Editorials
See related article, pages 976 –983
NO Contest
Nitrite Versus S-Nitroso-Hemoglobin
Mark T. Gladwin, Alan N. Schechter
A
This commentary will not specifically focus on this newly
proposed mechanism for microvascular pathology based on
impaired NO delivery from blood, but on the two competing
mechanisms for that delivery in the context of normal
physiology—a topic far from resolved in the work of James
et al.4 We begin by reviewing what we believe are the
currently accepted principles, supported by both laboratory
and clinical studies, for the interaction between NO and
hemoglobin in blood.
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recent flurry of research papers and commentaries in
this journal1– 4 has highlighted a current major controversy in cardiovascular biochemistry and physiology: how is nitric oxide (NO) transported in the bloodstream.
Two views have arisen. First, that S-nitrosated hemoglobin
and albumin serve as stable storage forms of intravascular
NO, and, in the case of S-nitrosated hemoglobin, as an
allosterically regulated delivery vehicle for NO.5,6 The second is that the anion nitrite, which is present in relative
abundance in both blood and tissue, subserves this function.7
The controversy is relevant to the study by James et al in
this issue of Circulation Research that examined the ability of
red blood cells treated with NO, from healthy subjects and
patients with diabetes, to vasodilate rabbit aortic rings.4 The
investigators report that exposure of oxygenated red blood
cells to NO in vitro results in increased levels of NO-modified
hemoglobin, specifically iron-nitrosyl-hemoglobin (NO
bound to the heme group) and S-nitroso-hemoglobin (NO
bound to the cysteine 93 residue of the ␤-globin chain), and
that these cells vasodilate rabbit aortic rings. The ability of
the NO-treated red blood cells to vasodilate increases with
greater NO exposure, raising intracellular S-nitrosohemoglobin levels and with progressive hypoxia. Additionally, they find that diabetic red blood cells form less
S-nitroso-hemoglobin with NO exposure than normal red
blood cells, dilate more at 1% oxygen concentration, and
dilate less at 2% oxygen. They ascribe these observations to
an impairment in NO delivery from glycohemoglobin (at 2%
oxygen) and suggest that the reduced NO bioavailability in
diabetes can be attributed to the red blood cells, in addition to
the generally accepted mechanism of reduced endothelial NO
production and NO inactivation by reactive oxygen species.
Indeed, the authors suggest that elevated levels of ironnitrosyl-hemoglobin may be a marker for poor glycemic
control.
Common Ground
NO Production From the Endothelium Accounts for
Basal Physiological NO-Dependent Vascular Tone
It has been appreciated for almost two decades that endothelial NO production by NO synthases produces tonic vasodilation (approximately 25% of basal blood flow in humans)
and accounts for the majority of both flow (shear stress)mediated vasodilation and acetylcholine-mediated vasodilation. Thus, any putative intravascular NO-donating species
cannot compensate for the loss of regional endothelial NO
synthesis. The dominance of endothelial-derived NO versus
blood-transported NO in basal blood flow regulation is
illustrated by the fact that NO gas inhalation in humans,8 and
recently demonstrated in cats,3 does not increase peripheral
blood flow under normal physiological conditions. It is only
with local inhibition of NO synthase activity (by infusion of
L-NMMA or L-NAME) that subtle peripheral blood flow
effects of blood-transported NO can be appreciated.8,9 Thus,
the physiological role for blood-transported NO is now
generally ascribed to that of hypoxic vasodilation.2,7
Hemoglobin, per se, Is an NO Destroyer
Nitric oxide reacts in a nearly diffusion-limited reaction with
oxyhemoglobin and deoxyhemoglobin to form methemoglobin and iron-nitrosyl-hemoglobin.10 These reactions inactivate NO and form part of the experimental basis supporting
the identity of endothelium-derived relaxing factor as NO:
investigators could show that addition of hemoglobin to
blood vessels inhibited acetylcholine-mediated vasodilation.
The NO scavenger and vasopressor effects of hemoglobin are
limited by compartmentalization of hemoglobin within the
erythrocyte. An unstirred diffusional barrier around the red
blood cell and perhaps unique submembrane properties limit
the rate of NO hemoglobin reactions by approximately
600-fold.11–14 Thus, erythrocyte-free or plasma hemoglobin
inactivates NO and produces vasoconstriction, an effect
consistently described in basic animal and clinical studies of
stroma-free hemoglobin blood substitutes and more recently
in patients with hemolytic disease.10,15–17 However, a paradoxical effect is observed with chemically S-nitrosated he-
The opinions expressed in this editorial are not necessarily those of the
editors or of the American Heart Association.
From the Critical Care Medicine Department (M.T.G.), Warren G.
Magnuson Clinical Center, National Institutes of Health; Cardiovascular
Branch (M.T.G.), National Heart, Lung, and Blood Institute, National
Institutes of Health; Laboratory of Chemical Biology (M.T.G., A.N.S.),
National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, Md.
Correspondence to Mark Gladwin, Critical Care Medicine Department, CC, Laboratory of Chemical Biology, NIDDK, National Institutes
of Health, Building 10/7D43, 10 Center Dr, Bethesda, MD 20892-1662.
E-mail [email protected]
(Circ Res. 2004;94:851-855.)
© 2004 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000126697.64381.37
851
852
Circulation Research
April 16, 2004
moglobin or with nitrite-treated deoxyhemoglobin, the NO
scavenger becomes an NO donor.
Blood, Red Blood Cells, and Hemoglobin Treated
With NO or Nitrite Can Mediate Vasodilation
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Inhalation of NO gas8 can produce distal vasodilation in the
human forearm if local NO synthesis is inhibited. Infusions of
NO-equilibrated buffer solutions have been shown to elicit
vasodilation distal to the site of infusion.18 Similar effects
have been observed in the feline intestine pretreated with NO
synthase inhibitors9 or after ischemia-reperfusion injury.3
Consistent with this observation, numerous laboratories have
used the experimental model of James et al4 and demonstrated that red blood cells treated with high concentrations of
NO,19 S-nitrosocysteine,20 or nitrite7 can export an NOderived species and produce vasodilation of rat or rabbit
aortic rings.
The principle that blood may carry an NO derivative in an
endocrine fashion to mediate distal vasodilation was first
proposed by Stamler and Loscalzo and ascribed to
S-nitrosated albumin.21 This paradigm was then extended to
S-nitrosated hemoglobin, and a role for the red blood cell in
oxygen-dependent NO delivery was further championed by
Stamler’s group.6 These groups deserve full credit for advancing the then novel principle of NO storage and transport
in blood. The relative balance of NO scavenging, limited by
compartmentalization of hemoglobin within the erythrocyte
membrane, and NO delivery, by S-nitroso-hemoglobin or the
nitrite reductase activity of deoxyhemoglobin, would determine whether the red blood cell can transport/deliver or
scavenge NO for a given physiological condition or vascular
bed.
The Heart of the Controversy
With a clear understanding that we recognize that red blood
cells can export NO activity under certain conditions and may
contribute to oxygen-dependent NO homeostasis, studies of
the mechanism for this effect have led to two alternative
models. In particular, is the storage form of NO in blood an
S-nitrosated protein or is it the anion nitrite? Is NO delivered
from the hemoglobin cysteine residue by an oxygen-linked
allosteric mechanism, or is the NO formed by the nitrite
reductase activity of deoxygenated hemoglobin? Distinguishing between these two mechanisms is an absolute requirement for translational application to human disease and
therapeutics. For example, will nitrite or S-nitrosohemoglobin serve as sensitive and specific biomarkers of
vascular dysfunction and NO deficiency? Which molecule
will provide a therapeutic target for ischemic tissue or limit
ischemia-reperfusion injury? Beyond a technical debate about
methodologies, hemoglobin allostery, and chemical reactions
lies the answer to a question with potentially broad therapeutic implications.
The Devil in the Details
The Problem With S-Nitroso-Proteins as a
Biological Storage Form for NO
Two issues pose significant problems for the S-nitrosohemoglobin paradigm: (1) It is not clear if significant
amounts of S-nitrosated proteins exist in the basal human
circulation. (2) There are no detectable arterial-to-venous
gradients of S-nitrosated hemoglobin in the human
circulation.
Prior studies by Stamler and colleagues first reported the
level of S-nitrosated albumin in human blood to be 7
␮mol/L21 and later lower at 700 nmol/L.22 However, multiple
groups using validated techniques with high sensitivity and
specificity, including the recent study by Ng et al in this
journal,3 found plasma levels less than 40 nmol/L NOmodified protein and this NO-protein complex is largely
mercury stable (consistent with an N-nitrosamine or ironnitrosyl complex and not an S-nitrosothiol).8,18,23–31 In our
hands, the levels of plasma S-nitroso-albumin are less than 5
nmol/L, and we have been unable to detect higher levels of
S-nitrosated albumin at rest or during NO gas inhalation in
humans using Cu/cysteine- and tri-iodide-based reductive
chemiluminescence, biotinylation assays, or the classic
Griess-Saville assays (even with collection of samples in
NEM and DTPA—required for stabilization of added standards). These nonphotolysis-based chemiluminescent methodologies used by multiple laboratories have been extensively validated. A recent editorial in this journal suggested
that S-nitrosated hemoglobin or albumin standards cannot be
used to validate analytic methods2; however, by all conventions, an oxyhemoglobin molecule with a 29 mass unit NO
group (subtracted proton in S-NO linkage) on the cysteine 93
residue of the ␤-chain (synthesized by a variety of methods)
must be considered a viable standard or the science cannot be
tested.
Using similar assays, our group and the Feelisch group
have reported that S-nitroso-hemoglobin levels are less than
40 nmol/L or undetectable in human red blood cells.20,31 No
arterial-to-venous gradients of S-nitroso-hemoglobin are observed by either group. Higher published values for S-nitrosoalbumin and hemoglobin under normal physiological conditions have largely been measured using high-energy UV
photolysis, a methodology that itself triggers conversion of
blood nitrate to NO.32 Thus, methods based on high-energy
UV photolysis of NO ligands from albumin or hemoglobin
yield measurements 100- to 1000-fold higher than other
high-sensitivity assays but are likely not specific for
S-nitrosated proteins.
Of particular concern, the levels of S-nitroso-hemoglobin
(0.0031 NO/Hb or 7.7 ␮mol/L in whole blood) and ironnitrosyl-hemoglobin (0.0088 NO/Hb or 22 ␮mol/L in whole
blood) reported in the present study by James and colleagues
are orders of magnitude higher than any values currently
measured in the field. While the chemiluminescent measurement of NO-modified proteins is the subject of considerable
controversy, the measurement of NO-heme by electron paramagnetic resonance spectroscopy (EPR) is an accepted,
highly specific gold standard with a sensitivity approaching
250 to 500 nmol/L in whole blood. Using this technique,
iron-nitrosyl-hemoglobin is not detectable in the human
circulation.40 The levels of iron-nitrosyl-hemoglobin of
22 000 nmol/L reported by James et al would provide a
massive signal by EPR that has quite simply never been
observed in human blood. We suggest that these authors
Gladwin and Schechter
perform these measurements using this gold standard to
validate their methodologies.
Is NO Delivery From S-Nitrosated Hemoglobin
Oxygen-Dependent and Under Hemoglobin
Allosteric (Oxygen-Linked) Control?
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The S-nitroso-hemoglobin model has been described as requiring three steps:
1. NO binds cooperatively to a deoxygenated heme group
of oxyhemoglobin (faster on R-state or oxyhemoglobin) to
preserve the NO molecule and protect it from reaction with
oxyhemoglobin.33 This step has been refuted by six independent groups27,34 –39 and the prior observations supporting
ascribed to an artifact of bolus addition of saturated NO
solutions.
2. As hemoglobin reoxygenates (presumably after deoxygenation in the peripheral circulation) the NO group “transfers” to the cysteine. The problem is that this transfer requires
a one-electron oxidation to an NO⫹ equivalent and has only
been demonstrated in the presence of excess nitrite.19 This
observation is in fact an effect of nitrite, not an oxygen-linked
heme to thiol NO transfer: Without added nitrite, NO migration
from heme to thiol is not observed, and with added nitrite,
isotopic labeling has proven that the source of iron-nitrosylhemoglobin is from nitrite and not S-nitroso-hemoglobin.40
3. Deoxygenated S-nitrosated hemoglobin releases the NO
group by transnitrosation to the anion exchange protein on the
red blood cell membrane for export, as a yet unidentified NO
species.41 This last step is invoked in the present study by
James et al4 to explain their observation that oxygenated red
blood cells treated with NO solutions vasodilate aortic rings
more during hypoxia than normoxia. This is an important
observation but may not be due to an allosteric function
intrinsic to hemoglobin. Importantly, James et al4 clearly
show that the S-nitroso-glutathione, a small peptide incapable
of oxygen-linked allosteric structural transitions, has the same
effect: greater vasodilation under hypoxic conditions. In fact,
all NO donors studied to date, S-nitrosothiols, NONOates,
nitroglycerin, nitroprusside, and the anion nitrite, vasodilate
more under hypoxia.7,42,43 The mechanism of this hypoxic
potentiation may involve intermediate reductive reactions
with deoxygenated heme proteins; nitrite and S-nitrosothiols
can be reduced by ferrous heme proteins to NO with
concomitant oxidation of the ferrous heme to ferric (metheme).7,44 It is therefore critically important to carefully
compare the oxygen-dependent vasodilatory effects of NOtreated red blood cells with other nonallosteric NO donors.
Furthermore, an allosteric function of S-nitrosated hemoglobin is limited by reversible second-order transnitrosation
reactions with intracellular glutathione.45 In the glutathionerich reductive red blood cell, S-nitrosated hemoglobin rapidly
decomposes independent of oxygen tension.20 Again, multiple groups have shown that S-nitrosated hemoglobin will
vasodilate under both normoxia and hypoxia in the presence
of greater than 100 ␮mol/L glutathione, ie, under normal
physiological glutathione concentrations.43,46 In this sense,
S-nitrosated red blood cells can be considered “leaky bags” of
NO, leaching NO out by transnitrosation, independent of
oxygen tension; the apparent hypoxic effect appears to be
NO Contest: Nitrite Versus S-Nitroso-Hemoglobin
853
secondary to the well-described hypoxic potentiation of all
NO donors and nitrite, not an intrinsic allosteric property of
S-nitroso-hemoglobin.
What Is Right About Nitrite
We recently proposed an alternative hypothesis that invokes
a simple reaction of nitrite with the heme group of deoxyhemoglobin, as well as other heme proteins, to generate NO and
produce vasodilation.7
Nitrite as the Ideal Vascular Storage Pool for NO
Nitrite is present in blood in concentrations of 500 to 1000
nmol/L and in 10 000 nmol/L in tissues.28,31,47,48 Nitrite is
stable in blood compared with NO and S-nitrosothiol, as it is
not as readily oxidized or reduced, respectively. Further,
nitrite is selectively taken up by deoxygenated red blood cells
and relatively stable with oxygenated red blood cells.49
Finally, nitrite is reduced to NO by deoxygenated hemoglobin.7,50,51 As such, nitrite is an ideal storage form for NO that
is conserved under normoxic conditions and utilized under
hypoxic conditions.
Nitrite Is a Vasodilator
Although some have argued that nitrite is not a vasodilator,52–54 in recent studies, we found that near-physiological
levels of nitrite vasodilate the human circulation, and levels
of nitrite as low as 500 nmol/L vasodilate rat aortic rings in
the presence of deoxygenating red blood cells.7 Whereas the
vasodilatory effects of nitrite are potentiated by hypoxia per
se by one order of magnitude, the vasodilation is potentiated
by four orders of magnitude in the presence of deoxygenated
hemoglobin. We and others have therefore proposed a function for hemoglobin as a hypoxic-regulated nitrite reductase
and suggest that this role might contribute to hypoxic vasodilation.7,51 Indeed, a recent study has demonstrated that
tissue stores of nitrite are rapidly converted to NO with
deoxygenation, suggesting a global role for this storage pool
in hypoxia-dependent NO generation.48
In the study by James et al,4 NO donors were added to
oxygenated red blood cells. Such additions produce predominantly nitrate and methemoglobin and to a lesser extent
nitrite and S-nitroso-hemoglobin. In fact, nitrite levels are
anticipated to be 97% higher than S-nitrosothiols for NO
additions to blood.18 Considering the fact that we have found
that 500 nmol/L nitrite vasodilates aortic ring bioassays in the
presence of deoxygenated erythrocytes, it would be of interest
to compare the results observed by James and colleagues with
NO donor additions versus equimolar additions of nitrite.
Are the Observed Vasodilatory Effects of
Nitrite Secondary to the Formation of
S-Nitrosated Hemoglobin?
Nitrite reactions with blood produce NO, iron-nitrosylhemoglobin, N-nitrosamines, nitrated tyrosine residues on
proteins, and S-nitroso-hemoglobin. In this context, one can
consider nitrite as the fundamental storage form for NO in the
vasculature capable of forming a vast range of reaction
products. This leads to the critical question as to whether
S-nitroso-hemoglobin actually mediates the nitrite-dependent
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Circulation Research
April 16, 2004
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vasodilation. We believe the answer is that it does not.
Definitive experiments using alkylated-blocked cysteine 93
or recombinant hemoglobins with cysteine to alanine, glycine, or leucine substitutions (mutants courtesy of Chien Ho,
Carnegie Mellon University; Pittsburgh, Pa) reveal that the
rate of nitrite reduction, NO generation, and vasodilation is
far from reduced and actually greatly increased (Z. Huang, J.
Crawford, M. Gladwin, and R. Patel, unpublished observations, 2004).
In conclusion, we have presented a critical and alternative
perspective on how erythrocytes modulate vascular responses
to hypoxia. While this function was initially ascribed to
S-nitrosated hemoglobin, we find that this function is subserved by nitrite and the nitrite-reductase activity of deoxyhemoglobin. Further studies by multiple research groups
evaluating the biochemistry, physiology, and the relative
therapeutic activities of nitrite versus S-nitroso-hemoglobin
will ultimately provide resolution to this debate. We can all
agree that a deeper understanding of the multiple facets of red
blood cell and NO biology will provide important insights
into the mechanisms of vascular homeostasis and will offer
novel therapeutic strategies for the treatment of vascularrelated pathologies.
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KEY WORDS: nitric oxide 䡲 hemoglobin
iron-nitrosyl-hemoglobin 䡲 nitrite
䡲
S-nitroso-hemoglobin
䡲
NO Contest: Nitrite Versus S-Nitroso-Hemoglobin
Mark T. Gladwin and Alan N. Schechter
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Circ Res. 2004;94:851-855
doi: 10.1161/01.RES.0000126697.64381.37
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