Inhibitors of the nitrite oxidation of hemoglobin

Different types of active inhibitors of the reaction of nitrite hemoglobin oxidation have been revealed and studied. The dependence of inhibition of methemoglobin formation, on concentration of inhibitors at pH 5.9 and 7.17 has been determined. Differential absorption spectra of the inhibitors in the presence sodium nitrite in UV and visual light has been studied. The values of oxidation-reduction potentials have been estimated. Possible mechanism of action of the inhibitors has been discussed.     

The reaction between nitrite and hemoglobin: the role of nitrite in hemoglobin-mediated hypoxic vasodilation

The reaction between nitrite and hemoglobin has been studied for over a century. However, recent evidence indicating nitrite is a latent vasodilatory agent that can be activated by its reaction with deoxyhemoglobin has led to renewed interest in this reaction. In this review we survey, in the context of our own recent studies, the chemical reactivity of nitrite with oxyhemoglobin, deoxyhemoglobin and methemoglobin, and place these reactions in both a physiological and pharmacological/therapeutic context.     

Effect of inositol hexaphosphate on hemoglobin oxidation by nitrite and ferricyanide

In the presence of inositol hexaphosphate (IHP), the rate of hemoglobin oxidation by nitrite was much inhibited; however, that of the hemoglobin oxidation by ferricyanide was much accelerated. The difference in the reaction mode was discussed in relation to the interaction of hemoglobin with IHP. The dissociation constant of IHP to oxyhemoglobin was estimated from the rate of the hemoglobin oxidation by ferricyanide in different concentrations of IHP under oxygen saturated conditions.     

Combined effect of gamma-irradiation and sodium nitrite on the oxidative sensitivity of rat hemoglobin

gamma-Irradiation has been defined to increase in the rats blood the methemoglobin level providing for shortening the initiation phase and accelerates the autocatalytic phase initiation, reduces the period of half transforming hemoglobin into methemoglobin and increases the velocity of its oxidation. Alongside with the latter there is observed a violation of methemoglobin concentration growth dependence on the animals irradiation dose (in the range of 0.16-0.50 Gr). The hemoglobin oxygenation reaction kinetics with the initial level of hemoglobin unexceeding 3% has been determined as having a biexponential character. The reaction kinetics parameters don't depend on ionizing radiation and number of sodium nitrite oxidized subunits formed in the process of reaction in the case if their composition unexceeds 50% of the total level.     

Effect of wall growth on the kinetic modeling of nitrite oxidation in a CSTR

A simple kinetic model was developed for describing nitrite oxidation by autotrophic aerobic nitrifiers in a continuous stirred tank reactor (CSTR), in which mixed (suspended and attached) growth conditions prevail. The CSTR system was operated under conditions of constant nitrite feed concentration and varying volumetric flow rates. Experimental data from steady-state conditions in the CSTR system and from batch experiments were used for the determination of the model's kinetic parameters. Model predictions were verified against experimental data obtained under transient operating conditions, when volumetric flow rate and nitrite feed concentration disturbances were imposed on the CSTR. The presented kinetic modeling procedure is quite simple and general and therefore can also be applied to other mixed growth biological systems.     

In vitro and in vivo kinetic handling of nitrite in blood: effects of varying hemoglobin oxygen saturation

Growing evidence suggests that nitrite, acting via reduction to nitric oxide by deoxyhemoglobin, may play an important role in local control of blood flow during hypoxia. To investigate the effect of hypoxia (65 Torr arterial Po(2)) on the kinetic properties of nitrite, a bolus injection of sodium nitrite (10 mg/kg iv) was given to normoxic or hypoxic newborn lambs, and the time course of plasma nitrite and methemoglobin (MetHb) concentrations was measured. The in vivo kinetics of nitrite disappearance from plasma were biphasic and were not affected by hypoxia. Changes in MetHb, a product of the nitrite-hemoglobin reaction, also did not differ with the level of oxygenation. Hypoxia potentiated the hypotensive effects of nitrite on pulmonary and systemic arterial pressures. The disappearance of nitrite from plasma was equivalent to the increase in MetHb on a molar basis. In contrast, nitrite metabolism in sheep blood in vitro resulted in more than one MetHb per nitrite equivalent under mid- and high-oxygenation conditions: oxyhemoglobin (HbO(2)) saturation = 50.3 +/- 1.7% and 97.0 +/- 1.3%, respectively. Under the low-oxygenation condition (HbO(2) saturation = 5.2 +/- 0.9%), significantly less than 1 mol of MetHb was produced per nitrite equivalent, indicating that a significant portion of nitrite is metabolized through pathways that do not produce MetHb. These data support the idea that the vasodilating effects of nitrite are potentiated under hypoxic conditions due to the reduction of nitrite to nitric oxide by deoxyhemoglobin.     

The biochemistry of nitric oxide, nitrite, and hemoglobin: role in blood flow regulation

Nitric oxide (NO) plays a fundamental role in maintaining normal vasomotor tone. Recent data implicate a critical function for hemoglobin and the erythrocyte in regulating the activity of NO in the vascular compartment. Intravascular hemolysis releases hemoglobin from the red blood cell into plasma (cell-free plasma hemoglobin), which is then able to scavenge endothelium-derived NO 600-fold faster than erythrocytic hemoglobin, thereby disrupting NO homeostasis. This may lead to vasoconstriction, decreased blood flow, platelet activation, increased endothelin-1 expression (ET-1), and end-organ injury, thus suggesting a novel mechanism of disease for hereditary and acquired hemolytic conditions such as sickle cell disease and cardiopulmonary bypass. Furthermore, therapy with NO gas inhalation or infusion of sodium nitrite during hemolysis may attenuate this disruption in vasomotor balance by oxidizing plasma cell-free hemoglobin, thereby preventing the consumption of endogenous NO and the associated pathophysiological changes. In addition to providing an NO scavenging role in the physiological regulation of NO-dependent vasodilation, hemoglobin and the erythrocyte may deliver NO as the hemoglobin deoxygenates. While this process has previously been ascribed to S-nitrosated hemoglobin, recent data from our laboratories suggest that deoxygenated hemoglobin reduces nitrite to NO and vasodilates the human circulation along the physiological oxygen gradient. This newly described role of hemoglobin as a nitrite reductase is discussed in the context of blood flow regulation, oxygen sensing, and nitrite-based therapeutics.     

Effect of pH and nitrite concentration on nitrite oxidation rate

The effect of pH and nitrite concentration on the activity of the nitrite oxidizing bacteria (NOB) in an activated sludge reactor has been determined by means of laboratory batch experiments based on respirometric techniques. The bacterial activity was measured at different pH and at different total nitrite concentrations (TNO?). The experimental results showed that the nitrite oxidation rate (NOR) depends on the TNO? concentration independently of the free nitrous acid (FNA) concentration, so FNA cannot be considered as the real substrate for NOB. NOB were strongly affected by low pH values (no activity was detected at pH 6.5) but no inhibition was observed at high pH values (activity was nearly the same for the pH range 7.5-9.95). A kinetic expression for nitrite oxidation process including switch functions to model the effect of TNO? concentration and pH inhibition is proposed. Substrate half saturation constant and pH inhibition constants have been obtained.     

Degradation of uric acid during autocatalytic oxidation of oxyhemoglobin induced by sodium nitrite.

The oxidation of oxyhemoglobin produced by sodium nitrite occurs in two stages: 1) an initial slow phase followed by 2) a rapid autocatalytic phase that carries the reaction to completion. The length of the slow phase is extended when uric acid is added to the reaction mixture. As the concentration of uric acid increases, the length of the slow phase increases until a concentration is reached at which the rate of methemoglobin formation is nearly linear until the reaction is complete. Further increases in the concentration of uric acid do not affect the rate of the reaction in the slow phase. At low concentrations of uric acid, where an autocatalytic phase is reached, uric acid is degraded during the reaction. At concentrations of uric acid that keep the reaction in the linear phase, the uric acid is not degraded. It is concluded that uric acid may protect oxyhemoglobin by reacting with HbO2H to yield [HbOH]+ and the urate radical. The urate radical may react with a second molecule of HbO2H and become oxidized. At higher concentrations, the radical may undergo electron transfer with oxyhemoglobin to regenerate the uric acid and form methemoglobin.     

Oxygen exchange between nitrate molecules during nitrite oxidation by Nitrobacter

During oxidation of nitrite, cells of Nitrobacter winogradskyi are shown to catalyze the active exchange of oxygen atoms between exogenous nitrate molecules (production of 15N16/18O3- during incubation of 14N16/18O3-, 15N16O3-, and 15N16O2- in H216O). Little, if any, exchange of oxygens between nitrate and water also occurs (production of 15N16/18O3- during incubation of 15N16O3- and 14N16O2- in H218O). 15N species of nitrate were assayed by 18O-isotope shift in 15N NMR. Taking into account the O-exchange reactions which occur during nitrite oxidation, H2O is seen to be the source of O in nitrate produced by oxidation of nitrite by N. winogradskyi. The data do not establish whether the nitrate-nitrate O exchange is catalyzed by nitrite oxidase (H2O + HNO2----HNO3 + 2H+ + 2e-) or nitrate reductase (HNO3 + 2H+ + 2e-----HNO2 + H2O) or both enzymes in consort. The nitrate-nitrate exchange reaction suggests the existence of an oxygen derivative of a H2O-utilizing oxidoreductase.