Measurement of nitrite in plasma and serum: still a challenging analytical task

To measure cerebrospinal fluid and plasma nitrite and nitrate concentrations as indicators of nitric oxide production in adults after severe closed-head injury. To determine if there is an association between cerebrospinal fluid and plasma nitrite and nitrate concentrations, and cerebral blood flow, arterio-jugular oxygen content difference, injury severity, and outcome after severe closed-head injury. A prospective, clinical study. Multidisciplinary intensive care unit. Fifteen comatose (Glasgow Coma Scale score of < or = 7) adult patients with severe closed-head injury were studied during the prospective, randomized evaluation of the effect of moderate hypothermia (32 degrees C for 24 hrs) on neurologic outcome after closed-head injury. Seven patients were in the hypothermic group and eight patients were in the normothermic treatment group. None. Patients were examined sequentially, every 12 hrs for 2 days. Intraventricular cerebrospinal fluid was assayed for nitrite and nitrate concentrations. Cerebral blood flow was measured by the 133xenon intravenous method. Simultaneous blood samples were obtained for measurements of arterio-jugular oxygen content difference and plasma nitrite and nitrate concentrations. Cerebral metabolic rate for oxygen was calculated. Cerebrospinal fluid nitrite and nitrate concentrations were highest at 30 to 42 hrs vs. 6 to 18, 18 to 30, and 42 to 54 hrs (26.4 +/- 3.3 vs. 17.3 +/- 2.1, 20.0 +/- 2.2, and 18.8 +/- 2.4 microM, respectively, p < .05). There was no difference over time in plasma nitrite and nitrate concentrations. Cerebral blood flow was increased and arterio-jugular oxygen content difference was reduced at 18 to 30, 30 to 42, and 42 to 54 hrs vs. 6 to 18 hrs (p < .05). At 30 to 42 hrs, cerebrospinal fluid nitrite and nitrate concentrations were 80% higher in patients who died vs. survivors (36.4 +/- 3.2 vs. 20.2 +/- 3.6, p < .05). Using a generalized, multivariate, linear regression model, both plasma nitrite and nitrate concentrations and injury Severity Score independently predicted cerebrospinal fluid nitrite and nitrate concentrations (p < .00001 and p = .0053, respectively). Cerebral blood flow and arterio-jugular oxygen content difference were not associated with cerebrospinal fluid or plasma nitrite and nitrate concentrations using this model. Cerebrospinal fluid nitrite and nitrate concentrations were increased over time in hypothermic vs. normothermic patients. But, where this difference occurred could not be determined by multiple comparisons (p = .03). The hypothermic patients had lower admission Glasgow Coma Scale scores than normothermic patients (p = .04) and tended to have higher injury Severity Scores (p = .09). Increases in cerebrospinal fluid nitrite and nitrate concentrations peaked at 30 to 42 hrs after severe closed-head injury. This increase in cerebrospinal fluid nitrite and nitrate concentrations was greater in nonsurvivors. Also, cerebrospinal fluid and plasma nitrite and nitrate concentrations were associated with injury Severity Score, suggesting that increased nitric oxide production in the brain is associated with injury severity and death. Hypothermia did not prevent the increase in cerebrospinal fluid nitrite and nitrate concentrations. Further study is required to determine the source of this increase in cerebrospinal fluid nitrite and nitrate concentrations and to further define the relationship to outcome and the effect of hypothermia on this process.

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. Venous blood samples were collected from healthy male subjects and plasma aliquots were then immediately removed and stored at -70 degrees C until analysed in duplicate for their nitrite and nitrate content using ozone-based chemiluminescence assays. Plasma cGMP levels were determined by using a commercial enzyme immunoassay. While we found no significant correlation between plasma nitrite and nitrate concentrations (P = 0.747), or between plasma nitrate and cGMP concentrations (P = 0.221), a significant positive correlation was found between plasma cGMP and nitrite concentrations (P = 0.017, r(s) = 0.270). The significant correlation we found between plasma nitrite and cGMP concentrations is consistent with the notion that nitrite or cGMP concentrations in plasma may be useful clinical markers of NO formation in healthy subjects.

BackgroundDietary nitrate consumption can increase concentrations of nitrate and nitrite in blood, saliva, and urine. Whether the change in concentrations is influenced by age is currently unknown. ObjectivesWe aimed to measure changes in nitrate and nitrite concentrations in plasma, urine, and saliva and exhaled NO concentrations after single incremental doses of dietary nitrate in young and older healthy adults. MethodsTwelve young (18–35 y old) and 12 older (60–75 y old) healthy, nonsmoking participants consumed single doses of 100 g, 200 g, 300 g whole beetroot (BR) and 1000 mg potassium nitrate (positive control) ≥7 d apart in a crossover, randomized clinical trial. Plasma nitrate and nitrite concentrations and exhaled NO concentrations were measured over a 5-h period. Salivary nitrate and nitrite concentrations were measured over a 12-h period and urinary nitrate over a 24-h period. Time, intervention, age, and interaction effects were measured with repeated-measures ANOVAs. ResultsDose-dependent increases were seen in plasma, salivary, and urinary nitrate after BR ingestion (all P ≤ 0.002) but there were no differences between age groups at baseline (all P ≥ 0.56) or postintervention (all P ≥ 0.12). Plasma nitrite concentrations were higher in young than older participants at baseline (P = 0.04) and after consumption of 200 g (P = 0.04; +25.7 nmol/L; 95% CI: 0.97, 50.3 nmol/L) and 300 g BR (P = 0.02; +50.3 nmol/L; 95% CI: 8.57, 92.1 nmol/L). Baseline fractional exhaled NO (FeNO) concentrations were higher in the younger group [P = 0.03; +8.60 parts per billion (ppb); 95% CI: 0.80, 16.3 ppb], and rose significantly over the 5-h period, peaking 5 h after KNO3 consumption (39.4 ± 4.5 ppb; P < 0.001); however, changes in FeNO were not influenced by age (P = 0.276). ConclusionsBR is a source of bioavailable dietary nitrate in both young and older adults and can effectively raise nitrite and nitrate concentrations. Lower plasma nitrite and FeNO concentrations were found in older subjects, confirming the impact of ageing on NO bioavailability across different systems.This trial was registered at www.isrctn.com as ISRCTN86706442.

To determine whether plasma nitrite and nitrate concentrations are associated with the development of sepsis-induced multiple organ failure. Prospective study. University children's hospital. Fifty-three consecutive children meeting criteria for sepsis and not receiving exogenous sources of nitric oxide. Plasma nitrite and nitrate concentrations were measured, and the number of organs failing was scored using an organ failure index on the first 3 days of sepsis. Children with three or more organs failing on day 3 of sepsis had higher plasma nitrite and nitrate concentrations than children who had resolution of failure of three or more organs by day 3 of sepsis (days 2 and 3) and children who never had three organs failing in the first 3 days of sepsis (days 1, 2, and 3). Children who developed sequential pulmonary/hepatic/renal organ failure had significantly higher plasma nitrite and nitrate concentrations (days 1, 2, and 3). Nonsurvivors had significantly higher plasma nitrite and nitrate concentrations (days 2 and 3) than survivors. Plasma nitrite and nitrate concentrations on day 1 predicted the development of persistent failure of three of more organs and sequential multiple organ failure but not mortality. Increased plasma nitrite and nitrate concentrations are associated with the development of multiple organ failure in pediatric sepsis.

Nitrate-rich beetroot juice supplementation has been shown to improve cardiovascular and cognitive function in younger and older adults via increased nitric oxide production. However, it is unclear whether the level of effects differs between the two groups. We hypothesized that acute supplementation with nitrate-rich beetroot juice would improve cardiovascular and cognitive function in older and younger adults, with the potential for greater improvements in older adults. Thirteen younger (18–30 years) and 11 older (50–70 years) adults consumed either 150 mL of nitrate-rich beetroot juice (BR; 10.5 mmol nitrate) or placebo (PL; 1 mmol nitrate) in a double-blind, crossover design, 2.25 h prior to a 30-min treadmill walk. Plasma nitrate and nitrite concentrations, blood pressure (BP), heart rate (HR), cognitive function, mood and perceptual tests were performed throughout the trial. BR consumption significantly increased plasma nitrate (p < 0.001) and nitrite (p = 0.003) concentrations and reduced systolic BP (p < 0.001) in both age groups and reduced diastolic BP (p = 0.013) in older adults. Older adults showed a greater elevation in plasma nitrite (p = 0.038) and a greater reduction in diastolic BP (p = 0.005) following BR consumption than younger adults. Reaction time was improved in the Stroop test following BR supplementation for both groups (p = 0.045). Acute BR supplementation increased plasma nitrite concentrations and reduced diastolic BP to a greater degree in older adults; whilst systolic BP was reduced in both older and younger adults, suggesting nitrate-rich BR may improve cardiovascular health, particularly in older adults due to the greater benefits from reductions in diastolic BP.

Dietary nitrate has been reported to lower oxygen consumption in moderate- and severe-intensity exercise. To date, it is unproven that sodium nitrate (NaNO3(-); NIT) and nitrate-rich beetroot juice (BR) have the same effects on oxygen consumption, blood pressure, and plasma nitrate and nitrite concentrations or not. The aim of this study was to compare the effects of different dosages of NIT and BR on oxygen consumption in male athletes. Twelve healthy, well-trained men (median [minimum; maximum]; peak oxygen consumption: 59.4 mL·min(-1)·kg(-1) [40.5; 67.0]) performed 7 trials on different days, ingesting different nitrate dosages and placebo (PLC). Dosages were 3, 6, and 12 mmol nitrate as concentrated BR or NIT dissolved in plain water. Plasma nitrate and nitrite concentrations were measured before, 3 h after ingestion, and postexercise. Participants cycled for 5 min at moderate intensity and further 8 min at severe intensity. End-exercise oxygen consumption at moderate intensity was not significantly different between the 7 trials (p = 0.08). At severe-intensity exercise, end-exercise oxygen consumption was ~4% lower in the 6-mmol BR trial compared with the 6-mmol NIT (p = 0.003) trial as well as compared with PLC (p = 0.010). Plasma nitrite and nitrate concentrations were significantly increased after the ingestion of BR and NIT with the highest concentrations in the 12-mmol trials. Plasma nitrite concentration between NIT and BR did not significantly differ in the 6-mmol (p = 0.27) and in the 12-mmol (p = 0.75) trials. In conclusion, BR might reduce oxygen consumption to a greater extent compared with NIT.

Stable-isotope dilution LC-MS/MS measurement of nitrite in human plasma after its conversion to S-nitrosoglutathione.

Aims: To investigate the effects of supplementation with high-nitrate and low-nitrate vegetables on plasma nitrate and nitrite concentrations, blood pressure and the oxygen demand of moderate-intensity exercise.&#x0D; Study Design: A randomized, cross-over design.&#x0D; Place and Duration of Study: Sport and Health Sciences, College of Life and Environmental Sciences, University of Exeter, between January 2011 and March 2012.&#x0D; Methodology: 15 non-smoking, physically active healthy men (age 25 ± 6 years, BMI 24 ± 4 kg/m2) were randomized to receive a 2-week supply of high-nitrate or low-nitrate vegetables, with a 2-week ‘wash-out’ period in between. Clinic blood pressure, plasma nitrate and nitrite concentrations and physiological responses to moderate-intensity exercise tests were measured before and after each 2-week intervention. Nitrate intake was calculated using nutritional analysis of reported vegetables consumed.&#x0D; Results: Participants consumed significantly more dietary nitrate on the high-nitrate diet (417 ± 139 mg/day) than the low-nitrate diet (26 ± 11 mg/day). The high-nitrate diet supplied 5.5 mg nitrate/kg body weight, exceeding the Acceptable Daily Intake (ADI) of 3.7 mg nitrate/kg body weight. Supplementation with high-nitrate vegetables significantly increased plasma nitrate concentrations (baseline; 30 ± 20 µM, after high-nitrate vegetables; 129 ± 87 µM) and plasma nitrite concentrations (baseline; 119 ± 35 nM, after high-nitrate vegetables; 227 ± 89 nM) but did not significantly change systolic blood pressure or the physiological response to moderate exercise. There were significant correlations between diastolic blood pressure and plasma nitrite concentrations (low-nitrate diet; r = 0.63, high-nitrate diet, r = 0.56).&#x0D; Conclusion: Supplementation with high-nitrate vegetables above the ADI significantly increased plasma nitrate and nitrate concentrations but did not significantly reduce systolic blood pressure or the physiological response to moderate exercise. Plasma nitrite concentrations significantly correlated with diastolic blood pressure after high-nitrate and low-nitrate diets.

Blood pressure-lowering effects of propofol or sevoflurane anaesthesia are not due to enhanced nitric oxide formation or bioavailability.

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.

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

Plasma nitrite concentrations reflect the degree of endothelial dysfunction in humans

Nitrate-Rich Vegetables Increase Plasma Nitrate and Nitrite Concentrations and Lower Blood Pressure in Healthy Adults

Context: Endothelial dysfunction has been suggested as a potential mechanism by which ambient air pollution may cause acute cardiovascular events. Recently, plasma nitrite has been developed as a marker of endothelial dysfunction.Objectives: We examined the changes in plasma nitrite concentration associated with increases in ambient air pollutant concentrations in the previous 7 d.Materials and methods: We linked up to three measurements of plasma nitrite concentrations obtained from 49 students to 24-h average concentrations of five criteria air pollutants [particle mass < 2.5 µm in aerodynamic diameter (PM2.5), carbon monoxide (CO), sulfur dioxide (SO2), nitrogen dioxide (NO2), and ozone (O3)] measured at two monitoring sites closest to Rutgers University campus (6–15 miles) in New Jersey during the years 2006–2009. We examined the change in plasma nitrite associated with each interquartile-range (IQR) increase in pollutant concentration in the previous 24 h and six preceding 24- h periods, using linear mixed models.Results: IQR increases in mean PM2.5 (7.0 µg/m3) and CO (161.7 parts per billion) concentrations in the first 24 h before the plasma nitrite measurement were associated with increased plasma nitrite concentrations (PM2.5: 15.5 nanomolar; 95% confidence interval (CI): 2.4, 28.5; CO: 15.6 nanomolar; 95% CI: 2.4, 28.9). Increased plasma nitrite associated with IQR increases in O3 and SO2 concentrations over longer lags were observed.Discussion and conclusion: Rapid increases in plasma nitrite following exposure to ambient air pollutants support the hypothesis that ambient air pollution is associated with inducible nitric oxide synthase-mediated systemic inflammation in humans.

Simultaneous GC-ECNICI-MS measurement of nitrite, nitrate and creatinine in human urine and plasma in clinical settings.