Correspondence
Hemoglobin and Nitric Oxide
N Engl J Med 2003; 349:402-405July 24, 2003
- Article
To the Editor:
The article by Schechter and Gladwin (April 10 issue)1 is flawed by a fundamental error. The conclusions that the authors draw from reports of measured S-nitrosohemoglobin levels and decomposition rates are not disciplined by quantitative standards of efficacy: “lower” does not imply “too low,” nor does “rapid” imply “too rapid.”
The authors cite studies in which S-nitrosohemoglobin levels (approximately 50 nmol per liter) are lower than those in previous reports (micromolar levels). They fail to mention, however, that Pawloski et al. demonstrated bioactivity of red cells with S-nitrosohemoglobin at 50 nmol per liter,2 and McMahon et al. showed bioactivity with native red cells (i.e., with ambient S-nitrosohemoglobin).3 Thus, even if the cited measurements are taken at face value (notwithstanding methodologic concerns), the lowest reported value is completely consistent with S-nitrosohemoglobin bioactivity.
Similarly, the authors, citing their own work,4 state that S-nitrosohemoglobin within red cells is “unstable.” However, even if a problematic kinetic analysis is set aside, the implicated physiologic lifetime of S-nitrosohemoglobin is 10 to 20 minutes. The authors describe this decay as rapid but provide no evidence that it is too rapid for S-nitrosohemoglobin to carry out its physiologic role. Indeed, their own observation with S-nitrosohemoglobin at a level of 50 nmol per liter in vivo demonstrates that erythrocytic S-nitrosohemoglobin is maintained at a biologically active level.
4 ReferencesJonathan S. Stamler, M.D.
Duke University Medical Center, Durham, NC 27710
staml001@mc.duke. edu 1
Schechter AN ,Gladwin MT . Hemoglobin and the paracrine and endocrine functions of nitric oxide. N Engl J Med 2003;348:1483-1485
Full Text | Web of Science | Medline2
Pawloski JR ,Hess DT ,Stamler JS . Export by red blood cells of nitric oxide bioactivity. Nature 2001;409:622-626
CrossRef | Web of Science | Medline3
McMahon TJ ,Moon RE ,Luschinger BP , et al. Nitric oxide in the human respiratory cycle. Nat Med 2002;8:711-717
Web of Science | Medline4
Gladwin MT ,Wang X ,Reiter CD , et al. S-nitrosohemoglobin is unstable in the reductive erythrocyte environment and lacks O2/NO-linked allosteric function. J Biol Chem 2002;277:27818-27828
CrossRef | Web of Science | Medline
To the Editor:
Schechter and Gladwin's opinions regarding S-nitrosohemoglobin do not provide an accurate or balanced picture of its role in physiology. First, as with every physiologic reaction involving hemoglobin, the reactivity of S-nitrosohemoglobin changes allosterically with iron–ligand binding.1,2 Deoxygenation allows transnitrosation from S-nitrosohemoglobin to anion-exchange protein 1 in the erythrocyte membrane. Indeed, erythrocyte deoxygenation forms stable, S-nitrosylated anion-exchange protein 1 at concentrations (in the low nanomolar range) that dilate blood vessels.2 Furthermore, transnitrosation from venous, but not arterial, erythrocytes to extraerythrocytic thiols, as measured by mass spectrometry, uniquely recapitulates normal respiratory physiology.3
Second, the distinction between a diffusion barrier and a red-cell–free layer is not relevant in the microcirculation, which has the largest capacity for dilatation and regulation of tissue perfusion in the vascular tree. In this part of the circulation, erythrocytes may contact the endothelium directly.4
Unfortunately, the authors dismiss these data and data from other publications that could have helped them substantially, both in the design of their experiments and in the composition of their article.
4 ReferencesBenjamin M. Gaston, M.D.
University of Virginia School of Medicine, Charlottesville, VA 22908
bmg3g@virginia.edu Joshua M. Hare, M.D.
Johns Hopkins University School of Medicine, Baltimore, MD 212871
Chan NL ,Rogers PH ,Arnone A . Crystal structure of the S-nitroso form of liganded human hemoglobin. Biochemistry 1998;37:16459-16464
CrossRef | Web of Science | Medline2
Pawloski JR ,Hess DT ,Stamler JS . Export by red blood cells of nitric oxide bioactivity. Nature 2001;409:622-626
CrossRef | Web of Science | Medline3
Lipton AJ ,Johnson MA ,Macdonald T ,Lieberman MW ,Gozal D ,Gaston B . S-nitrosothiols signal the ventilatory response to hypoxia. Nature 2001;413:171-174
CrossRef | Web of Science | Medline4
De Backer D ,Creteur J ,Preiser JC ,Dubois MJ ,Vincent JL . Microvascular blood flow is altered in patients with sepsis. Am J Respir Crit Care Med 2002;166:98-104
CrossRef | Web of Science | Medline
To the Editor:
Native human erythrocytes exploit the allostery of hemoglobin to deliver the vasodilator nitric oxide remotely, in concert with oxygen delivery, thus coupling regional blood flow to metabolic demand (and effecting hypoxic vasodilation).1 Schechter and Gladwin overlook or misinterpret important findings in speculating on the basis of this activity. The molecular key to this dynamic is the reactive, highly conserved cysteine residue at position 93 of the hemoglobin beta chain; this residue binds to nitric oxide in hemoglobin's oxygenated R (relaxed) structure (forming S-nitrosohemoglobin) and releases nitric oxide equivalents in its deoxygenated T (tense) structure. Contrary to the authors' claims, only S-nitrosohemoglobin, among nitric-oxide–hemoglobin reaction products, actually delivers nitric oxide–associated bioactivity. The suggestion that nitrite (at native concentrations) causes vasodilation in humans has been refuted experimentally.2 Iron-nitrosyl hemoglobin — which, like nitrite, is a precursor in S-nitrosohemoglobin formation3 — actually causes vasoconstriction.
S-nitrosohemoglobin is “unstable” only under extreme conditions: the authors raised red-cell S-nitrosohemoglobin levels 10,000-fold.4 Disagreement exists over the levels of circulating S-nitrosohemoglobin, but the release of even small fractions of the approximately 50 nmol of hemoglobin-bound S-nitrosothiol per liter (the low end of published estimates) that was evidently stable enough to be measured in native red cells4 suffices to regulate blood flow meaningfully.1
4 ReferencesTimothy J. McMahon, M.D., Ph.D.
Duke University Medical Center, Durham, NC 277101
McMahon TJ ,Moon RE ,Luschinger BP , et al. Nitric oxide in the human respiratory cycle. Nat Med 2002;8:711-717
Web of Science | Medline2
Rassaf T ,Preik M ,Kleinbongard P , et al. Evidence for in vivo transport of bioactive nitric oxide in human plasma. J Clin Invest 2002;109:1241-1248
CrossRef | Web of Science | Medline3
Luchsinger BP ,Rich EN ,Gow AJ ,Williams EM ,Stamler JS ,Singel DJ . Routes to S-nitroso-hemoglobin formation with heme redox and preferential reactivity in the beta subunits. Proc Natl Acad Sci U S A 2003;100:461-466
CrossRef | Web of Science | Medline4
Gladwin MT ,Wang X ,Reiter CD , et al. S-nitrosohemoglobin is unstable in the reductive erythrocyte environment and lacks O2/NO-linked allosteric function. J Biol Chem 2002;277:27818-27828
CrossRef | Web of Science | Medline
To the Editor:
Schechter and Gladwin rely on their prior study1 to assert that inactivation of luminal nitric oxide by circulating free hemoglobin is pathophysiologically germane in sickle cell disease. Invoking this mechanism as the cause of pulmonary hypertension of sickle cell disease without explaining the absence of vasoconstriction in systemic vessels exposed to the same milieu is incongruous, at best. In fact, patients with sickle cell disease have lower blood pressure both at base line2 and during hemolytic crises3 than matched controls. Schechter and Gladwin also claim that free hemoglobin outcompetes erythrocytes for nitric oxide in sickle cell disease, but they removed erythrocytes before their nitric oxide–consumption experiments,1 leaving this assertion untested and thus untenable.
The authors also propose that in red cells, iron-nitrosyl hemoglobin (in which nitric oxide is bound to heme) and nitrite ions may serve as sources of nitric oxide but provide no references for either contention. This is because the former has not been demonstrated to be a source of nitric oxide, and the latter has been shown to lack vasoactivity under physiologic conditions.4
Finally, Figure 1 of the article suggests that blood-vessel nitric oxide is subphysiologic under all pathologic conditions. In fact, the opposite is true in endotoxemia and septic shock.5
5 ReferencesJohn R. Pawloski, M.D., Ph.D.
Duke University Medical Center, Durham, NC 277101
Reiter CD ,Wang X ,Tanus-Santos JE , et al. Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease. Nat Med 2002;8:1383-1389
CrossRef | Web of Science | Medline2
Pegelow CH ,Colangelo L ,Steinberg M , et al. Natural history of blood pressure in sickle cell disease: risks for stroke and death associated with relative hypertension in sickle cell anemia. Am J Med 1997;102:171-177
CrossRef | Web of Science | Medline3
Ernst AA ,Weiss SJ ,Johnson WD ,Takakuwa KM . Blood pressure in acute vaso-occlusive crises of sickle cell disease. South Med J 2000;93:590-592
Web of Science | Medline4
Lauer T ,Preik M ,Rassaf T , et al. Plasma nitrite rather than nitrate reflects regional endothelial nitric oxide synthase activity but lacks intrinsic vasodilator action. Proc Natl Acad Sci U S A 2001;98:12814-12819
CrossRef | Web of Science | Medline5
Gomez-Jimenez J ,Salgado A ,Mourelle M , et al. L-arginine: nitric oxide pathway in endotoxemia and human septic shock. Crit Care Med 1995;23:253-258
CrossRef | Web of Science | Medline
To the Editor:
Schechter and Gladwin argue that the narrow paradigm of nitric oxide biology must be extended to include the packaging of the nitric oxide moiety in “ways that stabilize and protect it.” This extended paradigm — exemplified by the S-nitrosohemoglobin hypothesis — was originally articulated more than a decade ago.1 The suggestion that this broader perspective is “adding to the controversy” represents a dizzying spin on actual trends in the field.
Schechter and Gladwin cite work from their laboratory2 as evidence against a physiologic function for S-nitrosohemoglobin, in particular one linked to hemoglobin allostery. Their assertions, however, are not upheld by their data, which are too sparse to support conclusions about reaction schemes and kinetic-rate laws. Extrapolation of their observations to physiologic conditions has no scientific justification.
Their test of allostery is grossly ill conceived. Under their conditions, acceleration of S-nitrosohemoglobin decomposition by loss of oxygen is limited by the rate of deoxygenation, which is similar to the S-nitrosothiol decomposition rate that they observe for oxygenated S-nitrosohemoglobin. The marginal acceleration they observed should have been expected; it is wholly compatible with allosteric modulation of S-nitrosohemoglobin reactivity.
2 ReferencesDavid J. Singel, Ph.D.
Montana State University, Bozeman, MT 59717
rchds@gemini.oscs. montana. edu 1
Stamler JS ,Singel DJ ,Loscalzo J . Biochemistry of nitric oxide and its redox-activated forms. Science 1992;258:1898-1902
CrossRef | Web of Science | Medline2
Gladwin MT ,Wang X ,Reiter CD , et al. S-nitrosohemoglobin is unstable in the reductive erythrocyte environment and lacks O2/NO-linked allosteric function. J Biol Chem 2002;277:27818-27828
CrossRef | Web of Science | Medline
Author/Editor Response
The purpose of our article is to highlight pathophysiologic and pharmacologic implications of the rapidly evolving concepts concerning the complex reactions of nitric oxide with hemoglobin. We describe, on the basis of work from many laboratories, how the hemoglobin present within red cells or released into the plasma would be likely to affect these reactions.
The letters from Dr. Stamler and his current or recent collaborators contain a spirited defense of their S-nitrosohemoglobin hypothesis. However, we believe that much new experimental work is inconsistent with this idea.1-5 As a result of this new work, which we recently reviewed,6,7 it is our view that although S-nitrosohemoglobin may be formed under special conditions, it is probably not a physiologic regulator of respiration, as originally envisioned.
In response to Dr. Stamler: we are pleased that he now accepts our measured values for S-nitrosohemoglobin levels. However, we emphasize that under basal conditions and during inhalation of nitric oxide in normal volunteers, we found no evidence of arterial–venous gradients — a result that is inconsistent with the concept of nitric oxide delivery from S-nitrosohemoglobin.2
Drs. Gaston and Hare discuss the postulated transfer of nitric oxide from hemoglobin to the anion-exchange protein, but this transfer has not been confirmed by others. In making their last point, they ignore the fact that the greatest barrier to the diffusion of nitric oxide is the “unstirred” layer involving the red-cell membrane itself,3 which would greatly attenuate nitric oxide–hemoglobin interactions.
Dr. McMahon generally restates the S-nitrosohemoglobin hypothesis but also takes issue with the suggestion that nitrite may physiologically generate bioactive nitric oxide. Such effects have long been observed in vitro. Rassaf and colleagues, whose work is cited by Dr. McMahon, did not see an effect with short infusions, but in current studies with R. Cannon of the National Heart, Lung, and Blood Institute, we are finding substantial effects on blood flow from nitrite infusion into the brachial artery in normal subjects with forearm nitrite concentrations as low as 1.5 μmol per liter.
Dr. Pawloski, in commenting on the lack of effects of circulating free hemoglobin on blood pressure in patients with sickle cell disease, ignores the multiple humoral mechanisms that regulate blood pressure; we recently reported on the importance of mediators other than nitric oxide in these patients.8 Our discussion of pathology focused on hemolysis, but we agree with Dr. Pawloski about septic shock, which (as we noted in our article) is generally characterized by very high levels of nitric oxide synthase activity.
Dr. Singel's defense of the S-nitrosohemoglobin hypothesis raises the more general question of how to extrapolate from experiments in vitro or experiments in animals to explanatory paradigms relevant to the human circulation. Results of laboratory experiments from many different groups have led to multiple models to describe how nitric oxide can be preserved, destroyed, or delivered by the circulatory system. Only studies in human subjects will ultimately sort out these issues and show whether a robust pharmacology based on nitric oxide delivery will be possible.
8 ReferencesAlan N. Schechter, M.D.
Mark T. Gladwin, M.D.
National Institutes of Health, Bethesda, MD 208921
Wolzt M ,MacAllister RJ ,Davis D , et al. Biochemical characterization of S-nitrosohemoglobin: mechanisms underlying synthesis, NO release, and biological activity. J Biol Chem 1999;274:28983-28990
CrossRef | Web of Science | Medline2
Gladwin MT ,Ognibene FP ,Pannell LK , et al. Relative role of heme nitrosylation and β-cysteine 93 nitrosation in the transport and metabolism of nitric oxide by hemoglobin in the human circulation. Proc Natl Acad Sci U S A 2000;97:9943-9948
CrossRef | Web of Science | Medline3
Han TH ,Hyduke DR ,Vaughn MW ,Fukuto JM ,Liao JC . Nitric oxide reaction with red blood cells and hemoglobin under heterogeneous conditions. Proc Natl Acad Sci U S A 2002;99:7763-7768[Erratum, Proc Natl Acad Sci U S A 2002;99:10227.]
CrossRef | Web of Science | Medline4
Herold S ,Rock G . Reactions of deoxy-, oxy-, and methemoglobin with nitrogen monoxide: mechanistic studies of the S-nitrosothiol formation under different mixing conditions. J Biol Chem 2003;278:6623-6634
CrossRef | Web of Science | Medline5
Crawford JH ,White CR ,Patel RP . Vasoactivity of S-nitrosohemoglobin: role of oxygen, heme, and NO oxidation states. Blood 2003;101:4408-4415
CrossRef | Web of Science | Medline6
Hobbs AJ ,Gladwin MT ,Patel RP ,Williams DL ,Butler AR . Haemoglobin: NO transporter, NO inactivator, or NOne of the above? Trends Pharmacol Sci 2002;23:406-411
CrossRef | Web of Science | Medline7
Gladwin MT ,Lancaster JR ,Freeman BA ,Schechter AN . Nitric oxide's reactions with hemoglobin: a view through the SNO-storm. Nat Med 2003;9:496-500
CrossRef | Web of Science | Medline8
Gladwin MT ,Schechter AN ,Ognibene FP , et al. Divergent nitric oxide bioavailability in men and women with sickle cell disease. Circulation 2003;107:271-278
CrossRef | Web of Science | Medline
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M. T. Gladwin, D. B. Kim-Shapiro. (2008) The functional nitrite reductase activity of the heme-globins. Blood 112:7, 2636-2647
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A. C. Frei, Y. Guo, D. W. Jones, K. A. Pritchard, K. A. Fagan, N. Hogg, N. J. Wandersee. (2008) Vascular dysfunction in a murine model of severe hemolysis. Blood 112:2, 398-405
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Maqsood M. Elahi, Khalid M. Naseem, Bashir M. Matata. (2007) Nitric oxide in blood. FEBS Journal 274:4, 906-923
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David J. Singel, Jonathan S. Stamler. (2005) CHEMICAL PHYSIOLOGY OF BLOOD FLOW REGULATION BY RED BLOOD CELLS:. Annual Review of Physiology 67:1, 99-145
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J. P. Wallis. (2005) Nitric oxide and blood: a review. Transfusion Medicine 15:1, 1-11
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Christian J Hunter, André Dejam, Arlin B Blood, Howard Shields, Daniel B Kim-Shapiro, Roberto F Machado, Selamawit Tarekegn, Neda Mulla, Andrew O Hopper, Alan N Schechter, Gordon G Power, Mark T Gladwin. (2004) Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator. Nature Medicine 10:10, 1122-1127
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John S. Olson, Erin W. Foley, Corina Rogge, Ah-Lim Tsai, Michael P. Doyle, Douglas D. Lemon. (2004) No scavenging and the hypertensive effect of hemoglobin-based blood substitutes. Free Radical Biology and Medicine 36:6, 685-697
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