Howard Shields

Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation.

Nitrite anions comprise the largest vascular storage pool of nitric oxide (NO), provided that physiological mechanisms exist to reduce nitrite to NO. We evaluated the vasodilator properties and mechanisms for bioactivation of nitrite in the human forearm. Nitrite infusions of 36 and 0.36 μmol/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, S-nitroso-hemoglobin. NO-modified hemoglobin formation was inversely proportional to oxyhemoglobin saturation. 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. This finding links tissue hypoxia, hemoglobin allostery and nitrite bioactivation. 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 hypoxic vasodilation.

Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator

The blood anion nitrite contributes to hypoxic vasodilation through a heme-based, nitric oxide (NO)–generating reaction with deoxyhemoglobin and potentially other heme proteins. We hypothesized that this biochemical reaction could be harnessed for the treatment of neonatal pulmonary hypertension, an NO-deficient state characterized by pulmonary vasoconstriction, right-to-left shunt pathophysiology and systemic hypoxemia. To test this, we delivered inhaled sodium nitrite by aerosol to newborn lambs with hypoxic and normoxic pulmonary hypertension. Inhaled nitrite elicited a rapid and sustained reduction (∼65%) in hypoxia-induced pulmonary hypertension, with a magnitude approaching that of the effects of 20 p.p.m. NO gas inhalation. This reduction was associated with the immediate appearance of NO in expiratory gas. Pulmonary vasodilation elicited by aerosolized nitrite was deoxyhemoglobin- and pH-dependent and was associated with increased blood levels of iron-nitrosyl-hemoglobin. Notably, from a therapeutic standpoint, short-term delivery of nitrite dissolved in saline through nebulization produced selective, sustained pulmonary vasodilation with no clinically significant increase in blood methemoglobin levels. These data support the concept that nitrite is a vasodilator acting through conversion to NO, a process coupled to hemoglobin deoxygenation and protonation, and evince a new, simple and inexpensive potential therapy for neonatal pulmonary hypertension.

Electron Spin Resonance of X‐Irradiated Single Crystals of Hydroxyurea

The electron spin resonance of x‐irradiated single crystals of hydroxyurea have been observed and analyzed for different orientations of the crystal in the magnetic field. The free radical responsible for the resonance has been identified as NH2CONH, and the measurements indicate that 41% of the unpaired electron is localized in the 2p orbital of the nitrogen atom. Principal values for the hyperfine coupling constants in gauss are −21.2, −13.5, and −1.5 for H, and 22.5, 1.5, and 1.2 for N. On the basis of these measurements the induced isotropic hyperfine constants for an electron entirely localized in a 2p orbital of the nitrogen atom in the R–N–H radical would be 20.2 G for nitrogen and −31 G for hydrogen. The principal g values are 2.0027, 2.0062, and 2.0108. The results indicate that the trapped radical retains the approximate orientation of the parent molecule in the lattice and that the radical is planar.

Nitrosylation of Sickle Cell Hemoglobin by Hydroxyurea

Hydroxyurea (1) represents a new treatment for sickle cell anemia,2 a disease that affects about one out of 600 people of African descent born in the United States.3 While some of the beneficial effects of hydroxyurea treatment appear to result from an increase in the production of fetal hemoglobin, some patients benefit from hydroxyurea treatment before their levels of fetal hemoglobin increase, indicating that the positive effects of this drug cannot be completely explained by the increase of this protein and suggesting other mechanisms of action.4 The detailed molecular mechanism of hydroxyurea’s action remains poorly described, and speculation regarding the possible involvement of nitric oxide (NO) in the biological actions of hydroxyurea exists.5 Identification of the characteristic decomposition products of NO during the oxidation of hydroxyurea with various combinations of oxidants supports this suggestion.5 Both NO and nitrosyl hemoglobin (HbNO, Fe2+-NO), which forms as a minor product upon reaction of hydroxyurea with oxyhemoglobin (HbO2, Fe2+, Scheme 1),6,7 have recently been proposed as potential therapies for sickle cell disease.8 The identification of HbNO from hydroxyureatreated rats also provides evidence for the in vivo metabolic production of NO from hydroxyurea.9 Under physiological conditions, HbNO exists in an oxygen-dependent equilibrium with S-nitrosohemoglobin (S-nitrosylated on the â-93 cysteine residues), a vasorelaxant protein whose formation could benefit sickle cell patients.10 Here, we report for the first time that the in vitro reaction of sickle cell oxyhemoglobin with hydroxyurea to form sickle cell nitrosylhemoglobin involves the specific transfer of NO from the NHOH group of the drug (Scheme 1). Evidence that the NO group from HbNO derives from the NH-OH group of hydroxyurea was obtained by the use of 15N-labeled hydroxyurea and observance of the isotope effect on the hyperfine splitting pattern of the EPR spectrum of HbNO. 15N-Hydroxyurea (0.76 M), prepared by the condensation of commercially available 15N-hydroxylamine and trimethylsilyl (TMS) isocyanate, was incubated at room temperature with sickle cell oxyhemoglobin (0.42 mM) in 0.1 M sodium phosphate buffer (pH 7.3). After 20 h, a distinct visible absorbance change indicated the primary conversion of HbO2 to methemoglobin (metHb, Fe3+, Scheme 1), and the presence of HbNO was confirmed by EPR spectroscopy (77 K).6,7 Addition of sodium dodecyl sulfate (40 mM final concentration) prior to sample freezing cleanly resolved the hyperfine splitting patterns, allowing for analysis of the nitrogen coupling. The EPR spectrum of the reaction of sickle cell HbO2 and 15N-hydroxyurea after denaturation displayed a two peak pattern (Figure 1, trace A) consistent with electronic coupling to 15N (I ) 1/2) and identical to a reported Hb15NO EPR spectrum.11 In contrast, the EPR spectrum of the HbNO formed by the reaction of sickle cell HbO2 and 14N-hydroxyurea demonstrated a threepeak pattern (Figure 1, trace B) consistent with electronic coupling to 14N (I ) 1). These results indicate that the NO group of the HbNO formed in the reaction of HbO2 and hydroxyurea specifically derives from the NHOH portion of hydroxyurea. Model oxidation of hydroxyurea with hydrogen peroxide followed byproduct analysis indicates that hydroxyurea decomposes with the formation of nitroxyl (HNO) and nitric oxide (NO). As indicated by EPR spectroscopy,6,12 these conditions produce the same nitroxide radical (2, Scheme 1) as the HbO2 oxidation of hydroxyurea in the absence of protein, which could complicate the detection of any reactive species formed. The hydrogen peroxide oxidation of hydroxyurea (1.0 equivalent) produced nitrous oxide (N2O, 17%) as the major nitrogen oxide, carbon dioxide (CO2, 22%), ammonia (NH3, 25%), and nitrite (NO2, 6%) after 24 h.13 (1) Wake Forest University, Department of Chemistry. Tel.: (336)7585774. Fax: (336)758-4656. E-mail: kingsb@wfu.edu. (b) Wake Forest University, Department of Physics. (c) The Cardeza Foundation. (2) Charache, S.; Terrin, M. L.; Moore, R. D.; Dover, G. J.; Barton, F. B.; Eckert, S. V.; McMahon, R. P.; Bonds, D. R. and the Investigators of the Multicenter Study of Hydroxyurea in Sickle Cell Anemia. N. Engl. J. Med. 1995, 332, 1317-1322. (3) Rucknagel, D. L. in Sickle Cell Anemia and Other Hemoglobinpathies; Levere, R. D., Ed; Academic Press: New York, 1975; p 1. (4) Charache, S.; Barton, F. B.; Moore, R. D.; Terrin, M. L.; Steinberg, M. H.; Dover: G. J.; Ballas, S. K.; McMahon, P.; Oswaldo, E. P. and the Investigators of the Multicenter Study of Hydroxyurea in Sickle Cell Anemia. Med. 1996, 75, 300-326. (b) Charache, S. Semin. Hematol. 1997, 34(3), Suppl 3, 15-21. (5) Pacelli, R.; Taira, J.; Cook, J. A.; Wink, D. A.; Krishna, M. C. Lancet 1996, 347, 900. (b) Kwon, N. S.; Stuehr, D. J.; Nathan, C. F. J. Exp. Med. 1991, 174, 761-767. (6) Stolze, K.; Nohl, H. Biochem. Pharmacol. 1990, 40, 799-802. (7) Kim-Shapiro, D. B.; King, S. B.; Bonifant, C. L.; Kolibash, C. P.; Ballas, S. K. Biochim. Biophys. Acta 1998, 1380, 64-74. (8) Head, C. A.; Brugnara, C.; Martinez-Ruiz, R.; Kacmarek, R. M.; Bridges, K. R.; Kuter, D.; Bloch, K. D.; Zapol, W. M. J. Clin Invest. 1997, 100, 2293-1198. (b) McDade, W. A.; Shaba, H. M.; Carter, N. Biophys. J. 1996, 72, A9. (9) Jiang, J.; Jordan, S. J.; Barr, D. P.; Gunther, M. R.; Maeda, H.; Mason, R. P. Mol. Pharmacol. 1997, 52, 1081-1086. (10) Gow, A. J.; Stamler, J. S. Nature 1998, 391, 169-173. (b) Stamler, J. S.; Jia, L.; Eu, J. P.; McMahon, T. J.; Demchenko, I. T.; Bonaventura, J.; Gernert, K.; Piantadosi, C. A. Science 1997, 276, 2034-2037. (11) Kon, H. J. Biol. Chem. 1968, 243, 4350-4357. (b) Ramsey, N. F. Nuclear Moments; John Wiley and Sons: New York, 1953; p 79. (12) Lassmann, G.; Liermann, B. Free Radical Biol. Med. 1989, 6, 241244. (13) Full details of these procedures can be found in the Supporting Information. Figure 1. EPR spectra of Hb15NO (trace A) and Hb14NO (trace B). The spectra are of samples in a liquid nitrogen Dewar observed at 9.1 GHz. The 2.009 g value arrow is at the center of the nitrogen hyperfine patterns. The hyperfine constants are 25 G for 15N and 17.5 G for 14N.

Urease enhances the formation of iron nitrosyl hemoglobin in the presence of hydroxyurea.

Although it has been shown that hydroxyurea (HU) therapy produces measurable amounts of nitric oxide (NO) metabolites, including iron nitrosyl hemoglobin (HbNO) in patients with sickle cell disease, the in vivo mechanism for formation of these is not known. Much in vitro data and some in vivo data indicates that HU is the NO donor, but other studies suggest a role for nitric oxide synthase (NOS). In this study, we confirm that the NO-forming reactions of HU with hemoglobin (Hb) or other blood constituents is too slow to account for NO production measured in vivo. We hypothesize that, in vivo, HU is partially metabolized to hydroxylamine (HA), which quickly reacts with Hb to form methemoglobin (metHb) and HbNO. We show that addition of urease, which converts HU to HA, to a mixture of blood and HU, greatly enhances HbNO formation.

The reaction of deoxy-sickle cell hemoglobin with hydroxyurea

In addition to its capacity to increase fetal hemoglobin levels, other mechanisms are implicated in hydroxyureas ability to provide beneficial effects to patients with sickle cell disease. We hypothesize that the reaction of hemoglobin with hydroxyurea may play a role. It is shown that hydroxyurea reacts with deoxy-sickle cell hemoglobin (Hb) to form methemoglobin (metHb) and nitrosyl hemoglobin (HbNO). The products of the reaction as well as the kinetics are followed by absorption spectroscopy and electron paramagnetic resonance (EPR) spectroscopy. Analysis of the kinetics shows that the reaction can be approximated by a pseudo-first order rate constant of 3.7x10(-4) (1/(s.M)) for the disappearance of deoxy-sickle cell hemoglobin. Further analysis shows that HbNO is formed at an observed average rate of 5.25x10(-5) (1/s), three to four times slower than the rate of formation of metHb. EPR spectroscopy is used to show that the formation of HbNO involves the specific transfer of NO from the NHOH group of hydroxyurea. The potential importance of this reaction is discussed in the context of metHb and HbNO being able to increase the delay time for sickle cell hemoglobin polymerization and HbNOs vasodilating capabilities through conversion to S-nitrosohemoglobin.

The reactions of myoglobin, normal adult hemoglobin, sickle cell hemoglobin and hemin with hydroxyurea

The kinetics of the reaction of hydroxyurea (HU) with myoglobin (Mb), hemin, sickle cell hemoglobin (HbS), and normal adult hemoglobin (HbA) were determined using optical absorption spectroscopy as a function of time, wavelength, and temperature. Each reaction appeared to follow pseudo-first order kinetics. Electron paramagnetic resonance spectroscopy (EPR) experiments indicated that each reaction produced an FeNO product. Reactions of hemin and the ferric forms of HbA, HbS, and myoglobin with HU also formed the NO adduct. The formation of methemoglobin and nitric oxide-hemoglobin from these reactions may provide further insight into the mechanism of how HU benefits sickle cell patients.

An electron paramagnetic resonance study of the affinity of nitrite for methemoglobin.

Recent data suggests that reactions of nitrite with ferric hemoglobin are potentially important in heme-protein dependent NO signaling. Our group and others are evaluating the role of reductive nitrosylation reactions in the generation of N(2)O(3) as a signaling molecule. The latter reaction is hypothesized to involve reactions on NO, nitrite and methemoglobin to form N(2)O(3) in an anhydrase reaction. Of potential importance to these reactions is the affinity of methemoglobin for nitrite and the reactivity of nitrite-bound methemoglobin with nitric oxide. In this paper, we review work related to the electronic structure of nitrite-bound methemoglobin and its dissociation constant. We present new data using electron paramagnetic resonance spectroscopy which confirm that methemoglobin has a much higher affinity for nitrite, under certain conditions, than reported in classical observations. Interestingly the affinity is greatest at lower pH and low nitrite:methemoglobin ratios. These data suggest additional interesting chemistry in the reaction of nitrite with ferric and ferrous heme species. Moreover, this reaction could serve as a paradigm for ferric heme reactions with nitrite.

The influence of crystal structure on radical formation in x‐irradiated amino acids

ESR spectra from the L and DL optical isomers of thirteen amino acids have been observed after x‐irradiation at 77 K. For most of the acids, spectra from the L and DL isomers are different at corresponding temperatures in a range from 77 to 250 K. These differences are attributed to differences in crystal structure, and specifically to the conformation of the amino group with respect to the adjacent carboxyl group. The temperature at which deamination occurs is described by the equation T=13+137 sin2ϑ, where ϑ is the dihedral angle between the C–N bond and the pz orbital occupied by the unpaired electron.

Electron Spin Resonance Studies of Radical Pairs Formed in Hydroxyurea by X Irradiation at Low Temperatures

Radical pairs have been identified in single crystals of hydroxyurea x irradiated at 5°, 30°, and 77°K. There are two chemically identical but magnetically nonequivalent pairs present and at least one other weak pair. The main pairs have an average separation between the unpaired spins of 6.38 A, and the radicals of the pairs are located in adjacent molecular layers. A separation of R ≤ 5.3 A was measured for the weak pair. The magnetically nonequivalent pairs were identified as NH2CONHO···OHNOCH2N. Each radical has the same structure as the stable room‐temperature radical. Decay of the main radical pairs with R = 6.38 A into isolated radicals is exponential and follows a first‐order rate law giving an activation energy of 0.4 eV per molecule.