Letter by Tsikas and Rossi Regarding Article, “Nitrite Anion Provides Potent Cytoprotective and Antiapoptotic Effects as Adjunctive Therapy to Reperfusion for Acute Myocardial Infarction”
To the Editor:
We read with interest the work by Gonzalez et al,1 which reports that nitrite infusion may enhance the efficacy of reperfusion therapy for acute myocardial infarction and have potential clinical impact. Gonzalez et al1 concluded that nitrite but not other nitric oxide (NO)–related species is the bioactive intravascular NO metabolite that accounts for cardioprotection. In our opinion, this conclusion lacks proof because of potential analytic and methodological shortcomings; in particular, the results shown in Figures 2A and 2B of the article1 deserve comment.
Considering the hematocrit (Hct), the relation between nitrite concentration in blood (CB), plasma (CP), and erythrocytes (CE) is described by the equation: CB=(1−Hct)×CP+Hct×CE. For dog,1 the Hct value is ≈50%, and CE can be calculated from the reported CB and CP as CE=2×CB−CP. Before nitrite infusion, the enrichment factors for nitrite (ie, the respective molar ratios of CE:CP) are 13:1 and 12:1 for the erythrocytic compartment. The data displayed in Figures 2A and 2B suggest that 5 minutes after the start of nitrite infusion the enrichment factor CE:CP fell drastically to 1:5 and −2:5, respectively, and did not increase for the next 30 minutes in the 60-minute group.
These data are difficult to interpret and seem to contradict current knowledge about nitrite handling by erythrocytes. If we assume that valid analytic procedures have been used,1–4 we must conclude that infusion of nitrite causes complete consumption of nitrite in erythrocytes in both groups. Unfortunately, the authors1 did not report the results from the control experiment or nitrate concentrations.
The same investigator group finds in other studies that nitrite is enriched within erythrocytes2,3,4 as compared with plasma. 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 bioactivity.1–4 However, the partition of nitrite between erythrocytes and plasma is controversial. In agreement with the results by Parks et al5 in animals, we find by gas chromatography-mass spectrometry analysis that nitrite is practically uniformly distributed between plasma and erythrocytes in humans. Moreover, accumulation of nitrite in erythrocytes per se does not necessarily award nitrite and erythrocytes a prominent role in storage and transport of NO bioactivity.
We suggest a more cautious examination of biochemical measures and consideration of potentially necessary analytic modifications when experimental conditions change (eg, at nitrite concentrations far above basal levels as a result of nitrite infusion). Interpretation of experimental observations obtained by using potentially suboptimal analytic methods might not form the basis for drawing far-ranging conclusions about underlying mechanisms. Even if this was not the primary aim of the study,1 the use of animals with 10- to 20-fold higher basal erythrocytic nitrite concentrations than reported previously by this investigator group would have deserved careful revalidation of sample processing and analytic methodology used for quantification. The role of nitrite in the circulation remains elusive and demands sophisticated investigations and use of reliable quantitative analytic methods for measuring nitrite in plasma and erythrocytes.
Gonzalez FM, Shiva S, Vincent PS, Ringwood LA, Hsu LY, Hon YY, Aletras AH, Cannon IIIRO, Gladwin MT, Arai AE. Nitrite anion provides potent cytoprotective and antiapoptotic effects as adjunctive therapy to reperfusion for acute myocardial infarction. Circulation. 2008; 117: 2986–2994.
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.
Parks NJ, Krohn KJ, Mathis CA, Chasko JH, Geiger KR, Gregor ME, Peek NF. Nitrogen-13 labeled nitrite and nitrate: distribution and metabolism after intratracheal administration. Science. 1981; 212: 58–60.