Response to Letter Regarding Article, “Nitrite Anion Provides Potent Cytoprotective and Antiapoptotic Effects as Adjunctive Therapy to Reperfusion for Acute Myocardial Infarction”
We thank Dr Tsikas and Dr Rossi for their comments and applaud their efforts to standardize measurements of nitric oxide (NO) metabolites. The focus of our current study was (1) to determine if very low doses of nitrite (NO2−) improve the efficacy of reperfusion therapy in a large-animal model of myocardial infarction and (2) to address whether nitrite is the reactive nitrogen species that transports endocrine NO bioactivity in blood, limiting ischemia-reperfusion injury.
The targeted concentration of plasma nitrite in this study was 10 μmol, based on studies showing efficacy against ischemia-reperfusion injury in mice and rats. The dose of nitrite was calculated on the basis of data from our experiments in many species.1 The resultant plasma levels in the current study were close to the target 10 μmol in plasma and thus support the fidelity of our experimental plan and measured levels of nitrite. The potent effect of nitrite infusions in limiting cardiac infarction volume and the dominant increase in nitrite over other nitroso-reactive nitrogen species support the hypothesis that nitrite is the active endocrine agent.
Drs Tsikas and Rossi state concerns over the higher levels of nitrite measured at baseline in the dog red cells compared with plasma and the reversal during nitrite infusion (ie, increases in the plasma during nitrite infusions). We certainly agree that despite many studies of nitrate, chloride, potassium, sodium, and bicarbonate transport, more research is needed to understand the rates, mechanisms, and in vivo production and transport of nitrite in mammalian species.
Although baseline levels of nitrite in red cells relative to plasma was not the focus of this study, we have worked on this question in humans and have on-going work in mice.2 The reasons for higher levels of nitrite in red cells may be interesting. Controversial yet fascinating work suggests that a functional red cell endothelial nitric oxide synthase may exist,3 and there may be membrane compartmentalization of NO and multi-copper oxidases.4,5 Nitrite rapidly equilibrates across red blood cells in humans but not so quickly in mice. Thus, one cannot immediately extrapolate to other species (such as dog) without more study.
The statement that “… nitrite enrichment in erythrocytes is the basis for the working hypothesis that nitrite but not other NO-related species plays a leading role in vascular NO biology …” oversimplifies a broad field. The hypothesis that nitrite serves such a role comes from the work of many groups over the last 10 years evincing that nitrite is a potent vasodilator, is converted to NO along the physiological oxygen gradient (as hemoglobin, myoglobin, and tissue hemeproteins deoxygenate), is a hypoxic vasodilator, regulates blood pressure at low physiological concentrations, limits ischemia-reperfusion injury, regulates hypoxic and ischemic mitochondrial respiration, and is the major intravascular NO reservoir.1
Our study clearly demonstrates that nitrite enhances the efficacy of reperfusion of acute myocardial infarction. The biochemical mechanisms underlying such effects are consistent with a primary role of nitrite, as we concluded from metabolite levels, but we welcome further work to more thoroughly understand this complex biology.
Source of Funding
Support was provided by the Intramural Program of National Heart, Lung, and Blood Institute.
Dr Gladwin and Dr Cannon are named as coinventors on a National Institutes of Health patent application for the use of nitrite salts in cardiovascular diseases.
Dejam A, Hunter CJ, Pelletier MM, Hsu LL, Machado RF, Shiva S, Power GG, Kelm M, Gladwin MT, Schechter AN. Erythrocytes are the major intravascular storage sites of nitrite in human blood. Blood. 2005; 106: 734–739.
Kleinbongard P, Schulz R, Rassaf T, Lauer T, Dejam A, Jax T, Kumara I, Gharini P, Kabanova S, Ozuyaman B, Schnurch HG, Godecke A, Weber AA, Robenek M, Robenek H, Bloch W, Rosen P, Kelm M. Red blood cells express a functional endothelial nitric oxide synthase. Blood. 2006; 107: 2943–2951.
Shiva S, Brookes PS, Patel RP, Anderson PG, Darley-Usmar VM. Nitric oxide partitioning into mitochondrial membranes and the control of respiration at cytochrome c oxidase. Proc Natl Acad Sci U S A. 2001; 98: 7212–7217.