Whole Blood Nitrite Research Articles

21 - Arginase Inhibitor Reacts with Hemoglobin to Form Nitrite

Endothelial nitric oxide synthase (eNOS) is found in endothelial cells and has recently been found to also be present in red blood cells (RBCs). eNOS can catalyze L-arginine conversion to citrulline and produce nitric oxide (NO). L-arginine can also be converted to L-ornithine and urea through the activity of arginase which consumes L-arginine in blood. Due to their having a common substrate (arginine), arginase would compete with eNOS and limit NO signaling. Inside the RBC, NO formed from NOS is expected to form nitrate through its reaction with oxygenated hemoglobin, but nitrite formation has also been reported. In our study, Nω-hydroxy-nor-Arginine (nor-NOHA, 0.1mM and 1mM), an arginase inhibitor, was added to either RBCs, whole blood, or isolated hemoglobin. An arginase inhibitor is expected to increases RBC nitrite and nitrate due to increases in arginine available for NOS and this has been shown by others. We observed that all the samples treated with nor-NOHA produce more nitrite and nitrate than without nor-NOHA. That nor-NOHA would increase nitrite and nitrate in RBCs and whole blood is to be expected, but Hb, by itself should not result in these NO metabolites if their increase is due to NOS activity. Thus, we suspect that nitrite and nitrate are formed from a direct reaction of hemoglobin and nor-NOHA. When 3 mM arginine was added to whole blood, nitrite yields significantly increased. However, when arginine was added to RBCs alone, there was no significant increase in nitrite. Addition of the NOS inhibitor L-NG-Nitroarginine methyl ester (L-NAME) resulted in significant reductions in nitrite, supporting some NOS activity. However, in the presence of nor-NOHA, L-NAME had little to no effect on RBC and whole blood formation of nitrite, consistent with the proposal that most of the nitrite formed is due to a direct reaction of hemoglobin with nor-NOHA. Our data suggest that addition of nor-NOHA to RBCs with the goal using increased nitrite and nitrate as a method to study NOS activity is unwarranted. These results have implications in past and future use of nor-NOHA that perhaps incorrectly purport to be studying RBC NOS.

Determination of Nitrite in Whole Blood by High-Performance Liquid Chromatography with Electrochemical Detection and a Case of Nitrite Poisoning.

Although nitrite is widely used in meat processing, it is a major toxicity hazard to children and is responsible for the blue-baby syndrome. A simple and effective method to determine nitrite in whole blood has been devised using ion chromatography with suppressed conductivity detection. The blood sample was deproteinized by adding acetonitrile and purified with mini-cartridges to remove hydrophobic compounds, chloride ions, and metal ions. An aliquot of the filtrate was injected onto the ion chromatography. The retention time for nitrite was 13.8 min and the detection limit of nitrite in whole blood was 0.4 μmol/L. The calibration curve was linear (r(2) = 0.9999) over the concentration working range. The blood nitrite concentration of a victim who attempted suicide by ingesting sodium nitrite powder was determined using the present method. The basal levels for nitrite in human blood was determined with 7.1 ± 0.9 μmol/L (n = 12).

Erythrocyte storage increases rates of NO and nitrite scavenging: implications for transfusion-related toxicity

Storage of erythrocytes in blood banks is associated with biochemical and morphological changes to RBCs (red blood cells). It has been suggested that these changes have potential negative clinical effects characterized by inflammation and microcirculatory dysfunction which add to other transfusion-related toxicities. However, the mechanisms linking RBC storage and toxicity remain unclear. In the present study we tested the hypothesis that storage of leucodepleted RBCs results in cells that inhibit NO (nitric oxide) signalling more so than younger cells. Using competition kinetic analyses and protocols that minimized contributions from haemolysis or microparticles, our data indicate that the consumption rates of NO increased ~40-fold and NO-dependent vasodilation was inhibited 2-4-fold comparing 42-day-old with 0-day-old RBCs. These results are probably due to the formation of smaller RBCs with increased surface area: volume as a consequence of membrane loss during storage. The potential for older RBCs to affect NO formation via deoxygenated RBC-mediated nitrite reduction was also tested. RBC storage did not affect deoxygenated RBC-dependent stimulation of nitrite-induced vasodilation. However, stored RBCs did increase the rates of nitrite oxidation to nitrate in vitro. Significant loss of whole-blood nitrite was also observed in stable trauma patients after transfusion with 1 RBC unit, with the decrease in nitrite occurring after transfusion with RBCs stored for >25 days, but not with younger RBCs. Collectively, these data suggest that increased rates of reactions between intact RBCs and NO and nitrite may contribute to mechanisms that lead to storage-lesion-related transfusion risk.

Stable-isotope dilution GC–MS approach for nitrite quantification in human whole blood, erythrocytes, and plasma using pentafluorobenzyl bromide derivatization: Nitrite distribution in human blood

Previously, we reported on the usefulness of pentafluorobenzyl bromide (PFB-Br) for the simultaneous derivatization and quantitative determination of nitrite and nitrate in various biological fluids by GC–MS using their 15N-labelled analogues as internal standards. As nitrite may be distributed unevenly in plasma and blood cells, its quantification in whole blood rather than in plasma or serum may be the most appropriate approach to determine nitrite concentration in the circulation. So far, GC–MS methods based on PFB-Br derivatization failed to measure nitrite in whole blood and erythrocytes because of rapid nitrite loss by oxidation and other unknown reactions during derivatization. The present article reports optimized and validated procedures for sample preparation and nitrite derivatization which allow for reliable quantification of nitrite in human whole blood and erythrocytes. Essential measures for stabilizing nitrite in these samples include sample cooling (0–4 °C), hemoglobin (Hb) removal by precipitation with acetone and short derivatization of the Hb-free supernatant (5 min, 50 °C). Potassium ferricyanide (K 3Fe(CN) 6) is useful in preventing Hb-caused nitrite loss, however, this chemical is not absolutely required in the present method. Our results show that accurate GC–MS quantification of nitrite as PFB derivative is feasible virtually in every biological matrix with similar accuracy and precision. In EDTA-anticoagulated venous blood of 10 healthy young volunteers, endogenous nitrite concentration was measured to be 486 ± 280 nM in whole blood, 672 ± 496 nM in plasma ( C P), and 620 ± 350 nM in erythrocytes ( C E). The C E-to- C P ratio was 0.993 ± 0.188 indicating almost even distribution of endogenous nitrite between plasma and erythrocytes. By contrast, the major fraction of nitrite added to whole blood remained in plasma. The present GC–MS method is useful to investigate distribution and metabolism of endogenous and exogenous nitrite in blood compartments under basal conditions and during hyperemia.

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.

Simultaneous Analysis of Nitrite and Nitrate in Whole Blood by Ion Chromatography

A simple and modern method for simultaneous analysis of nitrite and nitrate in whole blood has been devised by ion chromatography with an autosuppressor and a conductivity detector. To our knowledge, this is the first report for such analysis dealing with whole blood as a specimen. A 1 mL volume of whole blood was mixed with 2 mL of Milli Q water, vortex‐mixed for hemolysis, and ultrafiltered to obtain a clear extract solution. It was passed through double connected mini-cartridges of silver resin and a silver absorbing material to remove a high concentration of chloride ion usually present in whole blood. An aliquot of the filtrate was analyzed on a Dionex DX‐500 instrument with a microbore IonPac® AS9‐HC column (2×250 mm). The present method gave very low backgrounds for blank whole blood at locations where nitrite should appear, and around the endogenous peak of nitrate. The spiked nitrite markedly decreased in whole blood, probably by the action of hemoglobin in vitro; it was only 10–20% 2 h after the addition. Therefore, quantitative validation had to be made only for nitrate. The recoveries of nitrate from whole blood were more than 90%; the linearity was confirmed in the range of 5–100 µg mL−1 for whole blood. Detection limit of nitrate in whole blood was about 0.5 µg mL−1. Coefficients of intra‐ and inter‐day variations were 3.66–7.14%. Using the present method, nitrite and nitrate concentrations were measured for human blood specimens after sniffing isobutyl nitrite gas in two male subjects. The endogenous nitrate concentrations without such sniffing were 1.27 to 7.60 µg mL−1 for seven subjects.

Erythrocytes are the major intravascular storage sites of nitrite in human blood

AbstractPlasma levels of nitrite ions have been used as an index of nitric oxide synthase (NOS) activity in vivo. Recent data suggest that nitrite is a potential intravascular repository for nitric oxide (NO), bioactivated by a nitrite reductase activity of deoxyhemoglobin. The precise levels and compartmentalization of nitrite within blood and erythrocytes have not been determined. Nitrite levels in whole blood and erythrocytes were determined using reductive chemiluminescence in conjunction with a ferricyanide-based hemoglobin oxidation assay to prevent nitrite destruction. This method yields sensitive and linear measurements of whole blood nitrite over 24 hours at room temperature. Nitrite levels measured in plasma, erythrocytes, and whole blood from 15 healthy volunteers were 121 plus or minus 9, 288 plus or minus 47, and 176 plus or minus 17 nM, indicating a surprisingly high concentration of nitrite within erythrocytes. The majority of nitrite in erythrocytes is located in the cytosol unbound to proteins. In humans, we found a significant artery-to-vein gradient of nitrite in whole blood and erythrocytes. Shear stress and acetylcholine-mediated stimulation of endothelial NOS significantly increased venous nitrite levels. These studies suggest a dynamic intravascular NO metabolism in which endothelial NOS-derived NO is stabilized as nitrite, transported by erythrocytes, and consumed during arterial-to-venous transit. (Blood. 2005;106:734-739)

Characterization of the Distribution of Nitrite in Human Blood.

Several groups have recently shown that blood can transport nitric oxide (NO) bioactivity so that NO can act as an endocrine signal transducer. The mechanisms of this process are uncertain. We have recently shown that nitrite ions can generate NO in the presence of deoxygenated hemoglobin (Cosby et al. Nature Medicine 2003). Thus, nitrite appears to be a major intravascular NO storage molecule and hemoglobin a nitrite reductase contributing to erythrocyte-dependent hypoxic vasodilation. Such a model requires a reaction of nitrite with intra-erythrocytic hemoglobin, however, published uptake rates of nitrite from plasma into erythrocytes are very slow and dynamic blood flow regulation requires more rapid responses. We therefore hypothesized that the red blood cell itself might be a reservoir for nitrite, with compartmentalization of nitrite and deoxyhemoglobin at the erythrocyte membrane; such a metabolon would allow for rapid nitrite reductase chemistry and NO export as hemoglobin deoxygenates. Nitrite was measured using reductive chemiluminescence in conjunction with a novel ferricyanide-based hemoglobin oxidation assay to rapidly preserve nitrite in the presence of hemoglobin following red cell lysis. We find that measurement of whole blood nitrite using ferricyanide-based hemoglobin oxidation to stabilize nitrite is a valid, sensitive, linear, and reproducible surrogate for RBC nitrite. Using the stabilization solution, nitrite in whole blood remained constant over 12 h at room temperature, whereas without it the half life of nitrite was 12 ± 4 min. Using this novel methodology, the nitrite levels measured in plasma, RBC, and calculated and measured in whole blood in normal volunteers were 121 ± 9, 620 ± 170, 245 ± 58, and 323 ± 44 nmol/L. The difference between measured and calculated blood nitrite levels reflects destruction of RBC nitrite by hemoglobin during sample processing. There was an artery-to-vein gradient for blood nitrite (A. vs V.: 169 ± 12 vs. 149 ± 11 nmol/L; p=0.03). Intraperitoneal injection of L-NAME (a potent NOS-inhibitor; 100 mg/kg) in mice caused a time dependent decrease of whole blood nitrite (0 h: 650 ± 29, 2h: 556 ± 26, 4h: 455 ± 11, 421 ± 19, 24 h: 551 ± 30 nmol/L; p=0.002). Differences of blood nitrite across the human forearm circulation correlated significantly (r2=0.94) with forearm bloodflow after infusion of acetylcholine (7.5 ug/min; n=5), consistent with nitrite formation across the vasculature with eNOS activation. We conclude that in humans, the RBC nitrite concentration is 5 times greater than plasma nitrite concentrations; A-V gradients in blood nitrite are consistent with nitrite utilization as a source of bioactive NO in the microvasculature and levels of blood nitrite are controlled by endogenous NO production. Levels of nitrite in the RBC may function as a locally available source of nitrite for deoxyhemoglobin-dependent nitrite reduction to NO, thus allowing for more efficient nitrite-deoxyhemoglobin mediated hypoxic vasodilation. Modulation of intraerythrocytic nitrite may be of therapeutic interest in diseases with a lack of bioavailable NO, such as sickle cell disease.

Solvent-free sample preparation by headspace solid-phase microextraction applied to the tracing of n-butyl nitrite abuse.

The most common alkyl nitrites encountered in forensic toxicology are iso-butyl, n-butyl and iso-pentyl(amyl) nitrites. All have become popular as an aphrodisiac, especially among the homosexual population. Alkyl nitrites are a volatile and unstable group of compounds, which hydrolyse in aqueous matrices to the alcohol and nitrite ion. Here we describe a fast, clean and sensitive procedure for the detection of hydrolysed n-butyl nitrite in whole human blood using a new, solvent-free sampling technique, the headspace solid-phase micro-extraction (HSPME), combined with GC/FID analysis. Sample preparation was investigated using two different stationary phases (100 microns polydimethylsiloxane and 85 microns polyacrylate), coating a fused silica fibre. The effect of different sampling times at fixed temperatures was also studied. Our results demonstrate that the HSPME/GC/FID procedure allows tracing of n-butyl nitrite abuse and detects hydrolysed n-butyl nitrite, i.e., released n-butanol, in whole blood at the 1 ng/mL level.

Nitrite and nitrate determinations in plasma: a critical evaluation

Plasma nitrite and nitrate determinations are increasingly being used in clinical chemistry as markers for the activity of nitric oxide synthase and the production of nitric oxide radicals. However, a systematic evaluation of the determination of nitrite and nitrate in plasma has not been performed. In this study the recovery and stability of nitrite and nitrate in whole blood and in plasma, the relation between nitrite and nitrate concentrations in plasma, and possible sources of artifacts were investigated. The main conclusions are: (a) Recovery of nitrite and nitrate from plasma is near-quantitative (87%) and reproducible; (b) nitrite and nitrate are stable in (frozen) plasma for at least 1 year; (c) nitrite in whole blood is very rapidly (> 95% in 1 h) oxidized to nitrate, and therefore plasma nitrite determination alone is meaningless; (d) the ranges of nitrite and nitrate concentrations in plasma samples of 26 healthy persons are 1.3-13 mumol/L (mean 4.2 mumol/L) and 4.0-45.3 mumol/L (mean 19.7 mumol/L), respectively; (e) plasma nitrite and nitrate concentrations were not correlated (nitrite as % of total nitrite + nitrate varied from 3.9% to 88% in plasma samples); and (f) plasma samples should be deproteinized, and background controls for each sample should be included in the assay, to avoid measuring artifactually high nitrite and nitrate concentrations in plasma.