Serum Nitrate Concentrations Research Articles

PSII-8 Impact of Grazing High Nitrate Forages on Serum Nitrate Concentration and Production Performance in Beef Heifers

Abstract The objective of this study was to compare nitrate concentrations in soil, forage, and the serum of animals grazing high (HNO3) and low (LNO3) nitrate forages. The fall before the study began one-half of a 6-ha field was amended with manure to increase nitrate levels in the soil. Forage barley (Hordeum vulgare L. var. Vernal) was planted in spring at 45 kg/ha. The 6-ha field was divided into twenty-seven 0.2-hectare paddocks (n = 13 HNO3; n =14 LNO3). Twenty-four crossbred yearling heifers were stratified by body weight (389.8 ± 2.8 kg) and randomly assigned to HNO3 (n = 12) or LNO3 (n = 12) with three replicates per treatment. Heifers entered paddocks when forage was 30 to 46 cm tall and removed when residual height was 10 cm. Heifers grazed for a total of 58 days. Each grazing period lasted 7-8 d. Soil samples were collected at the beginning of the study and sent to a commercial laboratory for soil analyses. Forage samples were collected prior to animal introduction to each grazing paddock, and before re-grazing. Heifers were weighed on d1 and every 14 d. Serum samples for nitrate analysis were collected on d 28, 42 and 57 of the trial. Forage samples were analyzed by near infrared spectrometry. Serum samples were analyzed for nitrate concentrations by ion chromatography. Data were analyzed using PROC GLM of SAS. Analysis of body weights and average daily gain included repeated measures. Soil, nitrate-nitrogen (ppm) and nitrate-nitrogen content (kg/ha) were greater (P < 0.001) in HNO3 compared to LNO3 paddocks. Soil P (P < 0.03) and K (P = 0.0002) levels were also greater in HNO3 than LNO3 paddocks. Average forage nitrate ion concentrations were greater (P < 0.0001) for HNO3 (1.58%) compared to LNO3 paddocks (0.42%). Forage nitrate ion concentrations increased (P < 0.002) with increasing grazing days. Peak nitrate ion concentration in HNO3 paddocks was 2.45%. Forage ADF, TDN, NDF, NEm, NEg were similar (P ≥ 0.07) between treatments. Forage crude protein was greater in HNO3 compared to LNO3 paddocks (P < 0.0001). Serum nitrate levels were undetectable (< 2 ppm) regardless of treatment. Heifer bodyweight and average daily gain were similar (P > 0.7) across treatments. Heifers gained an average of 1.1 kg/d resulting in a linear increase (P < 0.04) in body weight. In summary, grazing vegetative forage containing toxic concentrations of nitrate did not result in an increase in the serum nitrate concentration. Animal performance also did not differ across dietary treatments. This is suggestive of adaptive changes that prevented toxicity and compromised production performance for heifers grazing high nitrate forages. Supported by the Mary A. Turner Research Endowment

Differences in serum markers of oxidative stress in well controlled and poorly controlled asthma in Sri Lankan children: a pilot study

BackgroundAsthma is a disease characterised by hyper responsiveness and bronchoconstriction of airways, and is a major health burden globally. A dysfunction of the oxidant-antioxidant balance, termed oxidative stress, has been implicated in the pathophysiology of asthma. The present study aims to assess the changes in oxidative stress markers, namely nitric oxide metabolites and antioxidant capacity, in children with poorly controlled and well controlled asthma, in comparison to healthy controls.MethodsThe present study enrolled 72 children (ages 5-15 years) classified into three groups: (1) poorly controlled asthma (n = 20), (2) well controlled asthma (n = 24) and (3) healthy controls (n = 27). An interviewer-administered questionnaire was used to record socio-demographic data of the participants. The serum concentrations of the oxidant markers (nitrite, nitrate and total nitric oxide metabolites [NOx]) were determined using the Griess test, and the total antioxidant capacity (TAOC) was determined using the ABTS decolorisation method. The concentrations of these markers were compared across the three groups.ResultsThe three study groups were similar in terms of socio-demographic data. The differences across the three groups were statistically significant for serum concentrations of nitrate and NOx (but not nitrite) and serum TAOC. Further analyses showed that the disparity for nitrate and NOx concentrations was greatest between poorly controlled asthma and healthy controls (p = 0.001 and p < 0.001) compared to the well-controlled asthmatics and healthy controls (p = 0.036 and p = 0.049). A significant difference in serum nitrate and NOx concentrations was not observed between the two asthma groups (p = 0.311 and 0.203). The TAOC were significantly lower in poorly controlled asthmatics as compared to well-controlled asthmatics (p = 0.003) and healthy controls (p < 0.001). However, there was no significant difference in the serum TAOC between healthy controls and well-controlled asthmatics (p = 0.496). These findings may indicate that it is perhaps the higher TAOC that contributes to the well controlled state of asthma.ConclusionsThe present study indicated that an imbalance of oxidants and antioxidants in the serum may have an underlying role in asthma pathophysiology, and how these markers may be effective in asthma management.

75-OR: Dietary Nitrate and Fecal Transplantation Prevent Cardiac Dysfunction and Attenuate Left Ventricular Mitochondrial Reactive Oxygen Species Emission in High-Fat Diet-Fed Mice

Cardiometabolic diseases are multifactorial conditions involving dysfunction within the left ventricle (LV), adipose tissue (AT), and gut microbiome. Nitrate supplementation has been shown to influence cardiac contractility, mitochondrial function, and microbial populations, effects which require commensal gut bacteria to metabolize nitrate. Therefore, we examined if dietary nitrate and fecal microbial transplant (FMT) from nitrate-fed mice were capable of preventing high fat diet (HFD) induced cardiac abnormalities. Male C57Bl/6N mice were fed a control diet, or HFD (60% fat) in the absence or presence of 4mM sodium nitrate in drinking water for 8 weeks. A further subset of HFD-fed mice were gavaged with feces from HFD+nitrate donors or HFD donors for 3 days at the beginning of week 7, and followed until week 8. Compared to controls, HFD mice presented with ∼25% reduction in end diastolic volume, stroke volume, and cardiac output, in association with an increase in cardiac fibrosis, AT inflammation, and serum lipids. In contrast, dietary nitrate prevented these detrimental effects. The improvement in cardiac function following dietary nitrate was not attributed to changes in LV mitochondrial respiration, however, nitrate prevented the increase in LV mitochondrial reactive oxygen species (ROS) emission associated with HFD. While FMT from HFD+nitrate donors did not influence AT inflammation or cardiac fibrosis, HFD+nitrate FMT decreased serum lipids, LV ROS, and improved cardiac function compared to FMT from HFD-fed donors, without increasing serum nitrate concentrations. Altogether, dietary nitrate prevents the detriments of HFD on cardiac function and LV mitochondrial ROS emission, effects which may not be directly dependent on increasing serum nitrate. Rather, the beneficial effects of nitrate may be linked to changes in the gut microbiome, as FMT from nitrate-fed donors improved LV function. Disclosure H.L. Petrick: None. H. Brunetta: None. A. Kirsh: None. P. Barbeau: None. L.M. Ogilvie: None. J. Simpson: None. J.D. Schertzer: None. G. Holloway: None. Funding Natural Sciences and Engineering Research Council of Canada (400362)

Effect of Quercetin Monoglycosides on Oxidative Stress and Gut Microbiota Diversity in Mice with Dextran Sodium Sulphate-Induced Colitis.

The pathogenesis of inflammatory bowel disease (IBD) is linked to an intricate association of environmental, microbial, and host-related factors. This study examined the potential effects of dietary addition of two preparations from onion, one comprising quercetin aglycone alone (Q: 0.15% polyphenols, quercetin aglycone:quercetin monoglycosides, 98:2) and another comprising quercetin aglycone with monoglycosides (Q+MQ: 0.15% total polyphenols, quercetin aglycone:quercetin monoglycosides, 69:31), on dextran sodium sulphate- (DSS-) induced colitis in mice. The results revealed a significant decrease in the body weight gain of the mice with DSS-induced colitis, which was counteracted by the dietary Q or Q+MQ supplementation. Meanwhile, the oxidative stress indicated by myeloperoxidase (MPO), reduced glutathione (GSH), malondialdehyde (MDA), and serum nitrate (NO) concentrations was higher in mice with DSS-induced colitis than in the control group mice, but dietary Q or Q+MQ supplementation counteracted this trend. The colitis mice demonstrated reduced Chao1, angiotensin-converting enzyme (ACE), and Shannon indices and an increased Simpson index, but the colitis mice receiving dietary Q or Q+MQ exhibited higher Chao1, ACE, and Shannon indices and a reduced Simpson index. In conclusion, this research showed that even at a low dose, dietary Q or Q+MQ supplementation counteracts DSS-induced colitis in mice, indicating that Q or Q+MQ may be used as an adjuvant therapy for IBD patients.

Hepatic cytochrome P450 metabolism suppressed by mast cells in type 1 allergic mice

Mast cells and Kupffer cells secrete interleukin (IL)-1β, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, which stimulate excess nitric oxide (NO) producing-inducible NO synthase (iNOS). Unlike Kupffer cells, immunoglobulin E-sensitized mast cells elicit sustained NO production. We investigated the participation of mast cell-released NO and cytokine-derived iNOS activation in type 1 allergy-suppressed hepatic cytochrome P450 (CYP) metabolism. Aminoguanidine, a selective iNOS inhibitor, completely suppressed serum nitrate plus nitrite (NOx) concentrations after primary and secondary sensitization of ICR mice and markedly attenuated allergy-suppressed hepatic CYP1A2, CYP2C, CYP2E1, and CYP3A activities. In the liver, primary and secondary sensitization enhanced iNOS-stimulating IFN-γ (5-15-fold) and TNF-α (3-5-fold) mRNA levels more than IL-1β (2-fold) and F4/80-positive Kupffer cell (2-fold) mRNA levels. When mast cell-deficient (−/−) mice were sensitized, hepatic CYP activities were not suppressed. Serum NOx levels in the sensitized −/− mice were similar with those in saline-treated ICR and −/− mice. In the liver of −/− mice, secondary sensitization markedly enhanced mRNA expression of iNOS (20-fold), IFN-γ (15-fold), and TNF-α (3-fold). However, hepatic total NOS activities in −/− mice were not significantly different between saline treatment and sensitization. Similarly, primary and secondary ICR mice did not significantly enhance total NOS activities in the liver and hepatocytes. The total NOS activities observed did not relate to the high levels of iNOS, IFN-γ, and TNF-α mRNA in the liver. Hepatic c-kit-positive mast cells in sensitized ICR mice were maintained at control levels. Therefore, our data suggest that mast cell-released NO participates in type 1 allergy-suppressed CYP1A2, CYP2C, CYP2E1, and CYP3A metabolism.

Dietary L-Arginine Intakes and the Risk of Metabolic Syndrome: A 6-Year Follow-Up in Tehran Lipid and Glucose Study.

This study was conducted to investigate whether regular dietary intake of L-arginine could affect the occurrence of metabolic syndrome (MetS). Eligible adult men and women (n=1,237), who participated in the Tehran Lipid and Glucose Study, were followed for a median of 6.3 years. Dietary intakes of L-arginine and serum nitrate and nitrite (NOx) concentration were assessed at baseline (2006~2008), and demographics, anthropometrics, and biochemical variables were evaluated at baseline and follow-up examinations. The occurrence of MetS was assessed in relation to total L-arginine, intakes of L-arginine from animal and plant sources, with adjustment of potential confounding variables. Participants who had higher intake of L-arginine also had higher serum NOx at baseline (35.0 vs. 30.5 μmol/L, P<0.05). After 6 years of follow-up, higher intakes of L-arginine from animal sources were accompanied with increased risk of MetS [odd ratios (OR)=1.49, 95% confidence interval (95% CI)=1.02~2.18]. Compared to the lowest, the highest intakes of L-arginine from plant sources were related to significantly reduced risk of MetS (OR=0.58, 95% CI=0.32~0.99). In conclusion, our findings suggest a potentially protective effect of plant derived L-arginine intakes against development of MetS and its phenotypes; moreover, higher intakes of L-arginine from animal sources could be a dietary risk factor for development of metabolic disorders.

Abstract P218: Role of UCP2 on Mitochondrial Dysfunction and Blood Pressure Regulation in the Renal Oxidative Stress-mediated Hypertension Associated With Dj-1 Depletion

DJ-1 and uncoupling protein 2 (UCP2) exert protective roles against mitochondrial (MT) oxidative stress. DJ-1 -/- mice have increased systolic blood pressure (BP) (+30±3% vs WT, n=6). This study determined the mechanisms involved in the oxidative stress-mediated hypertension due to DJ-1 germline deletion. There were no differences in sodium excretion, renal renin expression, NADPH oxidase activity and serum creatinine between DJ-1 -/- and WT mice (n=5). However, renal expression of nitro-tyrosine was increased in DJ-1 -/- mice (+176.8±31% vs WT mice, n=5). Tempol, a radical scavenger, normalized the BP (tempol: 118±2% vs 100±1% vs WT, n=4) and renal malondialdehyde (tempol: 160±23% vs 109±15% vs WT, n=4) in DJ-1 -/- mice. Tempol-treated DJ-1 -/- mice had higher serum nitrite/nitrate levels than placebo-treated (172±30% vs WT, n=4). Heat shock protein mtHSP60 was increased in DJ-1 -/- mice (2.9±0.1-fold increase vs WT, n=4), indicating MT stress. However, there were no changes in the renal mRNA expression of mitophagy, MT fusion and MT biosynthesis markers indicating that MT function was not altered. Renal expression of UCP2 was increased in DJ-1 -/- mice (4.1±1.1-fold change vs WT, n=4), and was partially normalized by tempol (1.8±0.2-fold change vs WT, n=4), UCP2 may have a protective role on MT function in this model. UCP2 was selectively silenced via sub-capsular infusion of UCP2 siRNA in the kidney (WT: 63%±7 vs control: DJ-1 -/- :60%±6 vs control; n=4). mRNA expression of mitophagy markers BNIP3 (-0.65±6-fold) and PINK1 (1.55±0.3-fold), MT fusion markers FIS1 (-0.29±0.03-fold) and NFN2 (1.42±0.06-fold), and MT biosynthesis marker PPRC1 (1.71±0.07-fold) were altered by UCP2 silencing in DJ-1 -/- mice (n=4). Renal-selective silencing of UCP2 normalized BP in DJ-1 -/- mice ( DJ-1 -/- mice: 122±5 vs 98±7 mmHg, n=4), and the serum nitrite and nitrate concentrations (-40±9% vs WT, n=4). In conclusion, deletion of DJ-1 leads to oxidative stress-induced hypertension associated with down-regulation of NO synthesis. UCP2 has protective properties against the development of MT dysfunction in MT oxidative stress conditions . However, excessive and chronic over expression of UCP2 could have deleterious consequences on BP regulation.

Effects of polymorphisms in endothelial nitric oxide synthase and folate metabolizing genes on the concentration of serum nitrate, folate, and plasma total homocysteine after folic acid supplementation: A double-blind crossover study

ObjectivesA number of studies have explored the effects of dietary nitrate on human health. Nitrate in the blood can be recycled to nitric oxide, which is an essential mediator involved in many important biochemical mechanisms. Nitric oxide is also formed in the body from l-arginine by nitric oxide synthase. The aim of this study was to investigate whether genetic polymorphisms in endothelial nitric oxide synthase (eNOS) and genes involved in folate metabolism affect the concentration of serum nitrate, serum folate, and plasma total homocysteine in healthy individuals after folic acid supplementation. MethodsIn a randomized double-blind, crossover study, participants were given either folic acid 800 μg/d (n = 52) or placebo (n = 51) for 2 wk. Wash-out period was 2 wk. Fasting blood samples were collected, DNA was extracted by salting-out method and the polymorphisms in eNOS synthase and folate genes were genotyped by polymerase chain reaction methods. Measurement of serum nitrate and plasma total homocysteine (p-tHcy) concentration was done by high-performance liquid chromatography. ResultsThe concentration of serum nitrate did not change in individuals after folic acid supplements (trial 1); however, the concentration of serum nitrate increased in the same individuals after placebo (P = 0.01) (trial 2). The individuals with three polymorphisms in eNOS gene had increased concentration of serum folate and decreased concentration of p-tHcy after folic acid supplementation. Among the seven polymorphisms tested in folate metabolizing genes, serum nitrate concentration was significantly decreased only in DHFR del 19 gene variant. A significant difference in the concentration of serum nitrate was detected among individuals with MTHFR C > T677 polymorphisms. ConclusionsPolymorphisms in eNOS and folate genes affect the concentration of serum folate and p-tHcy but do not have any effect on the concentration of NO3 in healthy individuals after folic acid supplementation. 2014 Elsevier Inc. All rights reserved.

Biochemical Alterations of Experimentally Induced Hypomagnesaemia in Male Baladi Goats

This study was performed to investigate the effect of experimental hypomagnesemia on glutathione redox cycle, nitric oxide, serum, glucose, calcium, inorganic phosphorus, magnesium, cortisone and insulin in baladi goats. Two groups of twelve healthy Baladi male goats were used; the first one contained 5 animals and kept as control group. The second group includes 7 animals administrated with potassium chloride and citric acid daily until the development of the characteristic signs of hypomagnesaemia observed. Blood samples were collected at 6, 12, 18 and 24 days of administration. The obtained results revealed that decreases in serum magnesium, calcium, inorganic phosphorus, insulin and nitrate concentration and erythrocytes glutathione peroxidase, reduced glutathione, glutathione reductase, glutathione-S-transferase and total superoxide dismutase activities were decreased whereas erythrocytes catalase activity, serum cortisone and glucose concentration were significantly increased in hypomagnesemic induced group when compared with the control group. From these results it could be conclude that nitric oxide release, glutathione redox cycle activities, insulin and cortisone levels were markedly affected by hypomagnesaemia. Such alterations may be considered as predisposing factors responsible for the tissue injury and vascular changes observed in hypomagnesaemia.

Serum and Urinary Nitrites and Nitrates and Doppler Sonography in Detection of Early Diabetic Complications

Objective: to evaluate serum and urinary nitrite and nitrate concentrations as an index of nitric oxide (NO) production in type1diabetes mellitus (T1DM) and to investigate the possible alteration and correlation with intra renal Doppler resistive indexes (RIs). Design: The study included 90 children and adolescent with T1DM divided into 2 groups: Group 1: included 45 prepubertal type1 diabetics who were defined as Tanner stage1. Their age ranged between 8-14 years. Group 2: included 45 pubertal type diabetics who were defined as Tanner stages 2-5. Their ages ranged between 15-19 years. They were compared to 45 age and sex matched healthy controls. Clinical examination was done. Laboratory investigations included; post-prandial blood glucose (PPBG), glycosylated heamoglobin (HbA1C), Fundus examination, urinary microalbumin and measurement of serum and urinary nitrite and nitrate levels. Doppler ultrasonographic registration of intrarenal RIs were performed. Results: Compared to controls, both diabetic groups had significantly increased concentrations of serum (P=0.001) and urinary NO (P=0.008).Doppler RIs values were significantly elevated in both diabetic groups compared to controls (P<0.001).A significant positive correlation was found between serum and urinary NO levels (P<0.001). Serum NO was positively correlated with Doppler RIs (P=0.002), HbA1c (P=0.012), PPBG (P=0.000) and diabetes duration (P=0.004). Doppler RIs were positively correlated with mean HbA1c (P=0.025), PPBG (P=0.001) and diabetes duration (P=0.000). Conclusion: In type 1 diabetes chronic hyperglycemia may act through a mechanism that involves increased NO production or action and contributes to generating intrarenal hemodynamic abnormalities which could be detectable by Doppler ultrasonography even before overt clinical nephropathy. Further follow up studies are needed to document the usefulness of Doppler ultrasound in detection of preclinical nephropathy.

The Role of Nitric Oxide in Radiation Damage

This study investigated the role of nitric oxide in radiation-induced damage by examining changes in mouse serum nitrate concentrations after irradiation. In addition, the contribution of S-2-aminoethylisothiourea 2HBr (AET) to the mechanisms of radiation damage protection was also clarified. The serum nitrate concentration increased as soon as 1.5 h after irradiation, and after 2.5 to 3.0 h the concentrations were significantly higher compared with normal levels. Normal levels were re-established after 12 h. Post-irradiation serum nitrate concentrations increased dose-dependently with irradiation dose (19.6-31.5 Gy). AET suppressed increases in the serum nitrate concentration following irradiation while 2-mercaptoethylamine HCl (MEA) did not. AET has an inhibitory effect on inducible nitric oxide synthase (iNOS); therefore, the increase in nitric oxide after irradiation may be produced by iNOS. Combined administration of irradiation and lipopolysaccharide (LPS) induced a significant increase in serum nitrate concentration, and a significant decrease in survival rate, compared with irradiation alone. The administration of AET or aminoguanidine increased survival rate following irradiation. In contrast to findings after LPS administration, IL-1beta and IFN-gamma were not determined in serum following irradiation. Existing iNOS is activated by irradiation, and nitric oxide production appears to increase without iNOS induction. Thus, the irradiation-induced increase in nitric oxide may be related to lethal injury.

Serum and Urinary Nitrites and Nitrates and Doppler Sonography in Children With Diabetes

The aim of the present study was to evaluate serum and urinary nitric oxide (NO) concentrations in children and adolescents with diabetes compared with age-matched healthy control subjects to find out whether Doppler ultrasonography could be used to detect changes in renal resistive indexes (RIs) in children with diabetes and to assess whether there are correlations between these parameters and NO excretion. We studied 42 children with type 1 diabetes and 41 matched healthy control subjects, both divided into prepubertal or pubertal children. Serum and urinary nitrite and nitrate (NO2-+NO3-) concentrations were evaluated as an index of NO production. Doppler ultrasonographic registration of intrarenal RI was performed. Compared with healthy control subjects, children with diabetes had significantly increased concentrations of serum (30.26 +/- 6.52 vs. 24.47 +/- 7.27 mmol/l, P = 0.001) and urinary NO2-+NO3- (345.07 +/- 151.35 vs. 245.86 +/- 80.25 mmol/l, P = 0.002); the same was true for Doppler RI values (0.64 +/- 0.03 vs. 0.60 +/- 0.04, P = 0.035). This occurs in both prepubertal and the pubertal children. A significant positive correlation was found between serum and urinary NO2-+NO3- levels (P = 0.002, r = 0.374). Serum NO2-+NO3- concentrations also correlated positively with Doppler RI (P = 0.032, r = 0.262) and HbA1c (A1C) (P = 0.004, r = 0.329); urinary NO2-+NO3- concentrations correlated positively with A1C (P = 0.001, r = 0.394). Doppler RI correlated positively with A1C (P = 0.000, r = 0.424). This study demonstrates that in children with diabetes, chronic hyperglycemia may act through a mechanism that involves increased NO production and/or action and contributes to generating intrarenal hemodynamic abnormalities, which are detectable by Doppler ultrasonography even in early diabetic nephropathy.

Higher serum nitrate levels are associated with poor survival in lung cancer patients

Objectives: Angiogenic factors induce tumour growth and angiogenesis which leads to tumour metastasis and a poor survival rate. This study aimed to assess the possible roles of nitric oxide (NO) and vascular endothelial growth factor-A (VEGF-A) in the overall survival of patients with late stage lung cancer. Design and Methods: The study was carried out with primary lung carcinoma patients ( n = 31) and healthy controls ( n = 15). Pre- and post-cisplatin-based chemotherapy serum nitrite/nitrate levels were measured as nitrite after enzymatic conversion followed by Griess reaction and serum VEGF-A analysis was performed using ELISA. After patient follow-up, survival rates were calculated by using the Kaplan–Meier method [Dudek et al. Cancer Invest 2005; 23(3):193–200]. Results: The serum nitrite/nitrate and VEGF-A levels of lung cancer patients and the control group were 93.7 ± 48.9 and 63.7 ± 32.2 μM ( p = 0.018), and 620 ± 491 and 255 ± 157 pg/mL ( p = 0.001), respectively. High nitrite/nitrate (> 67.2 μM) concentration had statistically significant effects on overall survival (Cox analysis, p = 0.026). The overall survival of the lung cancer patients with higher serum nitrate concentrations was significantly less than the ones with lower serum nitrite/nitrate (Kaplan–Meier survival functions test, log rank significance = 0.0007). Conclusion: Our results suggest that having a high serum nitrite/nitrate concentration is a strong indicator of poor survival for late stage lung cancer patients. However, this conclusion deserves to be elucidated further by using a larger sample size.

Conservation of whole body nitric oxide metabolism in human alcoholic liver disease: Implications for nitric oxide production

Objective. Patients with advanced liver diseases tend to develop a hyperdynamic circulation which complicates cirrhosis. Impairment of nitric oxide (NO) metabolism has been implicated in the pathogenesis of portal hypertension. The aim of this study was to determine nitric oxide synthase (NOS)-dependent whole body NO production in patients with decompensated liver cirrhosis and portal hypertension. Material and methods. Ten patients with decompensated alcoholic liver disease and portal hypertension (Child-Pugh Classifications B and C with no signs of infection) and 10 age- and gender-matched control subjects received an intravenous infusion of L-[15N]2-arginine (50 µmol/min for 30 min). Urine and serum nitrite and nitrate concentrations were determined using ion chromatography-mass spectrometry. Results. NOS-dependent whole body NO synthesis was estimated by the conversion of [15N]guanidino nitrogen of arginine to urine 15N-nitrite and 15N-nitrate. The amount of 15N-nitrite and 15N-nitrate in the urine of patients and control subjects was significantly correlated with the amount of urine nitrite and nitrate over 36 h (r=0.91 and 0.77, respectively, p<0.0001). However, neither a median of 12 h 15N-nitrite and 15N-nitrate nor nitrite and nitrate excretion in the urine was different between patients and control subjects, 46.4 (9.4–152.2) versus 98.7 (29.9–146.5) nmol/mmol creatinine and 20.6 (2.1–69.0) versus 40.0 (27.0–70.1) µmol/mmol creatinine, respectively. No differences were found in serum nitrite and nitrate concentrations and glomerular filtration rates between patients and control subjects, 111.4 (73.2–158.8) versus 109.3 (83.5–176.4) µmol/l. Conclusion. Our results contraindicate a greater basal NOS-dependent whole body NO production in patients with decompensated liver disease and portal hypertension.

Spontaneous recovery of injured Achilles tendon in inducible nitric oxide synthase gene knockout mice

To determine if inducible nitric oxide synthase (iNOS) gene could affect Achilles tendon healing using iNOS gene knockout mice. 21 iNOS knockout (iNOS(-/-)) mice and 8 of the wild type (iNOS(+/+)) mice were utilized in this study. Group 1: iNOS(+/+) mice (n = 8), group 2: iNOS(-/-) mice (n = 11) and group 3: iNOS(-/-) with a NOS inhibitor, (aminoguanidine, 500 mg/kg/day, via an intraperitoneal mini-osmotic pump for 7 days, n = 10). The right Achilles tendon was transected in all mice and harvested on day 7 for cross-sectional area and biomechanical properties. Serum nitrate concentration of the mice was measured by gas chromatography mass spectrometry (GC/MS). A significant reduction in cross-sectional area of the healing Achilles tendon was observed in group 3 mice compared to group 2 mice (p < 0.01). The serum nitrate concentration in both group 2 and group 3 mice was lower than that in group 1 mice (p < 0.01) iNOS gene deletion and inhibition of NOS did not affect the biomechanical properties of the healing tendons. iNOS gene is not solely responsible for the beneficial effects of nitric oxide (NO) on tendon healing.

Repeated Sauna Therapy Increases Arterial Endothelial Nitric Oxide Synthase Expression and Nitric Oxide Production in Cardiomyopathic Hamsters

Vascular endothelial dysfunction is involved in the pathophysiology of chronic heart failure (CHF). It has been reported that sauna therapy, which allows thermal vasodilation, improves vascular endothelial dysfunction in patients with CHF. The present study investigates the mechanisms through which sauna therapy improves endothelial dysfunction induced by CHF. Normal control and male TO-2 cardiomyopathic hamsters were used. Thirty-week-old TO-2 hamsters were treated daily with an experimental far infrared-ray dry sauna system for 15 min at 39 degrees C followed by 20 min at 30 degrees C. This procedure raised the rectal temperatures by about 1 degrees C. Arterial endothelial nitric oxide (NO) synthase (eNOS) mRNA and protein expressions were examined, and serum concentrations of nitrate were measured. The expression of eNOS mRNA in the aortas of normal controls did not change, whereas those of the TO-2 hamsters decreased with age. Four weeks of sauna therapy significantly increased eNOS mRNA expression in the aortas of TO-2 hamsters compared with those that did not undergo sauna therapy. Sauna therapy also upregulated aortic eNOS protein expression. Serum nitrate concentrations of the TO-2 hamsters were increased by 4 weeks of sauna therapy compared with those that did not undergo sauna. Repeated sauna therapy increases eNOS expression and NO production in cardiomyopathic hamsters with heart failure.

Sauna therapy increases nitric oxide production through increasing arterial endothelial, but not inducible, nitric oxide synthase in heart failure

Purpose: Recent studies demonstrated that vascular endothelial dysfunction is involved in pathophysiology of chronic heart failure (CHF), and is mainly due to the decreased nitric oxide (NO) production by the decreased endothelial NO synthase (eNOS) expression and the increased oxidative stress. We previously reported that sauna therapy, using far infrared-ray dry sauna at 60 degrees centigrade, improves hemodynamics, vascular endothelial and cardiac function and clinical symptoms in CHF. The purpose of this study is to investigate the mechanisms by which sauna therapy improves endothelial function. Methods: Male TO-2 cardiomyopathic hamsters were used and were treated daily by sauna in which their rectal temperature raised almost 1 degree centigrade as clinical setting. Immunohistochemistry, Western blotting, reverse transcription-polymerase chain reaction and Griess regent reaction assay were performed to assess aortic eNOS, inducible NO synthase (iNOS) and concentration of serum nitrate. Results: Both eNOS mRNA and protein expression in aortas of TO-2 decreased with age. Four-week sauna therapy significantly increased eNOS mRNA and protein expression about 2 folds compared with those without sauna, but not modulated iNOS protein expression. Serum nitrate concentrations were increased by 4-week sauna therapy compared with those without sauna. Conclusions: Repeated sauna therapy increases NO production by the upregulation of the decreased eNOS expression in cardiomyopathic hamsters with heart failure, suggesting the improvement of endothelial function in CHF.

Enhanced nitric oxide production is closely associated with serum lipid concentrations in adolescents

Background: To investigate whether nitric oxide (NO) production is associated with serum lipid concentrations and body mass index (BMI), we measured serum nitrate and nitrites (NOx) concentrations, serum lipid profiles, and anthropometric parameters in 319 adolescents. Methods: Serum NOx concentrations were determined using the Griess reaction. Serum concentrations of triglyceride, total cholesterol, and low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were measured by standard enzymatic procedures. Results: Subjects with increased serum cholesterol or triglyceride concentrations exhibited remarkably high NOx levels. Total cholesterol and triglyceride averaged 161.5±27.4 and 205.9±107.8 mg/dl in males with NOx >92.8 μmol/l (upper 20%), which were significantly above the values (132.4±17.2 and 58.1±20.3 mg/dl) in those with NOx <15.6 μmol/l (lower 20%). The prevalences of male adolescents with increased concentrations of cholesterol and triglyceride were significantly higher in the subjects with NOx ≥51.2 μmol/l than in those with NOx <51.2 μmol/l (8.9% and 22.2% vs. 1.6% and 2.3%, p<0.05, respectively). Correlation coefficients of serum lipid concentrations and anthropometric parameters vs. serum NOx concentrations were higher in males than in females for cholesterol ( r=0.28 vs. 0.23), triglyceride ( r=0.51 vs. 0.42), HDL-C ( r=−0.25 vs. −0.16), and BMI ( r=0.39 vs. 0.27). Conclusions: NO production is closely associated with serum lipid concentrations in adolescents, and these associations are stronger in males than in females.

Increases in Serum Nitrite and Nitrate of a Few-Fold Adversely Affect the Outcome of Pregnancy in Rats

The objective of this study was to evaluate serum nitrite and nitrate (nitrite/nitrate) concentrations that affect adversely pregnancy outcome. Pregnant rats, from day 2 to day 8 of pregnancy, were daily given subcutaneously several doses (5, 10, and 30 mg/rat) of diethylenetriamine-nitric oxide (DETA/NO). Serum nitrite/nitrate concentrations were measured using an HPLC system. Serum nitrite/nitrate concentrations increased dose-dependently with DETA/NO. Effects of DETA/NO on pregnancy outcome were assessed on day 14 of pregnancy. In rats given 5 mg DETA/NO, there was a significant increase in serum nitrite/nitrate concentrations (49.2 vs 24.6 μmol/l, P<0.001), and both placental weight and fetal weight decreased compared to control rats. Macroscopic bleeding in placenta was frequently observed in rats given DETA/NO. We further studied effects of DETA/NO on cultured trophoblastic BeWo cells. DETA/NO added to the culture medium increased nitrite/nitrate concentrations in the medium in a dose-dependent manner. Nitrite/nitrate concentrations in the medium over four times the concentration of control decreased progesterone in the medium at 24 h after the application of DETA/NO. The hormonal secretion was not affected by DETA only. This study shows for the first time nitrite/nitrate concentrations affecting adversely pregnancy outcome and function of the trophoblastic cells.

Inhibition of reactive nitrogen species effects in vitro and in vivo by isoflavones and soy-based food extracts.

Recent studies have shown that soy isoflavone inhibits inducible nitric oxide (NO) synthase activities and is reported to have peroxynitrite scavenging ability. Consequently, we investigated whether isoflavones (daidzein and genistein) and extracts from soy-based products (miso, soymilk, tofu, soy sprout, black soybean, soybean, and yuba) would inhibit the reactive nitrogen species (RNS) effect in vitro and in vivo. In the in vitro experiments [including the protection of cellular DNA from peroxynitrite or sodium nitroprusside damage, an inhibitory effect on nitric oxide production from lipopolysaccharide (LPS)-induced RAW 264.7 cells, and nitric oxide scavenging ability], extracts from soy-based foods showed a potent antioxidant activity and an inhibiting effect on RNS activity. These effects were correlated with total isoflavone content. In the in vivo experiments, rats were given isoflavones (4.0 mg/kg bw) or soy-based product extracts (1.0 g/kg bw) orally for 1 week and were injected with vehicle H(2)O (1 mL/kg bw) or LPS (10 mg/kg bw) on the day 7. Twelve hours after treatment, the rats were killed, and blood serum was collected for analysis. The intraperitoneal administration of LPS resulted in an increase in serum nitrite, nitrate, and nitrotyrosine concentrations. These are stable metabolite end products of nitric oxide, to 4-, 16-, and 5-fold levels, (4, 10 microM and 58 +/- 14 pmol/mL), of the placebo control, respectively. Results showed that oral administration of isoflavones and extracts from soy-based products significantly decreased serum nitrite, nitrate, and nitrotyrosine levels in LPS-induced rats. This study demonstrates that soy isoflavone supplementation may inhibit RNS-induced oxidation both in vitro and in vivo.