Nitrite augments tolerance to ischemia/reperfusion injury via the modulation of mitochondrial electron transfer.

Nitrite (NO(2)(-)) is an intrinsic signaling molecule that is reduced to NO during ischemia and limits apoptosis and cytotoxicity at reperfusion in the mammalian heart, liver, and brain. Although the mechanism of nitrite-mediated cytoprotection is unknown, NO is a mediator of the ischemic preconditioning cell-survival program. Analogous to the temporally distinct acute and delayed ischemic preconditioning cytoprotective phenotypes, we report that both acute and delayed (24 h before ischemia) exposure to physiological concentrations of nitrite, given both systemically or orally, potently limits cardiac and hepatic reperfusion injury. This cytoprotection is associated with increases in mitochondrial oxidative phosphorylation. Remarkably, isolated mitochondria subjected to 30 min of anoxia followed by reoxygenation were directly protected by nitrite administered both in vitro during anoxia or in vivo 24 h before mitochondrial isolation. Mechanistically, nitrite dose-dependently modifies and inhibits complex I by posttranslational S-nitrosation; this dampens electron transfer and effectively reduces reperfusion reactive oxygen species generation and ameliorates oxidative inactivation of complexes II-IV and aconitase, thus preventing mitochondrial permeability transition pore opening and cytochrome c release. These data suggest that nitrite dynamically modulates mitochondrial resilience to reperfusion injury and may represent an effector of the cell-survival program of ischemic preconditioning and the Mediterranean diet.

Deoxymyoglobin is a nitrite reductase that generates nitric oxide and regulates mitochondrial respiration.

Previous studies have revealed a novel interaction between deoxyhemoglobin and nitrite to generate nitric oxide (NO) in blood. It has been proposed that nitrite acts as an endocrine reservoir of NO and contributes to hypoxic vasodilation and signaling. Here, we characterize the nitrite reductase activity of deoxymyoglobin, which reduces nitrite approximately 36 times faster than deoxyhemoglobin because of its lower heme redox potential. We hypothesize that physiologically this reaction releases NO in proximity to mitochondria and regulates respiration through cytochrome c oxidase. Spectrophotometric and chemiluminescent measurements show that the deoxymyoglobin-nitrite reaction produces NO in a second order reaction that is dependent on deoxymyoglobin, nitrite and proton concentration, with a bimolecular rate constant of 12.4 mol/L(-1)s(-1) (pH 7.4, 37 degrees C). Because the IC(50) for NO-dependent inhibition of mitochondrial respiration is approximately 100 nmol/L at physiological oxygen tensions (5 to 10 mumol/L); we tested whether the myoglobin-dependent reduction of nitrite could inhibit respiration. Indeed, the addition of deoxymyoglobin and nitrite to isolated rat heart and liver mitochondria resulted in the inhibition of respiration, while myoglobin or nitrite alone had no effect. The addition of nitrite to rat heart homogenate containing both myoglobin and mitochondria resulted in NO generation and inhibition of respiration; these effects were blocked by myoglobin oxidation with ferricyanide but not by the xanthine oxidoreductase inhibitor allopurinol. These data expand on the paradigm that heme-globins conserve and generate NO via nitrite reduction along physiological oxygen gradients, and further demonstrate that NO generation from nitrite reduction can escape heme autocapture to regulate NO-dependent signaling.

Measurement of circulating nitrite and S-nitrosothiols by reductive chemiluminescence.

Considerable disparities in the reported levels of basal human nitrite and S-nitrosothiols (RSNO) in blood have brought methods of quantifying these nitric oxide (NO) metabolites to the forefront of NO biology. Ozone-based chemiluminescence is commonly used and is a robust method for measuring these species when combined with proper reductive chemistry. The goal of this article is to review existing methodologies for the measurement of nitrite and RSNO by reductive chemiluminescence. Specifically, we discuss in detail the measurement of nitrite and RSNO in biological matrices using tri-iodide and copper(I)/cysteine-based reduction methods coupled to chemiluminescence. The underlying reaction mechanisms, as well as the potential pitfalls of each method are discussed.

Measurement of nitric oxide levels in the red cell: validation of tri-iodide-based chemiluminescence with acid-sulfanilamide pretreatment.

The tri-iodide-based chemiluminescence assay is the most widely used methodology for the detection of S-nitrosothiols (RSNOs) in biological samples. Because of the low RSNO levels detected in a number of biological compartments using this assay, criticism has been raised that this method underestimates the true values in biological samples. This claim is based on the beliefs that (i) acidified sulfanilamide pretreatment, required to remove nitrite, leads to RSNO degradation and (ii) that there is auto-capture of released NO by heme in the reaction vessel. Because our laboratories have used this assay extensively without ever encountering evidence that corroborated these claims, we sought to experimentally address these issues using several independent techniques. We find that RSNOs of glutathione, cysteine, albumin, and hemoglobin are stable in acidified sulfanilamide as determined by the tri-iodide method, copper/cysteine assay, Griess-Saville assay and spectrophotometric analysis. Quantitatively there was no difference in S-nitroso-hemoglobin (SNOHb) or S-nitroso-albumin (SNOAlb) using the tri-iodide method and a recently described modified assay using a ferricyanide-enhanced reaction mix at biologically relevant NO:heme ratios. Levels of SNOHb detected in human blood ranged from 20-100 nM with no arterial-venous gradient. We further find that 90% of the total NO-related signal in blood is caused by erythrocytic nitrite, which may partly be bound to hemoglobin. We conclude that all claims made thus far that the tri-iodide assay underestimates RSNO levels are unsubstantiated and that this assay remains the "gold standard" for sensitive and specific measurement of RSNOs in biological matrices.