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Emerging role of nitrite in human biology
Blood Cells, Molecules, and Diseases 32 (2004) 423 – 429 www.elsevier.com/locate/ybcmd
Emerging role of nitrite in human biology Andre´ Dejam, a Christian J. Hunter, a,b Alan N. Schechter, a and Mark T. Gladwin a,b,* a
Laboratory of Chemical Biology, National Institute of Diabetes, Digestive and Kidney Disease, National Institute of Health, Bethesda, MD 20892, USA b Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA Submitted 3 February 2004 (Communicated by J. Hoffman, Ph.D., 5 February 2004) Available online 14 March 2004
Abstract Nitric oxide (NO) plays a fundamental role in maintaining normal vascular function. NO is produced by endothelial cells and diffuses both into smooth muscle causing vasodilation and into the vessel lumen where the majority of this highly potent gas is rapidly inactivated by dioxygenation reaction with oxyhemoglobin to form nitrate. Diffusional barriers for NO around the erythrocyte and along the endothelium in laminar flowing blood reduce the inactivation reaction of NO by hemoglobin, allowing sufficient NO to escape for vasodilation and also to react in plasma and tissues to form nitrite anions (NO2 ) and NO-modified peptides and proteins (RX-NO). Several recent studies have highlighted the importance of the nitrite anion in human biology. These studies have shown that measurement of plasma nitrite is a sensitive index of constitutive NO synthesis, suggesting that it may be useful as a marker of endothelial function. Additionally, recent evidence suggests that nitrite represents a circulating storage pool of NO and may selectively donate NO to hypoxic vascular beds. The conversion of nitrite to NO requires a reaction with a deoxygenated heme protein, suggesting a novel function of hemoglobin as a deoxygenation-dependent nitrite reductase. This review focuses on the role of nitrite as a circulating NO donor, its potential as an index of NO synthase (NOS) activity and endothelial function, and discusses potential diagnostic and therapeutic applications. Published by Elsevier Inc. Keywords: Nitric oxide; Vasodilation; Endothelial function; Nitrite; Nitric oxide stores
Introduction Nitric oxide (NO) is a gas which is continuously synthesized in endothelial cells from the amino acid L-arginine by the constitutive calcium and calmodulin-dependent enzyme nitric oxide synthase [1]. This heme-containing enzyme catalyzes a five-electron oxidation of one of the basic guanidine nitrogen groups of L-arginine in the presence of multiple cofactors and molecular oxygen [2] (Fig. 1). In seminal experiments, Furchgott and Zawadzki [3] found that strips of aorta with intact endothelium relaxed in response to acetylcholine, but constricted in response to the same agonist when the endothelium had been rubbed off indicating an
essential role for endothelium in smooth muscle vascular reactivity. The substance responsible for this acetylcholinestimulated relaxation was initially called endothelium-derived relaxing factor, and subsequently identified by Ignarro et al. [4] and Palmer et al. [5] to be NO. Further studies showed that NO released from the endothelium diffuses into vascular smooth muscle where it activates soluble guanylyl cyclase by binding to its heme group, which results in increased cyclic guanosine monophosphate (cGMP) levels [6]. Cyclic GMP activates GMP-dependent kinases and downstream signaling that ultimately decreases intracellular calcium concentration leading to vasodilation [7].
Cardiovascular functions of NO * Corresponding author. Critical Care Medicine Department, Warren G. Magnuson Clinical Center, National Institutes of Health, Room 7D43, Building 10, 10 Center Drive, Bethesda, MD 20892. Fax: +1-301-4021213. E-mail address: [email protected] (M.T. Gladwin). 1079-9796/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.bcmd.2004.02.002
The importance of NO in the regulation of coronary and systemic vasodilator tone has been repeatedly demonstrated experimentally by inhibiting its synthesis [8 –10]. The drug
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NN-monomethyl-L-arginine (L-NMMA) inhibits endogenous NO production by competing with L-arginine as the substrate for NO synthase (NOS) [11]. Intra-coronary infusion of L-NMMA in normal human volunteers results in increased epicardial coronary vascular resistance and decreased blood flow as a result of arteriolar vasoconstriction [8]. These results indicate that NO contributes to basal coronary vasodilator tone and blood flow in humans [12,13]. In contrast, patients with endothelial dysfunction (due to coronary artery disease or risk factors for atherosclerosis) show minimal vasoconstriction responses to LNMMA, suggesting deficient NO release from the coronary endothelium in these patients [8]. NO also contributes to regulation of vascular tone in the systemic circulation. Recently, Lauer et al. used strain gauge venous plethysmography to measure forearm blood flow and resistance in human volunteers before and after inhibition or stimulation of NO synthesis (by infusion of L-NMMA or acetylcholine into the brachial artery). These workers showed that changes in regional vascular resistance correlated directly with concomitant changes in regional NO formation [9]. Akin to the coronary circulation, the vasoconstricting effects of L-NMMA are reduced in peripheral vascular beds of patients with hypertension, diabetes, hypercholesterolemia, obesity, and tobacco use, suggesting reduced endothelial-derived NO production in these conditions [10,14,15]. In addition to the regulation of basal vascular tone, NO is involved in systemic [16] and cerebral [17] hypoxic vasodilation. NO inhibits platelet activation,
leukocyte recruitment, smooth muscle cell proliferation, cell respiration, and has antioxidative and oxidative properties. (see Ref. [18] for review) Thus, NO is an endotheliumderived mediator that contributes substantially to homeostatic vascular function. Therapeutically, it seems highly desirable to selectively modulate NO synthesis and degradation in disease states with endothelial dysfunction and resulting reduction in NO bioavailability [19].
Fate of NO in human blood: the existence of circulating NO stores NO that diffuses into the vessel lumen and into the erythrocyte reacts at a nearly diffusion-limited rate with oxyhemoglobin yielding methemoglobin and nitrate [20] (Fig. 1). This reaction appears to limit the intravascular transport and half-life of NO considerably. The reactions of NO and hemoglobin are so fast and the intravascular hemoglobin concentration so high (10 mM heme) that all available NO would be consumed if hemoglobin was not compartmentalized within the erythrocyte. Several factors such as the erythrocyte membrane and submembrane [21,22], the unstirred layer surrounding the erythrocyte [20,23], and the presence of an erythrocyte-free zone of plasma along the surface of vascular endothelium [24 – 26] create diffusional barriers between NO and intra-erythrocytic hemoglobin. These barriers are estimated to decrease the rate of NO scavenging by intra-erythrocytic hemoglobin
Fig. 1. Potential role of nitric oxide and nitrite in human biology. The nitrite anion (NO2 ) unites two unique properties: It is a marker of constitutive nitric oxide (NO) synthase activity (NOS) and a circulating storage and delivery source of NO. Possible applications of this dual role of nitrite in human biology are depicted. NO2 entering the red blood cell can react either with oxyhemoglobin (oxy Hb) to form nitrate (NO3 ) and methemoglobin (met Hb) or with deoxyhemoglobin (deoxy Hb) to form NO, nitrosylhemoglobin (NO-Hb), and NO adducts (RX-NO). It is unknown whether NO formed from the reaction of NO2 with deoxyhemoglobin is directly exported from the red blood cell or may be transformed into an NO adduct (RX-NO) to cause NO2 induced vasodilation. Dotted lines represent degradation pathways.
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greater than 600-fold. This compartmentalization model of hemoglobin allows for the existence of a sufficient diffusional gradient for NO between endothelium and smooth muscle to allow local paracrine activity (endothelium to smooth muscle communication) but limit distant endocrine bioactivity (recently reviewed in Refs. [27,28]). Thus, in addition to the NO, which diffuses into vascular smooth muscle and activates soluble guanylyl cyclase, a portion of intravascular NO can escape hemoglobin scavenging and reacts with oxygen in plasma to form nitrite or nitrosate free thiols, amines, and unidentified constituents in blood to form mercury-labile and stable NO adducts [29]. The importance of these diffusional barriers is illustrated in patients with sickle cell disease. These patients have high levels of plasma cell-free hemoglobin that increases nitric oxide consumption and may be partially responsible for pulmonary hypertension and other pathophysiological manifestations [30]. In addition, infusion of cell-free hemoglobin in both animals and humans rapidly results in increased vascular resistance and blood pressure [31].
The nature of circulating NO stores Oxidative and nitrosative products of NO could represent a circulating NO storage pool, which may exert ‘‘endocrine’’ effects distal from its site of production. In support of this concept, the infusion of authentic NO solution into the human circulation produces systemic vasodilation of the micro- and macrovasculature, which is accompanied by an increase in plasma NO-protein adducts and nitrite [32,33]. Similar distal vasodilating effects of NO were observed in the human forearm vasculature during inhalation of NO gas in normal volunteers [34]. This vasodilating effect was associated with the formation of the nitrite anion and ironnitrosylated hemoglobin. Nitrite, the primary oxidative NO metabolite, has long been considered inert under physiologic conditions [9,35]. However, several factors would position nitrite as a major intravascular storage pool for NO and suggest that it may be bioactive. First, plasma nitrite is present in substantial concentrations in both plasma [36] and tissue [37]. Second, nitrite is relatively stable because it is not readily reduced by cellular reductants as are the S-nitrosothiol NO adducts [38]. Third, nitrite’s reaction rate with heme proteins is 10,000 times less than that of authentic NO which allows for action beyond its site of formation. Finally, recent data from our group, summarized at the end of this review, suggest that nitrite is vasoactive both in vitro and in vivo [39]. Therefore, we hypothesize that nitrite might serve as the fundamental intravascular storage pool of NO that can react with deoxyhemoglobin and the deoxy form of other heme proteins such as myoglobin, neuroglobin, and cytoglobin to form NO, nitrosylhemoglobin (NO-Hb), and to a lesser extent N-nitrosamines (RX-NO), S-nitrosothiols, and nitrated lipids (L-NO2) (Fig. 1).
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Nitrosative metabolites likewise have been implicated to serve as NO stores in vivo [32]. Putative candidates are Nnitrosamines [56]. Recently, it was shown that ingestion of a potent plant antioxidant alleviated endothelial dysfunction in patients at risk for atherosclerosis and was associated with an increase in RX-NO, likely representing an N-nirosamine [40]. Other NO-derived species such as S-nitrosothiols have been extensively studied. During the last decade, S-nitrosothiol albumin [41] and S-nitrosothiol hemoglobin [42] have been proposed as major circulating NO storage pools. Pharmacologic modulation of these species inhibits platelet function reducing asymptomatic embolization after carotid endaterectomy [43], diminish ischemia – reperfusion injury [44], and normalize pharmacologically induced endothelial dysfunction [34]. However, the role of endogenous S-nitrosothiols in the regulation of blood flow and as a circulating stable NO storage pool is intensely debated (as has been reviewed in Ref. [28]). Central to this controversy are methodological problems inherent in the detection of SNO species in blood [45 – 48]. We recently found that techniques use to identify high levels of S-nitrosothiol in plasma invariably use UV photolysis, which can artefactually generate NO signal from nitrate in the presence of reduced thiols [49]. Future work will be required to establish the relative contribution and importance of nitrite, RX-NO, or S-nitrosothiols of albumin or hemoglobin in the regulation of blood flow and storage of NO bioactivity in blood.
Measurement of plasma nitrite as a marker of constitutive NO synthase activity There is considerable interest in indirectly determining NO synthase activity by assaying the metabolites of NO in blood [29]. Due to its ultra-short half-life and its radical character, direct measurement of nitric oxide poses considerable difficulties under basal conditions [50]. In vitro studies reveal that in the presence of oxygen, NO is rapidly oxidized to nitrite, following pseudo-first-order kinetics with a strict 1:1 stoichiometry [51]. In blood, as discussed earlier, NO also reacts with oxyhemoglobin to form nitrate. Thus, endogenous NO formation may be measured via the determination of its oxidative products, nitrite, and nitrate. However, the reported basal nitrite concentrations in plasma of humans in the literature range from ‘‘nondetectable’’ [52], over nanomolar (450 nmol/l) [53], to micromolar (26 Amol/l) [54] levels. These enormous differences in nitrite levels can be rationalized considering the confounding factors and variations in blood sampling and sample processing as well as the methodological problems inherent to the analytical procedures used [36]. Some methods (such as the Griess reagent combined with spectrophotometric detection) simply do not possess the sensitivity to allow a precise measurement of nitrite in the proposed physiological concentration range. In addition, the analysis might be affected by many factors such as proteins, varying redox
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conditions, and trace contamination with ubiquitously present nitrite and nitrate during sample processing [55]. To address these methodological problems, Kelm et al. have measured plasma nitrite concentration of nine different mammal species including humans using three independent methodologies. Results of these study demonstrate the uniform occurrence of nanomolar levels of plasma nitrite (100 –600 nmol/l) [36,45,56,57]. Validation of methodologies to measure plasma nitrite concentration [36,45,56– 60] have led to studies testing the hypothesis that plasma nitrite is an index of constitutive NOS activity [9,61 – 63]. This hypothesis is principally supported by the observation that approximately 70– 90% of circulating plasma nitrite is derived from eNOS activity in humans and other mammals [36,64]. In addition, nitrite and nitrate levels are reduced up to 70% in eNOS knockout mice as compared to control mice [36,65] and both stimulation and inhibition of eNOS have been shown to produce acute changes in plasma nitrite concentrations in the human forearm vasculature [9]. Thus, basal plasma nitrite concentrations reflect constitutive NOS activity and NO availability uniformly in several mammalian species [36]. In aggregate, these data suggest that the measurement of plasma nitrite, using carefully validated methodologies that avoid ex vivo decay or nitrite contamination, can serve as a biomarker of NO synthase activity and thus endothelial function. Caution should be exercised when changes in plasma nitrate (NO3 ) are evaluated as an index of NO synthesis. Unlike plasma nitrite, plasma nitrate concentration does not change significantly during acute NOS inhibition and in addition are significantly affected by both diet and renal function [9,36,66].
The nitrite anion as a potential physiologic source of NO Nitrite has long been believed to form NO non-enzymatically only under certain conditions such as severe ischemia or acidosis [67]. Under physiologic levels of acidity, as occurs during cardiac ischemia and infarction, nitrite forms nitrous acid, which can react with nitrite again or an electron donor (such as ascorbate) to form dinitrogentrioxide (N2O3). This reactive nitrogen species can then nitrosate thiols (which are vasoactive) or, in the presence of an electron donor, produce NO gas [68]. This non-enzymatic NO generation has been documented in certain vascular tissue preparations [69,70] and in ischemic myocardium ex vivo; it requires low tissue pH (approximately 6.5) [71] and tissue reductants [67,71]. Alternatively, the conversion of nitrite to NO gas could be catalyzed by a metal or enzyme. For example, recent studies suggest that xanthine oxidoreductase, which is present in abundance in vascular endothelium, may reduce nitrite to NO [72,73], an effect that increases with decreasing pH [71], increasing NADH concentration, and hypoxia [74]. However, these hypothesized mechanisms
for nitrite reduction—nitrite disproportionation and xanthine oxidoreductase activity—both require extremely low pO2 and pH not readily found under physiologic conditions [35] casting doubt on these mechanisms contributing to the role of this anion as a physiologic vasodilator [35,75]. However, oral nitrite administration had been shown to have a sustained antihypertensive effect in spontaneous hypertensive rats [76] and several case series have shown that infusion of a high dose of nitrite (300 mg), as a methemoglobin-forming agent to treat cyanide poisoning, caused hypotension and increased cerebral blood flow [77,78]. In addition, the demonstration of an arterial-venous difference of plasma nitrite concentration [45] and the observation that inhalation of NO induced peripheral vascular effects associated with a selective increase in nitrite concentration [34] led us to the hypothesis that nitrite may be reduced to NO under physiologic conditions. Based on these studies, we evaluated the vasodilator properties and mechanisms for bioactivation of nitrite in the human forearm. Nitrite infusions of both 36 and 0.36 Amol/ min into the forearm brachial artery resulted in supra- and near-physiologic intravascular nitrite concentrations, respectively, and increased forearm blood flow before and during exercise, with or without NO synthase inhibition. Nitrite infusions were associated with rapid formation of erythrocyte iron-nitrosylated hemoglobin and, to a lesser extent, Snitrosohemoglobin. NO-modified hemoglobin formation was inversely proportional to oxyhemoglobin saturation, suggesting that a reaction with deoxygenated hemoglobin or tissues accounted for the mechanism of vasodilation. We subsequently found that vasodilation of rat aortic rings and formation of both NO gas and NO-modified hemoglobin resulted from the nitrite reductase activity of deoxyhemoglobin and deoxygenated erythrocytes (Fig. 1). Doyle et al. [79] first reported in 1981 that nitrite reacts with deoxyhemoglobin (and H+) to form methemoglobin and NO (and OH ), a chemistry consistent with our in vivo and in vitro observations. Such chemistry is ideally suited for hypoxic generation of NO from nitrite, as it requires hemoglobin deoxygenation, thus is linked to the physiological oxygen gradient, and protons connecting it to the physiological pH/CO2 gradient. We hypothesize that this nitrite chemistry takes advantage of the Bohr and Haldane effects to link local tissue hypoxia and acidosis and hemoglobin deoxygenation to nitrite bioactivation leading to NO formation. These results suggest that nitrite represents a major bioavailable pool of NO and describe a new physiological function for hemoglobin as a nitrite reductase, potentially contributing to systemic hypoxic vasodilation [39].
Summary Nitrite is a marker of NO synthase activity and endothelial function in humans and may constitute a novel storage and delivery source of NO. The observation that deoxygenated
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erythrocytes reduce nitrite to form NO sheds new light on the role of red blood cells and hemoglobin in NO physiology: Hemoglobin is not merely a sink of NO but rather a modulator of NO availability adapting NO levels in the circulation to the oxygen needs of the surrounding tissue [39]. This principle was first ascribed to S-nitrosohemoglobin, but our data suggest this process is primarily served by nitrite reduction to NO by deoxyheme proteins. This dual role of nitrite has potential diagnostic and therapeutic implications. Nitrite as a marker of constitutive NOS activity may index NO availability in atherosclerosis and associated risk factors. This may be of diagnostic and prognostic value and provide guidance towards more individualized cardiovascular therapy. Nitrite delivery to humans via infusion or inhalation may counterbalance pathophysiologic processes occurring in disease states with a relative or absolute lack of NO, such as atherosclerosis, hypertension, diabetes, hypercoaguable states caused by endothelial dysfunction, acute respiratory distress syndrome of the newborn, delayed-onset vasospasm due to subarachnoidal hemorrhage, and sickle cell disease. Further study will be required to establish the role of nitrite in physiology and its application for diagnostics and therapeutics.
Acknowledgments This work was supported by the Clinical Center and the Medical Scientist Training Program T32GM08361. Christian J. Hunter is supported by the Clinical Research Training Program of the National Institutes of Health. This paper is based on a presentation at the Red Cell Club Meeting held at Yale University School of Medicine on October 24, 2003.
References [1] J.S. Beckman, W.H. Koppenol, Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly, Am. J. Physiol.: Cell Physiol. 271 (1996) C1424 – C1437. [2] J.L. Boucher, C. Moali, J.P. Tenu, Nitric oxide biosynthesis, nitric oxide synthase inhibitors and arginase competition for L-arginine utilization, Cell. Mol. Life Sci. 55 (1999) 1015 – 1028. [3] R.F. Furchgott, J.V. Zawadzki, The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 288 (1980) 373 – 376. [4] L.J. Ignarro, R.E. Byrns, G.M. Buga, K.S. Wood, Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical, Circ. Res. 61 (1987) 866 – 879. [5] R.M.J. Palmer, A.G. Ferrige, S. Moncada, Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor, Nature 327 (1987) 524 – 526. [6] L.J. Ignarro, J.B. Adams, P.M. Horwitz, K.S. Wood, Activation of soluble guanylate cyclase by NO-hemoproteins involves NO – heme exchange. Comparison of heme-containing and heme-deficient enzyme forms, J. Biol. Chem. 261 (1986) 4997 – 5002. [7] L.J. Ignarro, Biosynthesis and metabolism of endothelium-derived nitric oxide, Annu. Rev. Pharmacol. Toxicol. 30 (1990) 535 – 560.
427
[8] A.A. Quyyumi, N. Dakak, N.P. Andrews, S. Husain, S. Arora, D.M. Gilligan, J.A. Panza, R.O. Cannon III, Nitric oxide activity in the human coronary circulation. Impact of risk factors for coronary atherosclerosis, J. Clin. Invest. 95 (1995) 1747 – 1755. [9] T. Lauer, M. Preik, T. Rassaf, B.E. Strauer, A. Deussen, M. Feelisch, M. Kelm, Plasma nitrite rather than nitrate reflects regional endothelial nitric oxide synthase activity but lacks intrinsic vasodilator action, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 12814 – 12819. [10] J.A. Panza, P.R. Casino, C.M. Kilcoyne, A.A. Quyyumi, Role of endothelium-derived nitric oxide in the abnormal endothelium-dependent vascular relaxation of patient with essential hypertension, Circulation 87 (1993) 1468 – 1474. [11] B.X. Mayer, C. Mensik, S. Krishnaswami, H. Derendorf, H.-G. Eichler, L. Schmetterer, M. Wolzt, Pharmacokinetic – pharmacodynamic profile of systemic nitric oxide-synthase inhibition with L-NMMA in humans, Br. J. Clin. Pharmacol. 47 (1999) 539 – 544. [12] M. Kelm, J. Schrader, Control of coronary vascular tone by nitric oxide, Circ. Res. 66 (1990) 1561 – 1575. [13] M. Kelm, J. Schrader, Nitric oxide release from the isolated guinea pig heart, Eur. J. Pharmacol. 155 (1988) 317 – 321. [14] K. Hishikawa, T. Nakaki, H. Suzuki, R. Kato, T. Saruta, Role of L-arginine-nitric oxide pathway in hypertension, J. Hypertens. 11 (1993) 639 – 645. [15] J. Loscalzo, J. Freedman, A. Inbal , J.F. Keaney Jr., A.D. Michelson, J.A. Vita, Nitric oxide insufficiency and arterial thrombosis, Trans. Am. Clin. Climatol. Assoc. 111 (2000) 158 – 163. [16] M.L. Blitzer, E. Loh, M.-A. Roddy, J. Stamler, M. Creager, Endothelium-derived nitric oxide regulates systemic and pulmonary vascular resistance during acute hypoxia in humans, J. Am. Coll. Cardiol. 28 (2003) 591 – 596. [17] C.J. Hunter, A.B. Blood, C.R. White, W.J. Pearce, G.G. Power, Role of nitric oxide in hypoxic cerebral vasodilation in the ovine fetus, J. Physiol. (London) 549 (2003) 625 – 633. [18] P. Vallance, N. Chan, Endothelial function and nitric oxide: clinical relevance, Heart 85 (2001) 342 – 350. [19] A.N. Schechter, M.T. Gladwin , R.O. Cannon III, NO solutions? J. Clin. Invest. 109 (2002) 1149 – 1151. [20] X. Liu, M.J.S. Miller, M.S. Joshi, H. Sadowska-Krowicka, D.A. Clark, J.R. Lancaster Jr., Diffusion-limited reaction of free nitric oxide with erythrocytes, J. Biol. Chem. 273 (1998) 18709 – 18713. [21] M.W. Vaughn, K.-T. Huang, L. Kuo, J.C. Liao, Erythrocytes possess an intrinsic barrier to nitric oxide consumption, J. Biol. Chem. 275 (2000) 2342 – 2348. [22] Z. Huang, J.G. Louderback, M. Goyal, F. Azizi, S.B. King, D.B. Kim-Shapiro, Nitric oxide binding to oxygenated hemoglobin under physiologic conditions, Biochim. Biophys. Acta 1568 (2001) 252 – 260. [23] J.T. Coin, J.S. Olsen, The rate of oxygen uptake by human red blood cells, J. Biol. Chem. 254 (1979) 1178 – 1190. [24] M.W. Vaughn, L. Kuo, J.C. Liao, Effective diffusion distance of nitric oxide in the microcirculation, Am. J. Physiol.: Heart Circ. Physiol. 274 (1998) H1705 – H1714. [25] A.R. Butler, I.L. Megson, P.G. Wright, Diffusion of nitric oxide and scavenging by blood in the vasculature, Biochim. Biophys. Acta 1425 (1998) 168 – 176. [26] J.C. Liao, T.W. Hein, M.W. Vaughn, K.-T. Huang, L. Kuo, Intravascular flow decreases erythrocyte consumption of nitric oxide, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 8757 – 8761. [27] A.N. Schechter, M.T. Gladwin, Hemoglobin and the paracrine and endocrine functions of nitric oxide, N. Engl. J. Med. 348 (2003) 1483 – 1485. [28] M.T. Gladwin, J.R. Lancaster, B.A. Freeman, A.N. Schechter, Nitric oxide’s reactions with hemoglobin: a view through the SNO-storm, Nat. Med. 9 (2003) 496 – 500. [29] M. Kelm, Nitric oxide metabolism and breakdown, Biochim. Biophys. Acta 1411 (1999) 273 – 289.
428
A. Dejam et al. / Blood Cells, Molecules, and Diseases 32 (2004) 423–429
[30] C.D. Reiter, X. Wang, J.E. Tanus-Santos, N. Hogg , R.O. Cannon III, A.N. Schechter, M.T. Gladwin, Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease, Nat. Med. 8 (2002) 1383 – 1389. [31] R.J. Rohlfs, E. Bruner, A. Chiu, A. Gonzales, M.L. Gonzales, D. Magde, M.D. Magde, K.D. Vandegriff, R.M. Winslow, Arterial blood pressure responses to cell-free hemoglobin solutions and the reaction with nitric oxide, J. Biol. Chem. 273 (1998) 12128 – 12134. [32] T. Rassaf, P. Kleinbongard, M. Preik, A. Dejam, P. Gharini, T. Lauer, J. Erckenbrecht, A. Duschin, R. Schulz, G. Heusch, M. Feelisch, M. Kelm, Plasma nitrosothiols contribute to the systemic vasodilator effects of intravenously applied NO; experimental and clinical study on the fate of NO in human blood, Circ. Res. 91 (2002) 470 – 477. [33] T. Rassaf, M. Preik, P. Kleinbongard, T. Lauer, C. Heiß, B.E. Strauer, M. Feelisch, M. Kelm, Evidence for in vivo transport of bioactive nitric oxide in human plasma, J. Clin. Invest. 109 (2002) 1241 – 1248. [34] R.O. Cannon III, A.N. Schechter, J.A. Panza, F.P. Ognibene, M.E. Pease-Fye, M.A. Waclawiw, J.H. Shelhamer, M.T. Gladwin, Effects of inhaled nitric oxide on regional blood flow are consistent with intravascular nitric oxide delivery, J. Clin. Invest. 108 (2001) 279 – 287. [35] D. Tsikas, J.C. Fro¨lich, Is circulating nitrite a directly acting vasodilator? Clin. Sci. 103 (2002) 107 – 110. [36] P. Kleinbongard, A. Dejam, T. Lauer, T. Rassaf, A. Schindler, O. Picker, T. Scheeren, A. Go¨decke, J. Schrader, R. Schulz, G. Heusch, G.A. Schaub, N. Bryan, M. Feelisch, M. Kelm, Plasma nitrite reflecting constitutive activity of nitric oxide synthase in mammals, Free Radical Biol. Med. 35 (2003) 790 – 796. [37] J. Rodriguez, R.E. Maloney, T. Rassaf, N.S. Bryan, M. Feelisch, Chemical nature of nitric oxide storage forms in rat vascular tissue, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 336 – 341. [38] M.T. Gladwin, X. Wang, C.D. Reiter, B.K. Yang, E.X. Vivas, C. Bonaventura, A.N. Schechter, S-nitrosohemoglobin is unstable in the reductive red cell environment and lacks O2/NO-linked allosteric function, J. Biol. Chem. 277 (2002) 27818 – 27828. [39] K. Cosby, K.S. Partovi, J.H. Crawford, R.P. Patel, C.D. Reiter, S. Martyr, B.K. Yang, M.A. Waclawiw, G. Zalos, X. Xu, K.T. Huang, H. Shields, D.B. Kim-Shapiro, A.N. Schechter, R.O. Cannon III, M.T. Gladwin, Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation, Nat. Med. 9 (2003) 1498 – 1505. [40] C. Heiß, A. Dejam, P. Kleinbongard, T. Schewe, H. Sies, M. Kelm, Vascular effects of cocoa rich in flavan-3-ols, JAMA 290 (2003) 1030 – 1031. [41] J.S. Stamler, O. Jaraki, J. Osborne, D.I. Simon, J. Keaney, J. Vita, D. Singel, C.R. Valeri, J. Loscalzo, Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 7674 – 7677. [42] L. Jia, C. Bonaventura, J. Bonaventura, J.S. Stamler, S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control, Nature 380 (1996) 221 – 226. [43] Z. Kaposzta, J.F. Martin, H.S. Markus, Switching off embolization from symptomatic carotid plaque using S-nitrosoglutathione, Circulation 105 (2002) 1480 – 1484. [44] S. Hallstro¨m, H. Gasser, C. Neumayer, A. Flu¨gl, J. Nanobashvili, A. Jakubowski, I. Huk, G. Schlag, T. Malinski, S-nitroso human serum albumin treatment reduces ischemia/reperfusion injury in skeletal muscle via nitric oxide release, Circulation 105 (2002) 3032 – 3038. [45] M.T. Gladwin, J.H. Shelhamer, A.N. Schechter, M.E. Pease-Fye, M.A. Waclawiw, J.A. Panza, F.P. Ognibene , R.O. Cannon III, Role of circulation nitrite and S-nitrosohemoglobin in the regulation of regional blood flow in humans, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 11482 – 11487. [46] T. Rassaf, N.S. Bryan, R.E. Maloney, V. Specian, M. Kelm, B. Kalyanaraman, J. Rodriguez, M. Feelisch, NO adducts in mammalian red blood cells: too much or too little? Nat. Med. 9 (2003) 481 – 483.
[47] D. Tsikas, Measurement of physiological S-nitrosothiols: a problem child and a challenge, Nitric Oxide 9 (2003) 53 – 55. [48] R. Rossi, D. Giustarini, A. Milzani, R. Colombo, I. Dalle-Donne, P. di Simplicio, Physiological levels of S-nitrosothiols in human plasma, Circ. Res. 89 (2001) E47. [49] A. Dejam, P. Kleinbongard, T. Rassaf, P. Gharini, S. Hamada, J. Rodriguez, M. Feelisch, M. Kelm, Thiols enhance NO formation from nitrate photolysis, Free Radical Biol. Med. 35 (2003) 1551 – 1559. [50] P. Vallance, S. Patton, K. Bhagat, R. MacAllister, M. Radomski, S. Moncada, T. Malinski, Direct measurement of nitric oxide in human beings, Lancet 345 (1995) 153 – 154. [51] P.C. Ford, D.A. Wink, D.M. Stanbury, Autoxidation kinetics of aqueous nitric oxide, FEBS Lett. 326 (1993) 1 – 3. [52] A. Meulemans, F. Delsenne, Measurement of nitrite and nitrate levels in biological samples by capillary electrophoresis, J. Chromatogr., B: Biomed. Appl. 660 (1994) 401 – 404. [53] A.M. Leone, P.L. Francis, P. Rhodes, S. Moncada, A rapid and simple method for the measurement of nitrite and nitrate in plasma by high performance capillary electrophoresis, Biochem. Biophys. Res. Commun. 200 (1994) 951 – 957. [54] M. Gorenflo, C. Zheng, A. Poge, M. Bettendorf, E. Werle, W. Fiehn, H.E. Ulmer, Metabolites of the L-arginine-NO pathway in patients with left-to-right shunt, Clin. Lab. 47 (2001) 441 – 447. [55] P. Kleinbongard, T. Rassaf, A. Dejam, S. Kerber, M. Kelm, Griess method for nitrite measurement of aqueous and protein containing sample, Methods Enzymol. 359 (2002) 158 – 168. [56] T. Rassaf, N.S. Bryan, M. Kelm, M. Feelisch, Concomitant presence of N-Nitroso and S-Nitroso Proteins in human plasma, Free Radical Biol. Med. 33 (2002) 1590 – 1596. [57] D. Tsikas, I. Fuchs, F.-M. Gutzki, J.C. Fro¨lich, Measurement of nitrite and nitrate in plasma, serum and urine of humans by highperformance liquid chromatography, the Griess assay, chemiluminescence and gas chromatography-mass spectrometry: interferences by biogenic amines and NG-nitro-L-arginine analogs, J. Chromatogr., B: Biomed. Appl. 715 (1998) 441 – 444. [58] M. Feelisch, T. Rassaf, S. Mnaimneh, N. Singh, N.S. Bryan, D. Jourd’heuil, M. Kelm, Concomitant S-, N-, and heme-nitros(yl)ation in biological tissues and fluids: implications for the fate of NO in vivo, FASEB J. 16 (2002) 1775 – 1785. [59] K. Schulz, S. Kerber, M. Kelm, A new technique for measurement of nitrite in human blood, Nitric Oxide: Biol. Chem. 2 (1998) 101. [60] H. Preik-Steinhoff, M. Kelm, Determination of nitrite in human blood by combination of a specific sample preparation with high performance anion-exchange chromatography and electrochemical determination, J. Chromatogr., B: Biomed. Appl. 685 (1996) 348 – 352. [61] M. Kelm, H. Preik-Steinhoff, M. Preik, B.E. Strauer, Serum nitrite sensitively reflects endothelial NO formation in human forearm vasculature: evidence for biochemical assessment of the endothelial L-arginine-NO pathway, Cardiovasc. Res. 41 (1999) 765 – 772. [62] T. Lauer, P. Kleinbongard, M. Preik, B.H. Rauch, A. Deussen, M. Feelisch, B.E. Strauer, M. Kelm, Direct biochemical evidence for eNOS stimulation by bradykinin in the human forearm vasculature, Basic Res. Cardiol. 98 (2003) 84 – 89. [63] T. Lauer, P. Kleinbongard, M. Kelm, Indexes of NO-bioavailibility in human blood, News Physiol. Sci. 17 (2002) 251 – 255. [64] P.M. Rhodes, A.M. Leone, P.L. Francis, A.D. Struthers, S. Moncada, The L-arginine:nitric oxide pathway is the major source of plasma nitrite in fasted humans, Biochem. Biophys. Res. Commun. 209 (1995) 590 – 596. [65] A. Go¨decke, U.K.M. Decking, Z. Ding, J. Hirchenhain, H.-J. Bidmon, S. Go¨decke, J. Schrader, Coronary hemodynamics in endothelial NO synthase knockout mice, Circ. Res. 82 (1998) 186 – 194. [66] T. Ishibashi, J. Yoshida, M. Nishio, New methods to evaluate endothelial function: a search for a marker of nitric oxide (NO) in vivo: reevaluation of NOx in plasma and red blood cells and a trial to detect nitrosothiols, J. Pharmacol. Sci. 93 (2003) 409 – 416.
A. Dejam et al. / Blood Cells, Molecules, and Diseases 32 (2004) 423–429 [67] J.L. Zweier, P. Wang, A. Samouilov, P. Kuppusamy, Enzyme-independent formation of nitric oxide in biological tissues, Nat. Med. 1 (1995) 804 – 809. [68] D.A. Wink, M.B. Grisham, J.B. Mitchell, P.C. Ford, Direct and indirect effects of nitric oxide in chemical reactions relevant to biology, Methods Enzymol. 268 (1996) 12 – 31. [69] L.J. Ignarro, H. Lippton, J.C. Edwards, W.H. Baricos, A.L. Hyman, P.J. Kadowitz, C.A. Gruetter, Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: evidence for the involvement of S-nitrosothiols as active intermediates, J. Pharmacol. Exp. Ther. 218 (1981) 739 – 749. [70] K. Matsunaga, R.F. Furchgott, Interactions of light and sodium nitrite in producing relaxation of rabbit aorta, J. Pharmacol. Exp. Ther. 248 (1989) 687 – 695. [71] E.A.G. Demoncheaux, T.W. Higenbottam, P.J. Foster, C.D.R. Borland, A.P.L. Smith, H.M. Marriott, D. Bee, S. Akamine, M.B. Davies, Circulating nitrite anions are a directly acting vasodilator and are donors for nitric oxide, Clin. Sci. 102 (2002) 77 – 83. [72] T.M. Millar, C.R. Stevens, D.R. Blake, Xanthine oxidase can generate nitric oxide from nitrate in ischaemia, Biochem. Soc. Trans. 25 (1997) 528. [73] T.M. Millar, C.R. Stevens, N. Benjamin, R. Eisenthal, R. Harrison, D.R. Blake, Xanthine oxidoreductase catalyses the reduction of
[74]
[75]
[76]
[77]
[78]
[79]
429
nitrates and nitrite to nitric oxide under hypoxic conditions, FEBS Lett. 427 (1998) 225 – 228. H. Li, A. Samouilov, X. Liu, J.L. Zweier, Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrite reduction. Evaluation of its role in nitric oxide generation in anoxic tissues, J. Biol. Chem. 276 (2001) 24482 – 24489. R.F. Moulds, R.A. Jauernig, J. Shaw, A comparison of the effects of hydrallazine, diazoxide, sodium nitrite and sodium nitroprusside on human isolated arteries and veins, Br. J. Clin. Pharmacol. 11 (1981) 57 – 61. S. Beier, H.G. Classen, K. Loeffler, E. Schumacher, H. Thoni, Antihypertensive effect of oral nitrite uptake in the spontaneous hypertensive rat, Arzneim.-Forsch. 45 (1995) 258 – 261. R. Bhattacharya, S.C. Pant, D. Kumar, S.N. Dube, Toxicity evaluation of two treatment regimens for cyanide poisoning, J. Appl. Toxicol. 15 (1995) 439 – 441. R. Klimmek, C. Roddewig, H. Fladerer, C. Krettek, N. Weger, Effects of 4-dimethylaminophenol, Co2EDTA, or NaNO2 on cerebral blood flow and sinus blood homeostasis of dogs in connection with acute cyanide poisoning, Toxicology 52 (1983) 210 – 216. M.P. Doyle, R.A. Pickering, T.M. DeWeert, J.W. Hoekstra, D. Pater, Kinetics and mechanism of the oxidation of human deoxyhemoglobin by nitrites, J. Biol. Chem. 256 (1981) 12393 – 12398.
Emerging role of nitrite in human biology Andre´ Dejam, a Christian J. Hunter, a,b Alan N. Schechter, a and Mark T. Gladwin a,b,* a
Laboratory of Chemical Biology, National Institute of Diabetes, Digestive and Kidney Disease, National Institute of Health, Bethesda, MD 20892, USA b Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA Submitted 3 February 2004 (Communicated by J. Hoffman, Ph.D., 5 February 2004) Available online 14 March 2004
Abstract Nitric oxide (NO) plays a fundamental role in maintaining normal vascular function. NO is produced by endothelial cells and diffuses both into smooth muscle causing vasodilation and into the vessel lumen where the majority of this highly potent gas is rapidly inactivated by dioxygenation reaction with oxyhemoglobin to form nitrate. Diffusional barriers for NO around the erythrocyte and along the endothelium in laminar flowing blood reduce the inactivation reaction of NO by hemoglobin, allowing sufficient NO to escape for vasodilation and also to react in plasma and tissues to form nitrite anions (NO2 ) and NO-modified peptides and proteins (RX-NO). Several recent studies have highlighted the importance of the nitrite anion in human biology. These studies have shown that measurement of plasma nitrite is a sensitive index of constitutive NO synthesis, suggesting that it may be useful as a marker of endothelial function. Additionally, recent evidence suggests that nitrite represents a circulating storage pool of NO and may selectively donate NO to hypoxic vascular beds. The conversion of nitrite to NO requires a reaction with a deoxygenated heme protein, suggesting a novel function of hemoglobin as a deoxygenation-dependent nitrite reductase. This review focuses on the role of nitrite as a circulating NO donor, its potential as an index of NO synthase (NOS) activity and endothelial function, and discusses potential diagnostic and therapeutic applications. Published by Elsevier Inc. Keywords: Nitric oxide; Vasodilation; Endothelial function; Nitrite; Nitric oxide stores
Introduction Nitric oxide (NO) is a gas which is continuously synthesized in endothelial cells from the amino acid L-arginine by the constitutive calcium and calmodulin-dependent enzyme nitric oxide synthase [1]. This heme-containing enzyme catalyzes a five-electron oxidation of one of the basic guanidine nitrogen groups of L-arginine in the presence of multiple cofactors and molecular oxygen [2] (Fig. 1). In seminal experiments, Furchgott and Zawadzki [3] found that strips of aorta with intact endothelium relaxed in response to acetylcholine, but constricted in response to the same agonist when the endothelium had been rubbed off indicating an
essential role for endothelium in smooth muscle vascular reactivity. The substance responsible for this acetylcholinestimulated relaxation was initially called endothelium-derived relaxing factor, and subsequently identified by Ignarro et al. [4] and Palmer et al. [5] to be NO. Further studies showed that NO released from the endothelium diffuses into vascular smooth muscle where it activates soluble guanylyl cyclase by binding to its heme group, which results in increased cyclic guanosine monophosphate (cGMP) levels [6]. Cyclic GMP activates GMP-dependent kinases and downstream signaling that ultimately decreases intracellular calcium concentration leading to vasodilation [7].
Cardiovascular functions of NO * Corresponding author. Critical Care Medicine Department, Warren G. Magnuson Clinical Center, National Institutes of Health, Room 7D43, Building 10, 10 Center Drive, Bethesda, MD 20892. Fax: +1-301-4021213. E-mail address: [email protected] (M.T. Gladwin). 1079-9796/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.bcmd.2004.02.002
The importance of NO in the regulation of coronary and systemic vasodilator tone has been repeatedly demonstrated experimentally by inhibiting its synthesis [8 –10]. The drug
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NN-monomethyl-L-arginine (L-NMMA) inhibits endogenous NO production by competing with L-arginine as the substrate for NO synthase (NOS) [11]. Intra-coronary infusion of L-NMMA in normal human volunteers results in increased epicardial coronary vascular resistance and decreased blood flow as a result of arteriolar vasoconstriction [8]. These results indicate that NO contributes to basal coronary vasodilator tone and blood flow in humans [12,13]. In contrast, patients with endothelial dysfunction (due to coronary artery disease or risk factors for atherosclerosis) show minimal vasoconstriction responses to LNMMA, suggesting deficient NO release from the coronary endothelium in these patients [8]. NO also contributes to regulation of vascular tone in the systemic circulation. Recently, Lauer et al. used strain gauge venous plethysmography to measure forearm blood flow and resistance in human volunteers before and after inhibition or stimulation of NO synthesis (by infusion of L-NMMA or acetylcholine into the brachial artery). These workers showed that changes in regional vascular resistance correlated directly with concomitant changes in regional NO formation [9]. Akin to the coronary circulation, the vasoconstricting effects of L-NMMA are reduced in peripheral vascular beds of patients with hypertension, diabetes, hypercholesterolemia, obesity, and tobacco use, suggesting reduced endothelial-derived NO production in these conditions [10,14,15]. In addition to the regulation of basal vascular tone, NO is involved in systemic [16] and cerebral [17] hypoxic vasodilation. NO inhibits platelet activation,
leukocyte recruitment, smooth muscle cell proliferation, cell respiration, and has antioxidative and oxidative properties. (see Ref. [18] for review) Thus, NO is an endotheliumderived mediator that contributes substantially to homeostatic vascular function. Therapeutically, it seems highly desirable to selectively modulate NO synthesis and degradation in disease states with endothelial dysfunction and resulting reduction in NO bioavailability [19].
Fate of NO in human blood: the existence of circulating NO stores NO that diffuses into the vessel lumen and into the erythrocyte reacts at a nearly diffusion-limited rate with oxyhemoglobin yielding methemoglobin and nitrate [20] (Fig. 1). This reaction appears to limit the intravascular transport and half-life of NO considerably. The reactions of NO and hemoglobin are so fast and the intravascular hemoglobin concentration so high (10 mM heme) that all available NO would be consumed if hemoglobin was not compartmentalized within the erythrocyte. Several factors such as the erythrocyte membrane and submembrane [21,22], the unstirred layer surrounding the erythrocyte [20,23], and the presence of an erythrocyte-free zone of plasma along the surface of vascular endothelium [24 – 26] create diffusional barriers between NO and intra-erythrocytic hemoglobin. These barriers are estimated to decrease the rate of NO scavenging by intra-erythrocytic hemoglobin
Fig. 1. Potential role of nitric oxide and nitrite in human biology. The nitrite anion (NO2 ) unites two unique properties: It is a marker of constitutive nitric oxide (NO) synthase activity (NOS) and a circulating storage and delivery source of NO. Possible applications of this dual role of nitrite in human biology are depicted. NO2 entering the red blood cell can react either with oxyhemoglobin (oxy Hb) to form nitrate (NO3 ) and methemoglobin (met Hb) or with deoxyhemoglobin (deoxy Hb) to form NO, nitrosylhemoglobin (NO-Hb), and NO adducts (RX-NO). It is unknown whether NO formed from the reaction of NO2 with deoxyhemoglobin is directly exported from the red blood cell or may be transformed into an NO adduct (RX-NO) to cause NO2 induced vasodilation. Dotted lines represent degradation pathways.
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greater than 600-fold. This compartmentalization model of hemoglobin allows for the existence of a sufficient diffusional gradient for NO between endothelium and smooth muscle to allow local paracrine activity (endothelium to smooth muscle communication) but limit distant endocrine bioactivity (recently reviewed in Refs. [27,28]). Thus, in addition to the NO, which diffuses into vascular smooth muscle and activates soluble guanylyl cyclase, a portion of intravascular NO can escape hemoglobin scavenging and reacts with oxygen in plasma to form nitrite or nitrosate free thiols, amines, and unidentified constituents in blood to form mercury-labile and stable NO adducts [29]. The importance of these diffusional barriers is illustrated in patients with sickle cell disease. These patients have high levels of plasma cell-free hemoglobin that increases nitric oxide consumption and may be partially responsible for pulmonary hypertension and other pathophysiological manifestations [30]. In addition, infusion of cell-free hemoglobin in both animals and humans rapidly results in increased vascular resistance and blood pressure [31].
The nature of circulating NO stores Oxidative and nitrosative products of NO could represent a circulating NO storage pool, which may exert ‘‘endocrine’’ effects distal from its site of production. In support of this concept, the infusion of authentic NO solution into the human circulation produces systemic vasodilation of the micro- and macrovasculature, which is accompanied by an increase in plasma NO-protein adducts and nitrite [32,33]. Similar distal vasodilating effects of NO were observed in the human forearm vasculature during inhalation of NO gas in normal volunteers [34]. This vasodilating effect was associated with the formation of the nitrite anion and ironnitrosylated hemoglobin. Nitrite, the primary oxidative NO metabolite, has long been considered inert under physiologic conditions [9,35]. However, several factors would position nitrite as a major intravascular storage pool for NO and suggest that it may be bioactive. First, plasma nitrite is present in substantial concentrations in both plasma [36] and tissue [37]. Second, nitrite is relatively stable because it is not readily reduced by cellular reductants as are the S-nitrosothiol NO adducts [38]. Third, nitrite’s reaction rate with heme proteins is 10,000 times less than that of authentic NO which allows for action beyond its site of formation. Finally, recent data from our group, summarized at the end of this review, suggest that nitrite is vasoactive both in vitro and in vivo [39]. Therefore, we hypothesize that nitrite might serve as the fundamental intravascular storage pool of NO that can react with deoxyhemoglobin and the deoxy form of other heme proteins such as myoglobin, neuroglobin, and cytoglobin to form NO, nitrosylhemoglobin (NO-Hb), and to a lesser extent N-nitrosamines (RX-NO), S-nitrosothiols, and nitrated lipids (L-NO2) (Fig. 1).
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Nitrosative metabolites likewise have been implicated to serve as NO stores in vivo [32]. Putative candidates are Nnitrosamines [56]. Recently, it was shown that ingestion of a potent plant antioxidant alleviated endothelial dysfunction in patients at risk for atherosclerosis and was associated with an increase in RX-NO, likely representing an N-nirosamine [40]. Other NO-derived species such as S-nitrosothiols have been extensively studied. During the last decade, S-nitrosothiol albumin [41] and S-nitrosothiol hemoglobin [42] have been proposed as major circulating NO storage pools. Pharmacologic modulation of these species inhibits platelet function reducing asymptomatic embolization after carotid endaterectomy [43], diminish ischemia – reperfusion injury [44], and normalize pharmacologically induced endothelial dysfunction [34]. However, the role of endogenous S-nitrosothiols in the regulation of blood flow and as a circulating stable NO storage pool is intensely debated (as has been reviewed in Ref. [28]). Central to this controversy are methodological problems inherent in the detection of SNO species in blood [45 – 48]. We recently found that techniques use to identify high levels of S-nitrosothiol in plasma invariably use UV photolysis, which can artefactually generate NO signal from nitrate in the presence of reduced thiols [49]. Future work will be required to establish the relative contribution and importance of nitrite, RX-NO, or S-nitrosothiols of albumin or hemoglobin in the regulation of blood flow and storage of NO bioactivity in blood.
Measurement of plasma nitrite as a marker of constitutive NO synthase activity There is considerable interest in indirectly determining NO synthase activity by assaying the metabolites of NO in blood [29]. Due to its ultra-short half-life and its radical character, direct measurement of nitric oxide poses considerable difficulties under basal conditions [50]. In vitro studies reveal that in the presence of oxygen, NO is rapidly oxidized to nitrite, following pseudo-first-order kinetics with a strict 1:1 stoichiometry [51]. In blood, as discussed earlier, NO also reacts with oxyhemoglobin to form nitrate. Thus, endogenous NO formation may be measured via the determination of its oxidative products, nitrite, and nitrate. However, the reported basal nitrite concentrations in plasma of humans in the literature range from ‘‘nondetectable’’ [52], over nanomolar (450 nmol/l) [53], to micromolar (26 Amol/l) [54] levels. These enormous differences in nitrite levels can be rationalized considering the confounding factors and variations in blood sampling and sample processing as well as the methodological problems inherent to the analytical procedures used [36]. Some methods (such as the Griess reagent combined with spectrophotometric detection) simply do not possess the sensitivity to allow a precise measurement of nitrite in the proposed physiological concentration range. In addition, the analysis might be affected by many factors such as proteins, varying redox
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conditions, and trace contamination with ubiquitously present nitrite and nitrate during sample processing [55]. To address these methodological problems, Kelm et al. have measured plasma nitrite concentration of nine different mammal species including humans using three independent methodologies. Results of these study demonstrate the uniform occurrence of nanomolar levels of plasma nitrite (100 –600 nmol/l) [36,45,56,57]. Validation of methodologies to measure plasma nitrite concentration [36,45,56– 60] have led to studies testing the hypothesis that plasma nitrite is an index of constitutive NOS activity [9,61 – 63]. This hypothesis is principally supported by the observation that approximately 70– 90% of circulating plasma nitrite is derived from eNOS activity in humans and other mammals [36,64]. In addition, nitrite and nitrate levels are reduced up to 70% in eNOS knockout mice as compared to control mice [36,65] and both stimulation and inhibition of eNOS have been shown to produce acute changes in plasma nitrite concentrations in the human forearm vasculature [9]. Thus, basal plasma nitrite concentrations reflect constitutive NOS activity and NO availability uniformly in several mammalian species [36]. In aggregate, these data suggest that the measurement of plasma nitrite, using carefully validated methodologies that avoid ex vivo decay or nitrite contamination, can serve as a biomarker of NO synthase activity and thus endothelial function. Caution should be exercised when changes in plasma nitrate (NO3 ) are evaluated as an index of NO synthesis. Unlike plasma nitrite, plasma nitrate concentration does not change significantly during acute NOS inhibition and in addition are significantly affected by both diet and renal function [9,36,66].
The nitrite anion as a potential physiologic source of NO Nitrite has long been believed to form NO non-enzymatically only under certain conditions such as severe ischemia or acidosis [67]. Under physiologic levels of acidity, as occurs during cardiac ischemia and infarction, nitrite forms nitrous acid, which can react with nitrite again or an electron donor (such as ascorbate) to form dinitrogentrioxide (N2O3). This reactive nitrogen species can then nitrosate thiols (which are vasoactive) or, in the presence of an electron donor, produce NO gas [68]. This non-enzymatic NO generation has been documented in certain vascular tissue preparations [69,70] and in ischemic myocardium ex vivo; it requires low tissue pH (approximately 6.5) [71] and tissue reductants [67,71]. Alternatively, the conversion of nitrite to NO gas could be catalyzed by a metal or enzyme. For example, recent studies suggest that xanthine oxidoreductase, which is present in abundance in vascular endothelium, may reduce nitrite to NO [72,73], an effect that increases with decreasing pH [71], increasing NADH concentration, and hypoxia [74]. However, these hypothesized mechanisms
for nitrite reduction—nitrite disproportionation and xanthine oxidoreductase activity—both require extremely low pO2 and pH not readily found under physiologic conditions [35] casting doubt on these mechanisms contributing to the role of this anion as a physiologic vasodilator [35,75]. However, oral nitrite administration had been shown to have a sustained antihypertensive effect in spontaneous hypertensive rats [76] and several case series have shown that infusion of a high dose of nitrite (300 mg), as a methemoglobin-forming agent to treat cyanide poisoning, caused hypotension and increased cerebral blood flow [77,78]. In addition, the demonstration of an arterial-venous difference of plasma nitrite concentration [45] and the observation that inhalation of NO induced peripheral vascular effects associated with a selective increase in nitrite concentration [34] led us to the hypothesis that nitrite may be reduced to NO under physiologic conditions. Based on these studies, we evaluated the vasodilator properties and mechanisms for bioactivation of nitrite in the human forearm. Nitrite infusions of both 36 and 0.36 Amol/ min into the forearm brachial artery resulted in supra- and near-physiologic intravascular nitrite concentrations, respectively, and increased forearm blood flow before and during exercise, with or without NO synthase inhibition. Nitrite infusions were associated with rapid formation of erythrocyte iron-nitrosylated hemoglobin and, to a lesser extent, Snitrosohemoglobin. NO-modified hemoglobin formation was inversely proportional to oxyhemoglobin saturation, suggesting that a reaction with deoxygenated hemoglobin or tissues accounted for the mechanism of vasodilation. We subsequently found that vasodilation of rat aortic rings and formation of both NO gas and NO-modified hemoglobin resulted from the nitrite reductase activity of deoxyhemoglobin and deoxygenated erythrocytes (Fig. 1). Doyle et al. [79] first reported in 1981 that nitrite reacts with deoxyhemoglobin (and H+) to form methemoglobin and NO (and OH ), a chemistry consistent with our in vivo and in vitro observations. Such chemistry is ideally suited for hypoxic generation of NO from nitrite, as it requires hemoglobin deoxygenation, thus is linked to the physiological oxygen gradient, and protons connecting it to the physiological pH/CO2 gradient. We hypothesize that this nitrite chemistry takes advantage of the Bohr and Haldane effects to link local tissue hypoxia and acidosis and hemoglobin deoxygenation to nitrite bioactivation leading to NO formation. These results suggest that nitrite represents a major bioavailable pool of NO and describe a new physiological function for hemoglobin as a nitrite reductase, potentially contributing to systemic hypoxic vasodilation [39].
Summary Nitrite is a marker of NO synthase activity and endothelial function in humans and may constitute a novel storage and delivery source of NO. The observation that deoxygenated
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erythrocytes reduce nitrite to form NO sheds new light on the role of red blood cells and hemoglobin in NO physiology: Hemoglobin is not merely a sink of NO but rather a modulator of NO availability adapting NO levels in the circulation to the oxygen needs of the surrounding tissue [39]. This principle was first ascribed to S-nitrosohemoglobin, but our data suggest this process is primarily served by nitrite reduction to NO by deoxyheme proteins. This dual role of nitrite has potential diagnostic and therapeutic implications. Nitrite as a marker of constitutive NOS activity may index NO availability in atherosclerosis and associated risk factors. This may be of diagnostic and prognostic value and provide guidance towards more individualized cardiovascular therapy. Nitrite delivery to humans via infusion or inhalation may counterbalance pathophysiologic processes occurring in disease states with a relative or absolute lack of NO, such as atherosclerosis, hypertension, diabetes, hypercoaguable states caused by endothelial dysfunction, acute respiratory distress syndrome of the newborn, delayed-onset vasospasm due to subarachnoidal hemorrhage, and sickle cell disease. Further study will be required to establish the role of nitrite in physiology and its application for diagnostics and therapeutics.
Acknowledgments This work was supported by the Clinical Center and the Medical Scientist Training Program T32GM08361. Christian J. Hunter is supported by the Clinical Research Training Program of the National Institutes of Health. This paper is based on a presentation at the Red Cell Club Meeting held at Yale University School of Medicine on October 24, 2003.
References [1] J.S. Beckman, W.H. Koppenol, Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly, Am. J. Physiol.: Cell Physiol. 271 (1996) C1424 – C1437. [2] J.L. Boucher, C. Moali, J.P. Tenu, Nitric oxide biosynthesis, nitric oxide synthase inhibitors and arginase competition for L-arginine utilization, Cell. Mol. Life Sci. 55 (1999) 1015 – 1028. [3] R.F. Furchgott, J.V. Zawadzki, The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 288 (1980) 373 – 376. [4] L.J. Ignarro, R.E. Byrns, G.M. Buga, K.S. Wood, Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical, Circ. Res. 61 (1987) 866 – 879. [5] R.M.J. Palmer, A.G. Ferrige, S. Moncada, Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor, Nature 327 (1987) 524 – 526. [6] L.J. Ignarro, J.B. Adams, P.M. Horwitz, K.S. Wood, Activation of soluble guanylate cyclase by NO-hemoproteins involves NO – heme exchange. Comparison of heme-containing and heme-deficient enzyme forms, J. Biol. Chem. 261 (1986) 4997 – 5002. [7] L.J. Ignarro, Biosynthesis and metabolism of endothelium-derived nitric oxide, Annu. Rev. Pharmacol. Toxicol. 30 (1990) 535 – 560.
427
[8] A.A. Quyyumi, N. Dakak, N.P. Andrews, S. Husain, S. Arora, D.M. Gilligan, J.A. Panza, R.O. Cannon III, Nitric oxide activity in the human coronary circulation. Impact of risk factors for coronary atherosclerosis, J. Clin. Invest. 95 (1995) 1747 – 1755. [9] T. Lauer, M. Preik, T. Rassaf, B.E. Strauer, A. Deussen, M. Feelisch, M. Kelm, Plasma nitrite rather than nitrate reflects regional endothelial nitric oxide synthase activity but lacks intrinsic vasodilator action, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 12814 – 12819. [10] J.A. Panza, P.R. Casino, C.M. Kilcoyne, A.A. Quyyumi, Role of endothelium-derived nitric oxide in the abnormal endothelium-dependent vascular relaxation of patient with essential hypertension, Circulation 87 (1993) 1468 – 1474. [11] B.X. Mayer, C. Mensik, S. Krishnaswami, H. Derendorf, H.-G. Eichler, L. Schmetterer, M. Wolzt, Pharmacokinetic – pharmacodynamic profile of systemic nitric oxide-synthase inhibition with L-NMMA in humans, Br. J. Clin. Pharmacol. 47 (1999) 539 – 544. [12] M. Kelm, J. Schrader, Control of coronary vascular tone by nitric oxide, Circ. Res. 66 (1990) 1561 – 1575. [13] M. Kelm, J. Schrader, Nitric oxide release from the isolated guinea pig heart, Eur. J. Pharmacol. 155 (1988) 317 – 321. [14] K. Hishikawa, T. Nakaki, H. Suzuki, R. Kato, T. Saruta, Role of L-arginine-nitric oxide pathway in hypertension, J. Hypertens. 11 (1993) 639 – 645. [15] J. Loscalzo, J. Freedman, A. Inbal , J.F. Keaney Jr., A.D. Michelson, J.A. Vita, Nitric oxide insufficiency and arterial thrombosis, Trans. Am. Clin. Climatol. Assoc. 111 (2000) 158 – 163. [16] M.L. Blitzer, E. Loh, M.-A. Roddy, J. Stamler, M. Creager, Endothelium-derived nitric oxide regulates systemic and pulmonary vascular resistance during acute hypoxia in humans, J. Am. Coll. Cardiol. 28 (2003) 591 – 596. [17] C.J. Hunter, A.B. Blood, C.R. White, W.J. Pearce, G.G. Power, Role of nitric oxide in hypoxic cerebral vasodilation in the ovine fetus, J. Physiol. (London) 549 (2003) 625 – 633. [18] P. Vallance, N. Chan, Endothelial function and nitric oxide: clinical relevance, Heart 85 (2001) 342 – 350. [19] A.N. Schechter, M.T. Gladwin , R.O. Cannon III, NO solutions? J. Clin. Invest. 109 (2002) 1149 – 1151. [20] X. Liu, M.J.S. Miller, M.S. Joshi, H. Sadowska-Krowicka, D.A. Clark, J.R. Lancaster Jr., Diffusion-limited reaction of free nitric oxide with erythrocytes, J. Biol. Chem. 273 (1998) 18709 – 18713. [21] M.W. Vaughn, K.-T. Huang, L. Kuo, J.C. Liao, Erythrocytes possess an intrinsic barrier to nitric oxide consumption, J. Biol. Chem. 275 (2000) 2342 – 2348. [22] Z. Huang, J.G. Louderback, M. Goyal, F. Azizi, S.B. King, D.B. Kim-Shapiro, Nitric oxide binding to oxygenated hemoglobin under physiologic conditions, Biochim. Biophys. Acta 1568 (2001) 252 – 260. [23] J.T. Coin, J.S. Olsen, The rate of oxygen uptake by human red blood cells, J. Biol. Chem. 254 (1979) 1178 – 1190. [24] M.W. Vaughn, L. Kuo, J.C. Liao, Effective diffusion distance of nitric oxide in the microcirculation, Am. J. Physiol.: Heart Circ. Physiol. 274 (1998) H1705 – H1714. [25] A.R. Butler, I.L. Megson, P.G. Wright, Diffusion of nitric oxide and scavenging by blood in the vasculature, Biochim. Biophys. Acta 1425 (1998) 168 – 176. [26] J.C. Liao, T.W. Hein, M.W. Vaughn, K.-T. Huang, L. Kuo, Intravascular flow decreases erythrocyte consumption of nitric oxide, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 8757 – 8761. [27] A.N. Schechter, M.T. Gladwin, Hemoglobin and the paracrine and endocrine functions of nitric oxide, N. Engl. J. Med. 348 (2003) 1483 – 1485. [28] M.T. Gladwin, J.R. Lancaster, B.A. Freeman, A.N. Schechter, Nitric oxide’s reactions with hemoglobin: a view through the SNO-storm, Nat. Med. 9 (2003) 496 – 500. [29] M. Kelm, Nitric oxide metabolism and breakdown, Biochim. Biophys. Acta 1411 (1999) 273 – 289.
428
A. Dejam et al. / Blood Cells, Molecules, and Diseases 32 (2004) 423–429
[30] C.D. Reiter, X. Wang, J.E. Tanus-Santos, N. Hogg , R.O. Cannon III, A.N. Schechter, M.T. Gladwin, Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease, Nat. Med. 8 (2002) 1383 – 1389. [31] R.J. Rohlfs, E. Bruner, A. Chiu, A. Gonzales, M.L. Gonzales, D. Magde, M.D. Magde, K.D. Vandegriff, R.M. Winslow, Arterial blood pressure responses to cell-free hemoglobin solutions and the reaction with nitric oxide, J. Biol. Chem. 273 (1998) 12128 – 12134. [32] T. Rassaf, P. Kleinbongard, M. Preik, A. Dejam, P. Gharini, T. Lauer, J. Erckenbrecht, A. Duschin, R. Schulz, G. Heusch, M. Feelisch, M. Kelm, Plasma nitrosothiols contribute to the systemic vasodilator effects of intravenously applied NO; experimental and clinical study on the fate of NO in human blood, Circ. Res. 91 (2002) 470 – 477. [33] T. Rassaf, M. Preik, P. Kleinbongard, T. Lauer, C. Heiß, B.E. Strauer, M. Feelisch, M. Kelm, Evidence for in vivo transport of bioactive nitric oxide in human plasma, J. Clin. Invest. 109 (2002) 1241 – 1248. [34] R.O. Cannon III, A.N. Schechter, J.A. Panza, F.P. Ognibene, M.E. Pease-Fye, M.A. Waclawiw, J.H. Shelhamer, M.T. Gladwin, Effects of inhaled nitric oxide on regional blood flow are consistent with intravascular nitric oxide delivery, J. Clin. Invest. 108 (2001) 279 – 287. [35] D. Tsikas, J.C. Fro¨lich, Is circulating nitrite a directly acting vasodilator? Clin. Sci. 103 (2002) 107 – 110. [36] P. Kleinbongard, A. Dejam, T. Lauer, T. Rassaf, A. Schindler, O. Picker, T. Scheeren, A. Go¨decke, J. Schrader, R. Schulz, G. Heusch, G.A. Schaub, N. Bryan, M. Feelisch, M. Kelm, Plasma nitrite reflecting constitutive activity of nitric oxide synthase in mammals, Free Radical Biol. Med. 35 (2003) 790 – 796. [37] J. Rodriguez, R.E. Maloney, T. Rassaf, N.S. Bryan, M. Feelisch, Chemical nature of nitric oxide storage forms in rat vascular tissue, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 336 – 341. [38] M.T. Gladwin, X. Wang, C.D. Reiter, B.K. Yang, E.X. Vivas, C. Bonaventura, A.N. Schechter, S-nitrosohemoglobin is unstable in the reductive red cell environment and lacks O2/NO-linked allosteric function, J. Biol. Chem. 277 (2002) 27818 – 27828. [39] K. Cosby, K.S. Partovi, J.H. Crawford, R.P. Patel, C.D. Reiter, S. Martyr, B.K. Yang, M.A. Waclawiw, G. Zalos, X. Xu, K.T. Huang, H. Shields, D.B. Kim-Shapiro, A.N. Schechter, R.O. Cannon III, M.T. Gladwin, Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation, Nat. Med. 9 (2003) 1498 – 1505. [40] C. Heiß, A. Dejam, P. Kleinbongard, T. Schewe, H. Sies, M. Kelm, Vascular effects of cocoa rich in flavan-3-ols, JAMA 290 (2003) 1030 – 1031. [41] J.S. Stamler, O. Jaraki, J. Osborne, D.I. Simon, J. Keaney, J. Vita, D. Singel, C.R. Valeri, J. Loscalzo, Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 7674 – 7677. [42] L. Jia, C. Bonaventura, J. Bonaventura, J.S. Stamler, S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control, Nature 380 (1996) 221 – 226. [43] Z. Kaposzta, J.F. Martin, H.S. Markus, Switching off embolization from symptomatic carotid plaque using S-nitrosoglutathione, Circulation 105 (2002) 1480 – 1484. [44] S. Hallstro¨m, H. Gasser, C. Neumayer, A. Flu¨gl, J. Nanobashvili, A. Jakubowski, I. Huk, G. Schlag, T. Malinski, S-nitroso human serum albumin treatment reduces ischemia/reperfusion injury in skeletal muscle via nitric oxide release, Circulation 105 (2002) 3032 – 3038. [45] M.T. Gladwin, J.H. Shelhamer, A.N. Schechter, M.E. Pease-Fye, M.A. Waclawiw, J.A. Panza, F.P. Ognibene , R.O. Cannon III, Role of circulation nitrite and S-nitrosohemoglobin in the regulation of regional blood flow in humans, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 11482 – 11487. [46] T. Rassaf, N.S. Bryan, R.E. Maloney, V. Specian, M. Kelm, B. Kalyanaraman, J. Rodriguez, M. Feelisch, NO adducts in mammalian red blood cells: too much or too little? Nat. Med. 9 (2003) 481 – 483.
[47] D. Tsikas, Measurement of physiological S-nitrosothiols: a problem child and a challenge, Nitric Oxide 9 (2003) 53 – 55. [48] R. Rossi, D. Giustarini, A. Milzani, R. Colombo, I. Dalle-Donne, P. di Simplicio, Physiological levels of S-nitrosothiols in human plasma, Circ. Res. 89 (2001) E47. [49] A. Dejam, P. Kleinbongard, T. Rassaf, P. Gharini, S. Hamada, J. Rodriguez, M. Feelisch, M. Kelm, Thiols enhance NO formation from nitrate photolysis, Free Radical Biol. Med. 35 (2003) 1551 – 1559. [50] P. Vallance, S. Patton, K. Bhagat, R. MacAllister, M. Radomski, S. Moncada, T. Malinski, Direct measurement of nitric oxide in human beings, Lancet 345 (1995) 153 – 154. [51] P.C. Ford, D.A. Wink, D.M. Stanbury, Autoxidation kinetics of aqueous nitric oxide, FEBS Lett. 326 (1993) 1 – 3. [52] A. Meulemans, F. Delsenne, Measurement of nitrite and nitrate levels in biological samples by capillary electrophoresis, J. Chromatogr., B: Biomed. Appl. 660 (1994) 401 – 404. [53] A.M. Leone, P.L. Francis, P. Rhodes, S. Moncada, A rapid and simple method for the measurement of nitrite and nitrate in plasma by high performance capillary electrophoresis, Biochem. Biophys. Res. Commun. 200 (1994) 951 – 957. [54] M. Gorenflo, C. Zheng, A. Poge, M. Bettendorf, E. Werle, W. Fiehn, H.E. Ulmer, Metabolites of the L-arginine-NO pathway in patients with left-to-right shunt, Clin. Lab. 47 (2001) 441 – 447. [55] P. Kleinbongard, T. Rassaf, A. Dejam, S. Kerber, M. Kelm, Griess method for nitrite measurement of aqueous and protein containing sample, Methods Enzymol. 359 (2002) 158 – 168. [56] T. Rassaf, N.S. Bryan, M. Kelm, M. Feelisch, Concomitant presence of N-Nitroso and S-Nitroso Proteins in human plasma, Free Radical Biol. Med. 33 (2002) 1590 – 1596. [57] D. Tsikas, I. Fuchs, F.-M. Gutzki, J.C. Fro¨lich, Measurement of nitrite and nitrate in plasma, serum and urine of humans by highperformance liquid chromatography, the Griess assay, chemiluminescence and gas chromatography-mass spectrometry: interferences by biogenic amines and NG-nitro-L-arginine analogs, J. Chromatogr., B: Biomed. Appl. 715 (1998) 441 – 444. [58] M. Feelisch, T. Rassaf, S. Mnaimneh, N. Singh, N.S. Bryan, D. Jourd’heuil, M. Kelm, Concomitant S-, N-, and heme-nitros(yl)ation in biological tissues and fluids: implications for the fate of NO in vivo, FASEB J. 16 (2002) 1775 – 1785. [59] K. Schulz, S. Kerber, M. Kelm, A new technique for measurement of nitrite in human blood, Nitric Oxide: Biol. Chem. 2 (1998) 101. [60] H. Preik-Steinhoff, M. Kelm, Determination of nitrite in human blood by combination of a specific sample preparation with high performance anion-exchange chromatography and electrochemical determination, J. Chromatogr., B: Biomed. Appl. 685 (1996) 348 – 352. [61] M. Kelm, H. Preik-Steinhoff, M. Preik, B.E. Strauer, Serum nitrite sensitively reflects endothelial NO formation in human forearm vasculature: evidence for biochemical assessment of the endothelial L-arginine-NO pathway, Cardiovasc. Res. 41 (1999) 765 – 772. [62] T. Lauer, P. Kleinbongard, M. Preik, B.H. Rauch, A. Deussen, M. Feelisch, B.E. Strauer, M. Kelm, Direct biochemical evidence for eNOS stimulation by bradykinin in the human forearm vasculature, Basic Res. Cardiol. 98 (2003) 84 – 89. [63] T. Lauer, P. Kleinbongard, M. Kelm, Indexes of NO-bioavailibility in human blood, News Physiol. Sci. 17 (2002) 251 – 255. [64] P.M. Rhodes, A.M. Leone, P.L. Francis, A.D. Struthers, S. Moncada, The L-arginine:nitric oxide pathway is the major source of plasma nitrite in fasted humans, Biochem. Biophys. Res. Commun. 209 (1995) 590 – 596. [65] A. Go¨decke, U.K.M. Decking, Z. Ding, J. Hirchenhain, H.-J. Bidmon, S. Go¨decke, J. Schrader, Coronary hemodynamics in endothelial NO synthase knockout mice, Circ. Res. 82 (1998) 186 – 194. [66] T. Ishibashi, J. Yoshida, M. Nishio, New methods to evaluate endothelial function: a search for a marker of nitric oxide (NO) in vivo: reevaluation of NOx in plasma and red blood cells and a trial to detect nitrosothiols, J. Pharmacol. Sci. 93 (2003) 409 – 416.
A. Dejam et al. / Blood Cells, Molecules, and Diseases 32 (2004) 423–429 [67] J.L. Zweier, P. Wang, A. Samouilov, P. Kuppusamy, Enzyme-independent formation of nitric oxide in biological tissues, Nat. Med. 1 (1995) 804 – 809. [68] D.A. Wink, M.B. Grisham, J.B. Mitchell, P.C. Ford, Direct and indirect effects of nitric oxide in chemical reactions relevant to biology, Methods Enzymol. 268 (1996) 12 – 31. [69] L.J. Ignarro, H. Lippton, J.C. Edwards, W.H. Baricos, A.L. Hyman, P.J. Kadowitz, C.A. Gruetter, Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: evidence for the involvement of S-nitrosothiols as active intermediates, J. Pharmacol. Exp. Ther. 218 (1981) 739 – 749. [70] K. Matsunaga, R.F. Furchgott, Interactions of light and sodium nitrite in producing relaxation of rabbit aorta, J. Pharmacol. Exp. Ther. 248 (1989) 687 – 695. [71] E.A.G. Demoncheaux, T.W. Higenbottam, P.J. Foster, C.D.R. Borland, A.P.L. Smith, H.M. Marriott, D. Bee, S. Akamine, M.B. Davies, Circulating nitrite anions are a directly acting vasodilator and are donors for nitric oxide, Clin. Sci. 102 (2002) 77 – 83. [72] T.M. Millar, C.R. Stevens, D.R. Blake, Xanthine oxidase can generate nitric oxide from nitrate in ischaemia, Biochem. Soc. Trans. 25 (1997) 528. [73] T.M. Millar, C.R. Stevens, N. Benjamin, R. Eisenthal, R. Harrison, D.R. Blake, Xanthine oxidoreductase catalyses the reduction of
[74]
[75]
[76]
[77]
[78]
[79]
429
nitrates and nitrite to nitric oxide under hypoxic conditions, FEBS Lett. 427 (1998) 225 – 228. H. Li, A. Samouilov, X. Liu, J.L. Zweier, Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrite reduction. Evaluation of its role in nitric oxide generation in anoxic tissues, J. Biol. Chem. 276 (2001) 24482 – 24489. R.F. Moulds, R.A. Jauernig, J. Shaw, A comparison of the effects of hydrallazine, diazoxide, sodium nitrite and sodium nitroprusside on human isolated arteries and veins, Br. J. Clin. Pharmacol. 11 (1981) 57 – 61. S. Beier, H.G. Classen, K. Loeffler, E. Schumacher, H. Thoni, Antihypertensive effect of oral nitrite uptake in the spontaneous hypertensive rat, Arzneim.-Forsch. 45 (1995) 258 – 261. R. Bhattacharya, S.C. Pant, D. Kumar, S.N. Dube, Toxicity evaluation of two treatment regimens for cyanide poisoning, J. Appl. Toxicol. 15 (1995) 439 – 441. R. Klimmek, C. Roddewig, H. Fladerer, C. Krettek, N. Weger, Effects of 4-dimethylaminophenol, Co2EDTA, or NaNO2 on cerebral blood flow and sinus blood homeostasis of dogs in connection with acute cyanide poisoning, Toxicology 52 (1983) 210 – 216. M.P. Doyle, R.A. Pickering, T.M. DeWeert, J.W. Hoekstra, D. Pater, Kinetics and mechanism of the oxidation of human deoxyhemoglobin by nitrites, J. Biol. Chem. 256 (1981) 12393 – 12398.