Letter by Stamler et al Regarding Article, “Nitrite and S-nitrosohemoglobin Exchange Across the Human Cerebral and Femoral Circulation: Relationship to Basal and Exercise Blood Flow Responses to Hypoxia” (original) (raw)

. Author manuscript; available in PMC: 2018 Jun 13.

To the Editor

We read with interest the article by Bailey et al.1 Based on measuring nitrite (NO2−), iron-nitrosyl hemoglobin (HbFeNO) and S-nitrosohemoglobin (SNO-Hb) in volunteers’ blood during hypoxia and exercise, they concluded that deoxyhemoglobin-mediated NO2− reduction to form HbFeNO is “…the dominant mechanism underlying hypoxic vasodilation”, with no role for SNO-Hb. We are concerned that this conclusion likely reflects confusion about the role of these species in blood flow regulation and limitations of the methodology used to measure them.

Classic experiments by Furchgott and Ignarro showed that hemoglobin scavenged endogenous nitric oxide (NO) by forming HbFeNO. This implied, paradoxically, that hemoglobin in RBCs would impair tissue blood flow. It was shown subsequently that RBCs actually dilate blood vessels at the lower PO2 characterizing tissues, despite the fact that NO cannot escape HbFeNO at any PO2.2 The solution to this conundrum required new understanding of hemoglobin chemistry and allostery, and came in the form of SNO-Hb, an O2-responsive, S-nitrosothiol (SNO) impervious to scavenging by heme-iron.2 It has since been shown conclusively that SNO-Hb mediates hypoxic vasodilation in the respiratory cycle,3 thereby re-defined as a three-gas system (O2/NO/CO2).

Implicating NO2− in physiological hypoxic vasodilation has been fraught with both methodological and conceptual problems. Proposals rely largely on extrapolations from pharmacology and arbitrary assignments of NO bioactivity. A case in point is the equating of NO2−-derived HbFeNO with NO bioactivity1. However, HbFeNO reflects NO sequestration by hemes, not NO bioactivity.

Bailey et al.1 report A>V gradients of NO2−/HbFeNO as indicative of nitrite vasoactivity. But again, NO cannot escape from HbFeNO during physiologically relevant A-V transit (~1 second)2; A-V sampling from an arm and leg1 (transit time ~20 sec) overlooks this critical chemical constraint. In addition, HbFeNO may derive from SNO-Hb under hypoxia (“NO re-capture”)2 and from endothelial NO; assignment of HbFeNO gradients to NO2− is arbitrary.

A role for SNO-Hb in vasodilation was dismissed based on V>A gradients.1 However, it was demonstrated that SNO-Hb is higher in human arterial than venous blood,2 only observed if PO2 is preserved in blood samples, because the disposition of NO within hemoglobin is regulated by PO2.2 Bailey et al exposed venous and “hypoxic” samples to room air, invalidating measurements of SNO-Hb gradients.

Analysis of NO2−/HbFeNO gradients1 is altogether mooted by use of the triiodide assay. Red blood cell Fe-NO is found principally in an Fe3+-NO-containing complex2,4 that cannot be detected by triiodide methodology.5 More generally, triiodide methodology converts HbFeNO to NO2−, and is subject to additional artifacts when applied to blood/red blood cells that prevent accurate measurement of NO2−, SNO-Hb or HbFeNO.5

The roles of SNO-Hb in vasoregulation2,3 and of SNOs in physiology generally are well established by strict genetic criteria, and anchor current understanding of NO biology, whereas a physiological role for NO2− remains to be demonstrated; proposals to date are challenged by chemical, biochemical and physiological constraints. It was recently shown that vasodilatory effects of oral NO2− are mediated by SNOs formed by acidification in the gut, whereas circulating NO2− is inactive. Generally, physiological roles for NO2− will likely follow a SNO-mediated formula.

Footnotes

Disclosures: Dr. Stamler has a financial interest in Nivalis Therapeutics, Adamas Pharma and LifeHealth. Dr. Reynolds has a financial interest in Miach Medical Innovations. Drs. Stamler and Reynolds also hold patents in this area that have been licensed for commercial development.

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