) period. Additionally, we will evaluate whether FMD correlates with nitrite concentration in both periods. METHODS: Eligibility criteria was healthy women with singleton pregnancy, gestational age >28 weeks, nonsmokers, and no personal or family history of vascular disease. Each women was examined during 3rdT and between 8 and 12 weeks PP to evaluate FMD and nitrite concentration in whole blood. Women not examined in both periods were excluded. Values between both periods were compared using paired t tests. Correlation between FMD and nitrite was examined by Pearson correlation coefficient. Significance level set as p < 0.05. RESULTS: We invited 42 pregnant women. Among them, 35 were eligible and 7 of them were excluded for not attending the PP evaluation resulting in 28 participants analyzed. We found no significant change in FMD (10.39 ± 5.57% vs. 8.42 ± 4.21%; p = 0.11; 3rdT vs. PP, respectively) and no significant change in nitrite concentration (257.41 ± 122.95 nmol/L vs. 237.16 ± 90.01 nmol/L; p = 0.28). Baseline brachial artery diameter had a significant reduction (3.11 ± 0.30 to 2.75 ± 0.34 mm; p < 0.01). No significant correlation between FMD and nitrite during 3rdT (r = -0.13; p = 0.50) or PP (r = 0.14; p = 0.48) was found. CONCLUSIONS: We did not observe significant changes in both FMD and nitrite concentration between third trimester and the PP period. FMD did not correlate with nitrite in both periods. More studies are needed to confirm our findings.

Low nitric oxide bioavailability is associated with better responses to sildenafil in patients with erectile dysfunction.

Erectile dysfunction (ED) is a multifactorial disease associated with vascular dysfunction, low nitric oxide (NO) bioavailability, and oxidative stress. However, it is not known whether low NO bioavailability and oxidative stress affect the responsiveness of ED patients to sildenafil. We tested this hypothesis by studying 28 healthy subjects (control group), 26 patients with ED without comorbidities (ED group), and 18 patients with ED and diabetes mellitus (ED/DM group). The International Index for Erectile Function (IIEF) questionnaire was used to assess the erectile function of all participants, and their responsiveness to sildenafil was assessed as the percentage of change in the five-item version of IIEF score before and after sildenafil treatment. Levels of whole blood nitrite, antioxidants markers (ferric reducing ability of plasma (FRAP) and reduced glutathione), and oxidative stress markers (thiobarbituric acid reactive substance and protein carbonyl) were determined. We found a negative correlation between whole blood nitrite levels and the responses to sildenafil in both ED groups (P<0.05). FRAP correlated negatively with the responses to sildenafil in the ED/DM group (P<0.05). No other significant associations were found. Our findings show evidence that low NO bioavailability is associated with better responses to sildenafil in patients with ED (with or without DM).

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 µm 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.

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

INTRODUCTION: The 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. METHODS: Thirty 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 Pearson's chi-square test or Fisher's 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. RESULTS: Storage 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. CONCLUSION: These 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.

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.

Antihypertensive effects exerted by enalapril in mild to moderate hypertension are not associated with changes in the circulating levels of nitric oxide-related markers.

PURPOSE: The antihypertensive effects of angiotensin-converting enzyme inhibitors (ACEi) are explained, at least in part, by enhanced bradykinin-dependent nitric oxide (NO) formation and decreased angiotensin II-induced oxidative stress and vasoconstriction. We examined for the first time whether treatment with enalapril increases the plasma levels of markers of NO formation and decreases oxidative stress in mild to moderate hypertensive patients. METHODS: Eighteen untreated hypertensive patients were treated with enalapril 10 mg/day (n=10) or 20 mg/day (n=8) for 60 days. Eighteen normotensive healthy controls were followed for the same period. Venous blood samples were collected at baseline and after 30/60 days of treatment with enalapril. Plasma NOx (nitrites + nitrates) concentrations were determined by using the Griess reaction. Plasma nitrite and whole blood nitrite concentrations were determined by using an ozone-based chemiluminescence assay. Plasma thiobarbituric acid-reactive species (TBARS) and 8-isoprostane concentrations were determined by a fluorimetric method and by ELISA, respectively. RESULTS: Treatment with enalapril decreased blood pressure in hypertensive patients. However, we found no significant changes in plasma NOx, nitrite, whole blood nitrite, and in the levels of markers of oxidative stress in both normotensive controls and hypertensive patients treated with enalapril. CONCLUSIONS: Our data show that enalapril 10-20 mg/day does not affect the concentrations of relevant markers of NO formation or markers of oxidative stress in mild to moderately hypertensive subjects, despite satisfactory blood pressure control. Our findings do not rule out the possibility that ACEi may produce such effects in more severely hypertensive patients treated with higher doses of ACEi.

eNOS haplotype associated with hypertension in obese children and adolescents.

OBJECTIVE: The aim of our study is to investigate whether genetic polymorphisms in the endothelial nitric oxide synthase (eNOS) gene (in the promoter region T(-786)C, in exon 7 (Glu298Asp) and in intron 4 (4b/4a)) or eNOS haplotypes are associated with hypertension in obese children and adolescents. METHODS: We genotyped 175 healthy (controls), 110 normotensive obese and 73 hypertensive obese children and adolescents. Genotypes were determined by Taqman allele discrimination assay and real-time PCR, and by PCR followed by fragment separation by electrophoresis. We compared the distribution of eNOS genotypes, alleles and haplotypes in the three study groups of subjects. We have also measured whole-blood nitrite concentrations. RESULTS: The 4a4a genotype for the intron 4 polymorphism was more common in normotensive obese and hypertensive obese (P<0.01). The AspAsp genotype for Glu298Asp polymorphism was less common in normotensive obese (P<0.02). No significant differences were found in allele distributions for the three eNOS polymorphisms. However, the haplotype combining the C, 4b and Glu variants for the three polymorphisms was more common in hypertensive obese than in normotensive obese or control children and adolescents (odds ratio=2.28 and 2.79, respectively; 95% confidence interval: 1.31-4.31 and 1.39-5.64, respectively; both P<0.00625). This haplotype was not associated with significantly different nitrite concentrations (P>0.05). CONCLUSIONS: Our findings suggest that the eNOS haplotype, C b Glu, is associated with hypertension in obese children and adolescents. Further studies examining the possible interactions of eNOS haplotypes with environmental factors and other genetic markers involved in the development of obesity and its complications are warranted.

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.

Effects of eNOS polymorphisms on nitric oxide formation in healthy pregnancy and in pre-eclampsia.

Pre-eclampsia (PE) is associated with decreased nitric oxide (NO) formation. However, no previous study has examined whether genetic variations in the endothelial NO synthase (eNOS) affect this alteration. We hypothesized that PE decreases NO formation depending on eNOS polymorphisms. We examined how three eNOS polymorphisms [T-786C, rs2070744; Glu298Asp, rs1799983; 27 bp variable number of tandem repeats (VNTR) in intron 4] affect plasma nitrite concentrations in 205 pregnant women [107 healthy pregnant (HP) and 98 PE]. Genotypes were determined and eNOS haplotypes were inferred using the PHASE 2.1 program. The plasma nitrite concentrations were determined using an ozone-based chemiluminescence assay. The Glu298Asp polymorphism had no effects on the plasma nitrite concentrations. Higher nitrite levels were found in HP women with the CC versus TT genotype for the T-786C polymorphism (277.9 +/- 19.5 versus 140.6 +/- 8.2 nM; P < 0.05). Lower nitrite levels were found in healthy women with the 4a4a versus 4b4b genotype for the VNTR polymorphism (95.1 +/- 3.3 versus 216.1 +/- 16.8 nM; P < 0.05). No effects of genotypes were found in PE women (all P > 0.05). The 'C Glu b' haplotype was more frequent in the HP group than in the PE group (20 versus 5; P = 0.0044). This haplotype was associated with higher nitrite concentrations than the other haplotypes in healthy pregnancies (P < 0.05). No differences in nitrite concentrations were found among PE women with different eNOS haplotypes (P > 0.05). These findings indicate that eNOS polymorphisms affect endogenous NO formation in normal pregnancy, but not in PE, and that the 'C Glu b' haplotype may protect against the development of PE by increasing endogenous NO formation.