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Cited by 157Nitric Oxide and Endothelin EffectsHemoglobin structure and functionRedox biology and oxidative stress

Mechanisms of nitrite bioactivation

Open access
Kim-Shapiro, Daniel B., Gladwin, Mark T.·2014·Nitric Oxide
DOI 10.1016/j.niox.2013.11.002PMC PMC3999231
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Abstract

It is now accepted that the anion nitrite, once considered an inert oxidation product of nitric oxide (NO), contributes to hypoxic vasodilation, physiological blood pressure control, and redox signaling. As such, its application in therapeutics is being actively testing in pre-clinical models and in human phase I–II clinical trials. Major pathways for nitrite bioactivation involve its reduction to NO by members of the hemoglobin or molybdopterin family of proteins, or catalyzed dysproportionation. These conversions occur preferentially under hypoxic and acidic conditions. A number of enzymatic systems reduce nitrite to NO and their activity and importance are defined by oxygen tension, specific organ system and allosteric and redox effectors. In this work, we review different proposed mechanisms of nitrite bioactivation, focusing on analysis of kinetics and experimental evidence for the relevance of each mechanism under different conditions.

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Written from the paper’s full text.

TL;DR

This review argues that nitrite bioactivation occurs mainly through reduction to NO by heme and molybdopterin proteins or catalyzed disproportionation, preferentially under hypoxic and acidic conditions. It compares kinetic estimates and experimental evidence across hemoglobin, myoglobin, neuroglobin, xanthine oxidoreductase, aldehyde oxidase, carbonic anhydrase, and other systems, concluding that different mechanisms likely dominate in different tissues.

Key findings

  • Nitrite bioactivation is favored under hypoxia and acidosis, with major pathways involving hemoglobin/molybdopterin-family proteins or catalyzed disproportionation to NO and other nitrogen oxides.
  • For hemoglobin, R-state vacant hemes react about 100 times faster than T-state hemes; estimated NO production is about 1 nM/s at 100 nM nitrite, 0.01 M heme, and pH 7.4, increasing to about 8 nM/s at pH 6.5 and up to 80 nM/s with high oral nitrate intake. NO made in red cells is rapidly scavenged by oxyHb, giving a lifetime of about 1 microsecond and diffusion of about 0.1 micrometers or less.
  • Myoglobin reduces nitrite with a bimolecular rate constant of 12 M−1s−1 at pH 7.4 and 37 °C, rising to 120 M−1s−1 at pH 6.4; in cardiomyocytes with 300 μM Mb and 1 μM nitrite, acidic deoxygenated conditions could yield about 36 nM/s, while at 1% oxygen about 30% deoxygenation gives about 10 nM/s.
  • Xanthine oxidoreductase has a reported Km of 2.5 mM under some conditions and 12–59 mM at 2%–21% oxygen, with Vmax about 10 nM/mg protein/s; aldehyde oxidase-mediated NO production is estimated to reach 1.5 nM/s at low pH in the heart.

Methods

  • Design: narrative review of proposed mechanisms of nitrite bioactivation, focusing on kinetics and experimental evidence for each mechanism under different conditions.
  • Data or sample: no primary cohort or new experimental sample; the paper synthesizes cited in vitro, animal, and human studies and kinetic equations.
  • Main analysis: qualitative comparison of reaction mechanisms, rate constants, estimated NO production rates, and inhibitor/knockout evidence across heme and molybdopterin enzyme systems.

Limitations

  • Stated: the mechanisms responsible for nitrite bioactivation remain controversial and not clearly defined; the biological significance of pathways involving sulfite oxidase, carbonic anhydrase, NOS, and mARC requires further study.
  • Stated: some cited evidence is preliminary or unpublished, including meeting-presented work on sulfite oxidase and mARC and unpublished carbonic anhydrase Km/Kcat results.
  • Evident: as a narrative review, it does not use a systematic search or meta-analysis, and it reports conflicting findings across models, such as differing conclusions about XOR versus Hb/Mb contributions and the effect of oxypurinol on nitrite-mediated blood flow.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.niox.2013.11.002

Cites

Statistics

Nothing to recompute.

Claims the paper makes

0 of 242 cited sources checked · 0 supported · 0 flagged

  • Findingcitation

    Taking a bimolecular rate constant of 1 M−1s−1, 0.01 M heme, and 100 nM nitrite, the maximum rate of NO production at the p50 oxygen pressure would be 1 nanomolar/sec.

    “The maximum rate of NO production at this oxygen pressure, taking a bimolecular rate constant of 1 M−1s−1 [36], 0.01 M heme, and 100 nM nitrite would be 1 nanomolar/sec.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [36] Huang Z Enzymatic function of hemoglobin as a nitrite reductase that produces Nitric oxide under allosteric control Journal of Clinical Investigation 2005 115 8 2099 2107 16041407 PMC1177999 source

      Not checked in the free run.

  • Findingreasoning

    With 20 mM Hb in the red cell (in heme), the lifetime of NO would be about 1 microsecond.

    “With 20 mM Hb in the red cell (in heme), the lifetime of NO would only be about τ = 1 microsecond.”
    Quote in the paperSubstantial claim
  • Findingreasoning

    NO could only diffuse about 0.1 micrometers or less in the red blood cell.

    “Thus, NO could only diffuse about 0.1 micrometers or less (taking a diffusion constant D = 1000 μm2/s with distance going as the square root of D τ).”
    Quote in the paperSubstantial claim
  • Findingreasoning

    In acidic conditions, NO could be produced in cardiomyocytes at a rate of about 36 nM/s, if all of the Mb is deoxygenated.

    “Thus, in acidic conditions, NO could be produced here at a rate of about 36 nM/s, if all of the Mb is deoxygenated.”
    Quote in the paperNot a substantial claim
  • Findingreasoning

    With a concentration of 0.05 μM eNOS in an arteriole, the rate of nitrite reduction to NO by eNOS in anoxia would be 0.18 nM/sec.

    “With a concentration of 0.05 μM in an arteriole [41], the rate of nitrite reduction to NO by eNOS in anoxia would be 0.18 nM/sec.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [41] Chen K Popel AS Theoretical analysis of biochemical pathways of nitric oxide release from vascular endothelial cells Free Radical Biology and Medicine 2006 41 4 668 680 16864000 source

      Not checked in the free run.

  • May combine two claims. 67
  • Classified as probably not a substantial claim. 29

  • May combine two claims.

    Findingreasoning

    The rate of NO production from Hb-mediated nitrite reduction increases linearly with a decrease in pH so that at pH 6.5 it would be about 8 nM/s.

    “This rate would increase linearly with a decrease in pH so that at a pH of 6.5 it would be about 8 nM/s.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    The rate of Mb-mediated nitrite reduction increases as pH decreases so that at pH 6.4 it would be 120 M−1s−1.

    “As predicted by Equation 1, the rate increases as the pH decreases so that it will be substantially faster under ischemic conditions. At pH 6.4 it would be 120 M−1s−1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    At 1% oxygen (7.6 torr), about 30% of the Mb hemes would be deoxygenated so the rate of NO production would be about 10 nM/s.

    “At 1% oxygen (7.6 torr), about 30% of the Mb hemes would be deoxygenated so the rate of NO production would be about 10 nM/s, which is still considerable.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Findingnone given

    With 300 μM Mb, the lifetime of NO would be about 70 μs in a cardiomyocyte and it could diffuse about 0.3 μm.

    “With 300 μM Mb, the lifetime of NO would be about 70 μs in a cardiomyocyte and it could diffuse about 0.3 μm.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingreasoning

    If nitrite concentrations are taken as 1 μM then in extreme hypoxia capable of making the Ngb completely deoxygenated, the rate of NO production would be about 0.005 nM/s in brain tissue and 0.5 nM/s in retina, taking a bimolecular rate constant of 5 M−1s−1.

    “If nitrite concentrations are taken as 1 μM then in extreme hypoxia capable of making the Ngb completely deoxygenated, the rate of NO production would be about 0.005 nM/s in brain tissue and 0.5 nM/s in retina, taking a bimolecular rate constant of 5 M−1s−1.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Nitrite contributes to hypoxic vasodilation, physiological blood pressure control, and redox signaling.

    “It is now accepted that the anion nitrite, once considered an inert oxidation product of nitric oxide (NO), contributes to hypoxic vasodilation, physiological blood pressure control, and redox signaling.”
    • Also stated in: § Introduction · boosted
      “bioactivation of nitrite through formation of NO and other nitrogen oxides is now widely recognized and the importance of this pathway in the physiological control of blood flow and pressure accepted [2, 3].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [2] Gladwin MT The emerging biology of the nitrite anion Nature Chemical Biology 2005 1 6 308 314 16408064 source

      Not checked in the free run.

    • cites [3] Lundberg JO Nitrate and nitrite in biology, nutrition and therapeutics Nature Chemical Biology 2009 5 12 865 869 PMC4038383 19915529 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The physiological effects of dietary nitrate are eliminated and nitrite levels decreased when volunteers either spit or use mouthwash.

    “These effects are eliminated and nitrite levels decreased when volunteers either spit or use mouthwash [22, 23]”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [22] Govoni M The increase in plasma nitrite after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash Nitric Oxide-Biology and Chemistry 2008 19 4 333 337 18793740 source

      Not checked in the free run.

    • cites [23] Webb AJ Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite Hypertension 2008 51 3 784 790 18250365 PMC2839282 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The observed effects of nitrite are consistent with nitrite being reduced to NO and mediating NO-dependent signal transduction.

    “All of these effects are consistent with nitrite being reduced to NO and mediating NO-dependent signal transduction.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Salivary nitrite increases gastric mucosal blood flow and mucous thickness.

    “Salivary nitrite has been show to increase gastric mucosal blood flow and mucous thickness [7].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [7] Bjorne H Nitrite in saliva increases gastric mucosal blood flow and mucus thickness Journal of Clinical Investigation 2004 113 1 106 114 14702114 PMC300767 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Given that NOS function decreases in hypoxia while nitrite reductase activity increases, NO production rates on the order of 1 nM/s or more may contribute to NO bioactivity while ones substantially lower would not.

    “Given that NOS function decreases in hypoxia (while nitrite reductase activity increases), it is reasonable to consider NO production rates that are on the order of 1 nM/s or more may contribute to NO bioactivity while ones that are substantially lower would not.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In humans, nitrite reduction to NO was discovered in expelled air and attributed to non-enzymatic reduction in the stomach.

    “In humans, nitrite reduction to NO was discovered in expelled air and attributed to non-enzymatic reduction in the stomach [28].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [28] Lundberg JON Intragastric Nitric-Oxide Production in Humans - Measurements in Expelled Air Gut 1994 35 11 1543 1546 7828969 PMC1375608 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationreasoning

    With higher plasma nitrate such as after high oral nitrate intake, one could envision an NO production rate of 80 nM/s.

    “With higher plasma nitrate (such as that achievable after high oral nitrate intake as occurs when drinking beet juice), one could envision an NO production rate of 80 nM/s.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Furchgott and Bhadrakhom demonstrated that nitrite relaxes pre-constricted aortic vessels, but the concentrations used were substantially higher than physiological.

    “In 1952 Furchgott and Bhadrakhom demonstrated the ability of nitrite to effect relaxation of pre-constricted aortic vessels, but the concentrations used where substantially higher than that found in physiological conditions (100 μM compared to normal levels of tens to hundreds of nanomolar) [27].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] Furchgott RF Bhadrakom S Reactions of strips of rabbit aorta to epinephrine, isopropylarterenol, sodium nitrite and other drugs Journal of Pharmacological and Experimental Therapeutics 1953 108 129 43 13062084 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Zweier and colleagues demonstrated reduction of nitrite to NO in an ischemic heart model and attributed the reduction to non-enzymatic processes.

    “In that same year, Zweier and colleagues demonstrated reduction of nitrite to NO in an ischemic heart model and attributed the reduction to non-enzymatic processes [31].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [31] Zweier JL Enzyme-Independent Formation of Nitric-Oxide in Biological Tissues Nature Medicine 1995 1 8 804 809 7585184 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Gladwin, Cannon and others observed an arterial-venous gradient in nitrite along with substantial consumption during exercise when NOS is blocked in humans.

    “In 2000 Gladwin, Cannon and others observed an arterial-venous gradient in nitrite along with a substantial consumption occurring during exercise when nitric oxide synthase (NOS) is blocked in humans [32].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Gladwin MT Role of circulating nitrite and S-nitrosohemoglobin in the regulation of regional blood flow in humans Proceedings of the National Academy of Sciences of the United States of America 2000 97 21 11482 11487 11027349 PMC17226 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Infusion of as little as a few micromolar nitrite increased blood flow in humans in a NOS independent manner and was potentiated by exercise.

    “Then, in 2003, we showed that infusion of as little of a few micromolar nitrite increased blood flow in the human blood flow in a NOS independent manner and this was potentiated when subjects performed exercise (Figure 2) [5].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [5] Cosby K Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation Nature Medicine 2003 9 12 1498 1505 14595407 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The mechanism by which myoglobin leads to nitrite bioactivation is the same as for Hb, but the reaction is substantially faster for Mb than Hb due to its lower heme redox potential.

    “The mechanism by which myoglobin (Mb) would lead to nitrite bioactivation is the same as for Hb (Equation 1), but the reaction was found to be about substantially faster for Mb than Hb due to its lower heme redox potential [54].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [54] Shiva S Deoxymyoglobin is a nitrite reductase that generates nitric oxide and regulates mitochondrial respiration Circulation Research 2007 100 5 654 661 17293481 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Mb-mediated nitrite reduction has been reported to be important in both cardiomyocytes and smooth muscle.

    “Mb-mediated nitrite reduction has been reported to be important in both cardiomyocytes [55] and smooth muscle [56].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [55] Hendgen-Cotta UB Nitrite reductase activity of myoglobin regulates respiration and cellular viability in myocardial ischemia-reperfusion injury Proceedings of the National Academy of Sciences 2008 105 29 10256 10261 PMC2481313 18632562 source

      Not checked in the free run.

    • cites [56] Totzeck M Nitrite Regulates Hypoxic Vasodilation via Myoglobin-Dependent Nitric Oxide Generation Circulation 2012 126 3 325 + 22685116 PMC3410747 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    With reported values of KHis being about 3000, the binding of the iron plays a major role in limiting the rate of nitrite reduction by Ngb, giving an overall bimolecular rate constant of about 0.1 M−1s−1 at neutral pH.

    “With reported values of being about 3000 for KHis [59], the binding of the iron plays a major role in limiting the rate of nitrite reduction by Ngb, giving an overall bimolecular rate constant of about 0.1 M−1s−1 at neutral pH [58].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [58] Tiso M Human Neuroglobin Functions as a Redox-regulated Nitrite Reductase Journal of Biological Chemistry 2011 286 20 18277 18289 21296891 PMC3093900 source

      Not checked in the free run.

    • cites [59] Kiger L Neuroglobin ligand binding kinetics Iubmb Life 2004 56 11–12 709 719 15804836 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    The diffusion distance in smooth muscle is likely greater than in cardiomyocytes.

    “The diffusion distance in the smooth muscle is likely greater.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    The k0 for Ngb is 500 fold faster than for Hb and approaching a rate constant characteristic of that which is rate-limited by how fast nitrite can get to the heme pocket.

    “This is 500 fold faster than for Hb and approaching a rate constant characteristic of that which rate-limited by how fast nitrite can get to the heme pocket [61, 62].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [61] Olson JS Rohlfs RJ Gibson QH Ligand Recombination to the Alpha-Subunits and Beta-Subunits of Human-Hemoglobin Journal of Biological Chemistry 1987 262 27 12930 12938 3654596 source

      Not checked in the free run.

    • cites [62] Olson JS Phillips GN Kinetic pathways and barriers for ligand binding to myoglobin Journal of Biological Chemistry 1996 271 30 17593 17596 8663556 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationnone given

    The rate of NO production via smooth muscle Mb depends on its concentration which has not been determined, but is likely to be substantially less than that of cardiomyocytes.

    “The rate of NO production via smooth muscle Mb depends on its concentration which has not been determined, but is likely to be substantially less than that of cardiomyocytes.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Ngb can reduce nitrite efficiently, approaching the rate for Hb, despite being predominately in the hexacoordinate form, suggesting that the intrinsic rate constant for the reaction of Ngb with HONO, k0, is very large.

    “The fact that Ngb can reduce nitrite so efficiently, approaching the rate for Hb, despite being predominately in the hexcoordinate form (generally less than 1%) suggests that the intrinsic rate constant for the reaction of Ngb with HONO, k0, is very large.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Cytoglobin is found in micromolar concentrations but can be increased about 10-fold under hypoxic conditions.

    “Cytoglobin is found in micromolar concentrations but can be increased about 10-fold under hypoxic conditions [75].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [75] Schmidt M Cytoglobin is a respiratory protein in connective tissue and neurons, which is up-regulated by hypoxia Journal of Biological Chemistry 2004 279 9 8063 8069 14660570 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    The rate of reduction of nitrite by Ngb in physiology depends on oxygenation state, pentacoordination, and pH.

    “The rate of reduction of nitrite by Ngb in physiology depends on oxygenation state (with Ngb having an oxygen affinity similar to Mb [64]), pentacoordination, and pH.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [64] Burmester T Hankeln T What is the function of neuroglobin? Journal of Experimental Biology 2009 212 10 1423 1428 19411534 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationnone given

    Heme oxygenation or oxidation would reduce the rates of Ngb-mediated nitrite reduction.

    “Of course, heme oxygenation or oxidation would reduce the rates.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    The heme iron of cytochrome c is predominately hexacoordinate, but can become pentacoordinate when the iron-methionine bond is ruptured which occurs when the methionine is oxidized, tyrosines are nitrated, or the cytochrome interacts with anionic phospholipids.

    “Similar to Ngb, the heme iron of cytochrome c is predominately hexacoordinate, but can become pentacoordinate when the iron-methionine bond is ruptured which occurs when the methionine is oxidized [65, 66], tyrosines are nitrated [67, 68], or the cytochrome interacts with anionic phospholipids such as those present in the inner membrane of the mitochondria [69–71].”
    Quote in the paperSubstantial claim0 of 7 sources checked
    • cites [65] Chen YR Protein oxidation of cytochrome c by reactive halogen species enhances its peroxidase activity Journal of Biological Chemistry 2002 277 33 29781 29791 12050149 source

      Not checked in the free run.

    • cites [66] Estevam ML Changes in the spin state and reactivity of cytochrome c induced by photochemically generated singlet oxygen and free radicals Journal of Biological Chemistry 2004 279 38 39214 39222 15247265 source

      Not checked in the free run.

    • cites [67] Batthyany C Time course and site(s) of cytochrome c tyrosine nitration by peroxynitrite Biochemistry 2005 44 22 8038 8046 15924423 source

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    • cites [68] Cassina AM Cytochrome c nitration by peroxynitrite Journal of Biological Chemistry 2000 275 28 21409 21415 10770952 source

      Not checked in the free run.

    • cites [69] Spooner PJR Watts A Cytochrome-C Interactions with Cardiolipin in Bilayers - a Multinuclear Magic-Angle Spinning Nmr-Study Biochemistry 1992 31 41 10129 10138 1327134 source

      Not checked in the free run.

    • cites [70] Belikova NA Peroxidase activity and structural transitions of cytochrome c bound to cardiolipin-containing membranes Biochemistry 2006 45 15 4998 5009 16605268 PMC2527545 source

      Not checked in the free run.

    • cites [71] Tuominen EKJ Wallace CJA Kinnunen PKJ Phospholipid-cytochrome c interaction - Evidence for the extended lipid anchorage Journal of Biological Chemistry 2002 277 11 8822 8826 11781329 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationnone given

    The Ngb-mediated NO production rates would increase at lower pH and could be much higher under conditions where the pentacoordinate form of the protein is achieved.

    “These rates would increase at lower pH and could be much higher under conditions where the pentacoordinate form of the protein is achieved.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    The rate constant for cytoglobin-mediated nitrite reduction was measured to be about 0.14 M−1s−1 (pH 7, 25°C) and NO production rates were estimated to be capable of reaching 0.35 nM/s under physiologically-relevant hypoxia and acidosis.

    “The rate constant was measured to be about 0.14 M−1s−1 (pH 7, 25°C and NO production rates were estimated to be capable of reaching 0.35 nM/s under physiologically-relevant hypoxia and acidosis [74].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [74] Li H Characterization of the Mechanism and Magnitude of Cytoglobin-mediated Nitrite Reduction and Nitric Oxide Generation under Anaerobic Conditions Journal of Biological Chemistry 2012 287 43 36623 36633 22896706 PMC3476328 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Cytochrome c oxidase has been proposed to reduce nitrite following a mechanism similar to Hb.

    “Cytochrome c oxidase has been proposed to reduce nitrite following a mechanism similar to Hb [78, 79].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [78] Castello PR Mitochondrial cytochrome oxidase produces nitric oxide under hypoxic conditions: Implications for oxygen sensing and hypoxic signaling in eukaryotes Cell Metabolism 2006 3 4 277 287 16581005 source

      Not checked in the free run.

    • cites [79] Castello PR Oxygen-regulated isoforms of cytochrome c oxidase have differential effects on its nitric oxide production and on hypoxic signaling Proceedings of the National Academy of Sciences 2008 105 8203 8208 PMC2448815 18388202 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Nitrite is reduced by the molybdenum of XOR which can itself be reduced by one of two pathways so that overall nitrite reduction has been described as occurring by two separate processes.

    “Nitrite is reduced by the molybdenum which can itself be reduced by one of two pathways so that overall nitrite reduction has been described as occurring by two separate processes [87].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [87] Li HT Characterization of the effects of oxygen on xanthine oxidase-mediated 44 nitric oxide formation Journal of Biological Chemistry 2004 279 17 16939 16946 14766900 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The rate of NO production by cytochrome c oxidase increases at lower oxygen concentration and depends on the subunit 5 isozyme.

    “The rate of NO production increases at lower oxygen concentration and depends on the subunit 5 isozyme [79].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [79] Castello PR Oxygen-regulated isoforms of cytochrome c oxidase have differential effects on its nitric oxide production and on hypoxic signaling Proceedings of the National Academy of Sciences 2008 105 8203 8208 PMC2448815 18388202 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In Process I, the FAD site is open and oxygen competes with nitrite for electrons so that process 1 is favored in hypoxia and anoxia.

    “In this process, the FAD site is open and oxygen competes with nitrite for electrons so that process 1 is favored in hypoxia and anoxia (Figure 5).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [87] Li HT Characterization of the effects of oxygen on xanthine oxidase-mediated 44 nitric oxide formation Journal of Biological Chemistry 2004 279 17 16939 16946 14766900 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The Michaelis constant Km for XOR has been reported to be 2.5 mM under some conditions and 12 mM to 59 mM with oxygen tensions of 2% to 21%.

    “The Michaelis constant Km has been reported to be 2.5 mM under some conditions [86]. and 12 mM to 59 mM with oxygen tensions of 2% to 21% [87].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [86] Li HT Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrite reduction - Evaluation of its role in nitric oxide generation in anoxic tissues Journal of Biological Chemistry 2001 276 27 24482 24489 11312267 source

      Not checked in the free run.

    • cites [87] Li HT Characterization of the effects of oxygen on xanthine oxidase-mediated 44 nitric oxide formation Journal of Biological Chemistry 2004 279 17 16939 16946 14766900 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisenone given

    XOR has an FAD binding site, iron-sulfur centers and a molybdenum center.

    “It has an FAD binding site, iron-sulfur centers and a molybdenum center.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Nitrite reduction by eNOS is most effective in the presence of calcium/calmodulin, tetrahydrobiopterin, and arginine, and is suggested to involve an additional active site besides the heme.

    “Nitrite reduction is most effective in the presence of calcium/calmodulin, tetrahydrobiopterin, and arginine, and is suggested to involve an additional active site besides the heme [81].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [81] Gautier C Endothelial nitric oxide synthase reduces nitrite anions to NO under anoxia Biochemical and Biophysical Research Communications 2006 341 3 816 821 16442076 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    In Process II, NADH occupies the FAD site so oxygen no longer competes with nitrite for electrons; Process II is independent of oxygen tension.

    “In Process II, NADH occupies the FAD site so oxygen no longer competes with nitrite for electrons [87]. Process II is then independent of oxygen tension (Figure 5).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [87] Li HT Characterization of the effects of oxygen on xanthine oxidase-mediated 44 nitric oxide formation Journal of Biological Chemistry 2004 279 17 16939 16946 14766900 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Endothelial NOS is the only NOS isoform that is capable of effectively reducing nitrite and it does so at a rate of 3.5 nM/sec per micromolar protein.

    “Slama-Schwok and colleagues showed that endothelial NOS is the only NOS isoform that is capable of effectively reducing nitrite and it does so at a rate of 3.5 nM/sec per micromolar protein [83].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [83] Mikula I Isoform-specific differences in the nitrite reductase activity of nitric oxide synthases under hypoxia Biochemical Journal 2009 418 673 682 19046140 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisenone given

    Xanthine oxidoreductase is a flavoprotein that reduces xanthine to hypoxanthine and can also reduce hypoxanthine to uric acid.

    “Xanthine oxidoreductase (XOR) is a flavoprotein that reduces xanthine to hypoxanthine and can also reduce hypoxanthine to uric acid.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Other studies have reported negligible XOR in blood.

    “Other studies have reported negligible XOR in blood [39].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] Li H Nitric Oxide Production from Nitrite Occurs Primarily in Tissues Not in the Blood: CRITICAL ROLE OF XANTHINE OXIDASE AND ALDEHYDE OXIDASE Journal of Biological Chemistry 2008 283 26 17855 17863 18424432 PMC2440597 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    RBC XOR has recently been proposed to play a particularly important role in producing NO from nitrite in patients with hypertension where XOR is suggested to be upregulated.

    “RBC XOR has recently been proposed to play a particularly important role in producing NO from nitrite in patients with hypertension where XOR is suggested to be upregulated [25].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [25] Ghosh SM Enhanced Vasodilator Activity of Nitrite in Hypertension Critical Role for Erythrocytic Xanthine Oxidoreductase and Translational Potential Hypertension 2013 61 5 1091 1102 23589565 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisenone given

    Aldehyde oxidase catalyzes oxidation of aldehyde to carboxylic acid and functions in metabolizing xenobiotics.

    “Aldehyde oxidase (AO) catalyzes oxidation of aldehyde to carboxylic acid and functions in metabolizing xenobiotics.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Like XOR, AO has an FAD binding site, iron-sulfur centers and a molybdenum center.

    “Like XOR, it has an FAD binding site, iron-sulfur centers and a molybdenum center [92].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [92] Calzi ML Purification, cDNA cloning, and tissue distribution of bovine liver aldehyde oxidase Journal of Biological Chemistry 1995 270 52 31037 31045 8537361 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The Vmax of XOR is constant over different oxygen tensions and equal to about 10 nM/mg protein/s.

    “The Vmax is constant over different oxygen tensions and equal to about 10 nM/mg protein/s [87].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [87] Li HT Characterization of the effects of oxygen on xanthine oxidase-mediated 44 nitric oxide formation Journal of Biological Chemistry 2004 279 17 16939 16946 14766900 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    NO scavenging by Hb would limit the effectiveness of NO generated by RBC XOR, but XOR localization to the RBC membrane could limit NO scavenging to some extent.

    “In addition, NO scavenging by Hb would also limit the effectiveness of NO generated by RBC XOR, but XOR localization to the RBC membrane could limit NO scavenging to some extent.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Levels of XOR vary by tissue and have been reported to be about 7g/g tissue in the heart and 80 g/g tissue in the liver but these levels can increase dramatically along with levels of NADPH in ischemia.

    “Levels of XOR vary by tissue and have been reported to be about 7g/g tissue in the heart and 80 g/g tissue in the liver but these levels can increase dramatically along with levels of NADPH in ischemia [39].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] Li H Nitric Oxide Production from Nitrite Occurs Primarily in Tissues Not in the Blood: CRITICAL ROLE OF XANTHINE OXIDASE AND ALDEHYDE OXIDASE Journal of Biological Chemistry 2008 283 26 17855 17863 18424432 PMC2440597 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    XOR has been proposed to be associated with endothelial cells and RBCs and reduce nitrite there.

    “In addition to XOR mediated NO generation from nitrite occurring in heart, liver, and smooth muscle tissue, XOR has been proposed to be associated with endothelial cells and RBCs and reduce nitrite there [88].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [88] Webb AJ Mechanisms Underlying Erythrocyte and Endothelial Nitrite Reduction to Nitric Oxide in Hypoxia Role for Xanthine Oxidoreductase and Endothelial Nitric Oxide Synthase Circulation Research 2008 103 9 957 U114 18818408 PMC2841343 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Under aerobic conditions, the NADH-dependent process of AO is favored as oxygen acts as a competitive inhibitor of nitrite reduction in the absence of NADH.

    “Under aerobic conditions, the NADH-dependent process is favored as oxygen acts as a competitive inhibitor of nitrite reduction in the absence of NADH [93].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [93] Li HT Kundu TK Zweier JL Characterization of the Magnitude and Mechanism of Aldehyde Oxidase-mediated Nitric Oxide Production from Nitrite Journal of Biological Chemistry 2009 284 49 33850 33858 19801639 PMC2797155 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

    In the case of Hb, nitrite anhydrase has been proposed whereby 2NO2− + HbFe2+ + H+ → N2O3 + HbFe2+ + OH−.

    “In the case of Hb, nitrite anhydrase has been proposed whereby (7)2NO2−+HbFe2++H+→N2O3+HbFe2++OH−”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisereasoning

    Several suggested pathways for nitrite bioactivation involve the formation of N2O3.

    “Several suggested pathways for nitrite bioactivation involve the formation of N2O3, as mentioned above in the case of potential Hb-mediated export of NO bioactivity from the red blood cell.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    The mechanism of nitrite reduction by AO is similar to that of XOR where nitrite is reduced at the molybdenum site and either an aldehyde can supply electrons at that same site, or NADH can supply electrons from the flavin site.

    “The mechanism of nitrite reduction by AO is similar to that of XOR where nitrite is reduced at the molybdenum site and either an aldehyde can supply electrons at that same site, or NADH can supply electrons from the flavin site [93].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [93] Li HT Kundu TK Zweier JL Characterization of the Magnitude and Mechanism of Aldehyde Oxidase-mediated Nitric Oxide Production from Nitrite Journal of Biological Chemistry 2009 284 49 33850 33858 19801639 PMC2797155 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Lowering pH increases the rate of AO-mediated reduction, but not as much as other pathways; about 35% in going from pH 7.4 to pH 6.0.

    “Like other nitrite reduction pathways, lowering pH increases the rate of AO-mediated reduction, but not as much as other pathways; about 35% in going from pH 7.4 to pH 6.0 [93].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [93] Li HT Kundu TK Zweier JL Characterization of the Magnitude and Mechanism of Aldehyde Oxidase-mediated Nitric Oxide Production from Nitrite Journal of Biological Chemistry 2009 284 49 33850 33858 19801639 PMC2797155 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Preliminary work with mARC presented at the meeting by the Gladwin group has demonstrated that it can reduce nitrite to NO and is capable of using NADH or cytochrome b5 and cytochrome b5 reductase as electron sources.

    “Preliminary work with this enzyme presented at the meeting by the Gladwin group has demonstrated that it too can reduce nitrite to NO and is capable of using NADH or cytochrome b5 and cytochrome b5 reductase as electron sources [95].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [95] Wang J Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to NO Nitric Oxide-Biology and Chemistry 2013 31 S39 S40 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In one proposed mechanism for nitrite anhydrase, an intermediate ferric nitrosyl species is formed and this then reacts with a second nitrite molecule.

    “In one proposed mechanism for nitrite anhydrase, an intermediate ferric nitrosyl species is formed (which is equivalent to a ferrous nitrosonium) and this then reacts with a second nitrite molecule [49, 97, 98].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [49] Salgado MT Nagababu E Rifkind JM Quantification of Intermediates Formed during the Reduction of Nitrite by Deoxyhemoglobin Journal of Biological Chemistry 2009 284 19 12710 12718 19270306 PMC2676000 source

      Not checked in the free run.

    • cites [97] Nagababu E Active nitric oxide produced in the red cell under hypoxic conditions by deoxyhemoglobin-mediated nitrite reduction Journal of Biological Chemistry 2003 278 47 46349 46356 12952953 source

      Not checked in the free run.

    • cites [98] Nagababu E Ramasamy S Rifkind JM Intermediates detected by visible Spectroscopy during the reaction of nitrite with deoxyhemoglobin: The effect of nitrite concentration and diphosphoglycerate Biochemistry 2007 46 11650 11659 17880185 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Formation of N2O3 from nitrite would facilitate export of NO activity from the red blood cell and evidence for the formation of this species has been reported.

    “Formation of N2O3 from nitrite would facilitate export of NO activity from the red blood cell and evidence for the formation of this species has been reported [45–47, 99].”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [45] Jeffers A Hemoglobin mediated nitrite activation of soluble guanylyl cyclase Comparative Biochemistry and Physiology a-Molecular & Integrative Physiology 2005 142 2 130 135 15936233 source

      Not checked in the free run.

    • cites [46] Robinson JM Lancaster JR Hemoglobin-mediated, hypoxia-induced vasodilation via nitric oxide - Mechanism(s) and physiologic versus pathophysiologic relevance American Journal of Respiratory Cell and Molecular Biology 2005 32 4 257 261 15778415 source

      Not checked in the free run.

    • cites [47] Basu S Catalytic generation of N2O3 by a concerted nitrite reductase and anhydrase activity of hemoglobin Nature Chemical Biology 2007 3 785 794 17982448 source

      Not checked in the free run.

    • cites [99] Roche CJ Friedman JM NO reactions with sol-gel and solution phase samples of the ferric nitrite derivative of HbA Nitric Oxide-Biology and Chemistry 2010 22 2 180 190 PMC2834230 19919854 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Another possibility is that nitrite binds to MetHb which subsequently reacts with NO to make N2O3.

    “Another possibility is that nitrite binds to MetHb which subsequently reacts with NO to make N2O3 [47].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [47] Basu S Catalytic generation of N2O3 by a concerted nitrite reductase and anhydrase activity of hemoglobin Nature Chemical Biology 2007 3 785 794 17982448 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    N2O3 can carry NO bioactivity through subsequent nitrosation or through NO formation.

    “N2O3 can carry NO bioactivity through subsequent nitrosation or through NO formation (6)N2O3↔NO+NO2.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Preliminary work presented at the Fifth International Meeting suggests that sulfite oxidase functions as a nitrite reductase both as an isolated enzyme and in cultured fibroblasts.

    “Preliminary work presented at the Fifth International Meeting on the Role of Nitrite and Nitrate in Physiology, Pathophysiology, and Therapeutics suggests that sulfite oxidase, which catalyzes the oxidation of sulfite to sulfate, functions as a nitrite reductase both as an isolated enzyme and in cultured fibroblasts [95].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [95] Wang J Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to NO Nitric Oxide-Biology and Chemistry 2013 31 S39 S40 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The low Km of AO for nitrite (about 3 mM) limits its NO production rate, but it has been estimated that AO mediated NO production from nitrite can reach 1.5 nM/s at low pH in the heart.

    “Again, the low Km of AO for nitrite (about 3 mM) limits its NO production rate, but it has been estimated that AO mediated NO production from nitrite can reach 1.5 nM/s at low pH in the heart [93].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [93] Li HT Kundu TK Zweier JL Characterization of the Magnitude and Mechanism of Aldehyde Oxidase-mediated Nitric Oxide Production from Nitrite Journal of Biological Chemistry 2009 284 49 33850 33858 19801639 PMC2797155 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The reaction of CA with nitrite was found to be faster at higher pH (7.2) but produce more total NO at lower pH (5.9).

    “The reaction was found to be faster at higher pH (7.2) but produce more total NO at lower pH (5.9) [103].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [103] Aamand R Generation of nitric oxide from nitrite by carbonic anhydrase: a possible link between metabolic activity and vasodilation American Journal of Physiology-Heart and Circulatory Physiology 2009 297 6 H2068 H2074 19820197 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The biggest challenges for the pathway involving a metHb-nitrite intermediate are that metHb has a low to moderate affinity for nitrite and the reaction with NO must compete with the fast reactions of NO and oxygenated and deoxygenated Hb.

    “The biggest challenges for the pathway involving a metHb-nitrite intermediate are that metHb has a low to moderate affinity for nitrite and the reaction with NO must compete with the fast reactions of NO and oxygenated and deoxygenated Hb.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Others have questioned whether N2O3 can and does form.

    “However, others have questioned whether this species can and does form [100, 101].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [100] Tui C Reactions of nitrite with hemoglobin measured by membrane inlet mass spectrometry Free Radical Biology and Medicine 2009 46 1 14 19 18848984 PMC2849169 source

      Not checked in the free run.

    • cites [101] Koppenol WH Nitrosation, Thiols, and Hemoglobin: Energetics and Kinetics Inorganic Chemistry 2012 51 10 5637 5641 22554003 PMC3357094 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Carbonic anhydrase is a zinc-containing enzyme that catalyzes the formation of carbonic acid from carbon dioxide and water.

    “Carbonic anhydrase (CA) is a zinc-containing enzyme that catalyzes the formation of carbonic acid from carbon dioxide and water, (12)CO2+H2O↔HCO3−+H+”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    The pH dependence of CA reactivity with nitrite is suggested to be due to the involvement of the nitrite anion in the anhydrase reaction balanced by a requirement for protons.

    “These results were suggested to be due to the involvement of the nitrite anion in the anhydrase reaction (rather than nitrous acid) balanced by a requirement for protons (Equation 7) [103].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [103] Aamand R Generation of nitric oxide from nitrite by carbonic anhydrase: a possible link between metabolic activity and vasodilation American Journal of Physiology-Heart and Circulatory Physiology 2009 297 6 H2068 H2074 19820197 source

      Not checked in the free run.

  • May combine two claims.

    Premisenone given

    Recent work in the Gladwin lab has shown that the Km of CA for nitrite is about 40 μM and the Kcat is very low at about 2 × 10−4 s−1 at pH 7.2 (unpublished results).

    “However, recent work in the Gladwin lab has shown that the Km of CA for nitrite is about 40 μM and the Kcat is very low at about 2 × 10−4 s−1 at pH 7.2 (unpublished results).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    It was suggested that CA catalyzes formation of N2O3 similar to the reverse of the reaction shown in Equation 12, with subsequent formation of NO and nitrogen dioxide via Equation 6.

    “It was suggested that CA catalyzes formation of N2O3 similar to the reverse of the reaction shown in Equation 12, (7)2NO2−+2H+↔N2O3+H2O With subsequent formation of NO and nitrogen dioxide via Equation 6 [103].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [103] Aamand R Generation of nitric oxide from nitrite by carbonic anhydrase: a possible link between metabolic activity and vasodilation American Journal of Physiology-Heart and Circulatory Physiology 2009 297 6 H2068 H2074 19820197 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Several studies have shown that oral nitrate decreases blood pressure and that this effect is dependent on reduction of nitrate to nitrite by commensal bacteria.

    “Several studies have shown that oral nitrate decreases blood pressure and that this effect is dependent on reduction of nitrate to nitrite by commensal bacteria [22–26].”
    Quote in the paperSubstantial claim0 of 5 sources checked
    • cites [22] Govoni M The increase in plasma nitrite after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash Nitric Oxide-Biology and Chemistry 2008 19 4 333 337 18793740 source

      Not checked in the free run.

    • cites [23] Webb AJ Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite Hypertension 2008 51 3 784 790 18250365 PMC2839282 source

      Not checked in the free run.

    • cites [24] Kapil V Inorganic Nitrate Supplementation Lowers Blood Pressure in Humans. Role for Nitrite-Derived NO Hypertension 2010 56 274 281 20585108 source

      Not checked in the free run.

    • cites [25] Ghosh SM Enhanced Vasodilator Activity of Nitrite in Hypertension Critical Role for Erythrocytic Xanthine Oxidoreductase and Translational Potential Hypertension 2013 61 5 1091 1102 23589565 source

      Not checked in the free run.

    • cites [26] Larsen FJ Effects of dietary nitrate on blood pressure in healthy volunteers New England Journal of Medicine 2006 355 26 2792 2793 17192551 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    NO production increases as Hb oxygenation is decreased from 100%, so that the balance tips towards relative NO production upon deoxygenation.

    “On the other hand, NO production increases as Hb oxygenation is decreased from 100%, so that the balance tips towards relative NO production upon deoxygenation.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Another study by Dalsgaard and coworkers using aortic rings has questioned whether Hb and red cells actually contribute to nitrite-mediated vasodilation.

    “Another study by Dalagaard and coworkers using aortic rings has questioned whether Hb and red cells actually contribute to nitrite-mediated vasodilation [111].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [111] Dalsgaard T Simonsen U Fago A Nitrite-dependent vasodilation is facilitated by hypoxia and is independent of known NO-generating nitrite reductase activities American Journal of Physiology-Heart and Circulatory Physiology 2007 292 6 H3072 H3078 17307993 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationreasoning

    All of these effects of nitrite can be explained by formation of NO bioactivity in the blood or blood vessels, but the mechanisms for bioactivation in these tissues has been the subject of considerable debate and active study.

    “All of these effects of nitrite can be explained by formation of NO bioactivity in the blood or blood vessels, but the mechanisms for bioactivation in these tissues has been the subject of considerable debate and active study.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Relaxation by nitrite and red blood cells was associated with cGMP formation and dependent on Hb oxygen affinity, with maximal effect occurring near the Hb p50, consistent with the notion that R-state Hb reduces nitrite more effectively than T-state Hb.

    “Relaxation by nitrite and red blood cells was associated with cGMP formation and dependent on Hb oxygen affinity, with maximal effect occurring near the Hb p50, consistent with the notion that R-state Hb reduces nitrite more effectively than T-state Hb [109].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [109] Crawford JH Hypoxia, red blood cells and nitrite regulate NO-dependent hypoxic vasodilatation Blood 2006 107 566 574 16195332 PMC1895612 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Erythrocytic XOR was found to increase in human patients with mild hypertension and in a rodent spontaneous hypertension model.

    “Erythrocytic XOR was found to increase in human patients with mild hypertension and in a rodent spontaneous hypertension model [25].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [25] Ghosh SM Enhanced Vasodilator Activity of Nitrite in Hypertension Critical Role for Erythrocytic Xanthine Oxidoreductase and Translational Potential Hypertension 2013 61 5 1091 1102 23589565 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    At oxygen tensions of 15 and 25 mmHg as little as 200 nM nitrite was subsequently shown to effect relaxation in aortic rings in the presence of red blood cells and relaxation was abrogated by the NO scavenger CPTIO.

    “At oxygen tensions of 15 and 25 mmHg as little as 200 nM nitrite was subsequently shown to effect relaxation in aortic rings in the presence of red blood cells and relaxation was abrogated by the NO scavenger CPTIO [109].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [109] Crawford JH Hypoxia, red blood cells and nitrite regulate NO-dependent hypoxic vasodilatation Blood 2006 107 566 574 16195332 PMC1895612 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    They also suggested that eNOS, both in the RBC and endothelial cells, played a role in vascular NO production from nitrite using a NOS blocker.

    “They also suggested that eNOS, both in the RBC and endothelial cells, played a role in vascular NO production from nitrite using a NOS blocker [88].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [88] Webb AJ Mechanisms Underlying Erythrocyte and Endothelial Nitrite Reduction to Nitric Oxide in Hypoxia Role for Xanthine Oxidoreductase and Endothelial Nitric Oxide Synthase Circulation Research 2008 103 9 957 U114 18818408 PMC2841343 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The importance of XOR-mediated nitrite reduction was emphasized in blood pressure reduction in hypertensive rat models and based on NO formation from human red cells from patients with hypertension.

    “The importance of XOR-mediated nitrite reduction was emphasized in blood pressure reduction in hypertensive rat models and based on NO formation from human red cells from patients with hypertension [25].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [25] Ghosh SM Enhanced Vasodilator Activity of Nitrite in Hypertension Critical Role for Erythrocytic Xanthine Oxidoreductase and Translational Potential Hypertension 2013 61 5 1091 1102 23589565 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The increase in XOR protein content was associated with increased ability to reduce nitrite to NO in erythrocytes but not in rodent arterial vessels and this erythrocytic ability to reduce nitrite was substantially reduced upon administration of the XOR inhibitor allopurinol in the rodent model and in erythrocytes from hypertensive patients.

    “The increase in protein content was associated with increased ability to reduce nitrite to NO in erythrocytes but not in rodent arterial vessels and this erythrocytic ability to reduce nitrite was substantially reduced upon administration of the XOR inhibitor allopurinol in the rodent model and in erythrocytes form hypertensive patients [25].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [25] Ghosh SM Enhanced Vasodilator Activity of Nitrite in Hypertension Critical Role for Erythrocytic Xanthine Oxidoreductase and Translational Potential Hypertension 2013 61 5 1091 1102 23589565 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    Nitrite could be an effective source of NO in cytoprotection because it is an ample source of NO bioactivity and can be bioactivated in ischemic conditions.

    “Nitrite could be an effective source of NO in cytoprotection due to its role an ample source of NO bioactivity and ability to be bioactivated in ischemic conditions [116].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [116] Murillo D Nitrite as a mediator of ischemic preconditioning and cytoprotection Nitric Oxide-Biology and Chemistry 2011 25 2 70 80 PMC3118399 21277988 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Nitrite-mediated cytoprotection is mediated by NO production from the molybdenum-containing enzyme XOR and to some extent SO.

    “Numerous studies have suggested that nitrite-mediated cytoprotection is mediated by NO production from the molybdenum containing enzyme XOR [8, 39, 86, 118, 127] and to some extent SO [39] as well.”
    Quote in the paperSubstantial claim0 of 5 sources checked
    • cites [8] Webb A Reduction of nitrite to nitric oxide during ischemia protects against myocardial ischemia-reperfusion damage PNAS 2004 101 37 13683 13688 15347817 PMC518813 source

      Not checked in the free run.

    • cites [39] Li H Nitric Oxide Production from Nitrite Occurs Primarily in Tissues Not in the Blood: CRITICAL ROLE OF XANTHINE OXIDASE AND ALDEHYDE OXIDASE Journal of Biological Chemistry 2008 283 26 17855 17863 18424432 PMC2440597 source

      Not checked in the free run.

    • cites [86] Li HT Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrite reduction - Evaluation of its role in nitric oxide generation in anoxic tissues Journal of Biological Chemistry 2001 276 27 24482 24489 11312267 source

      Not checked in the free run.

    • cites [118] Baker JE Nitrite confers protection against myocardial infarction: Role of xanthine oxidoreductase, NADPH oxidase and K-ATP channels channels Journal of Molecular and Cellular Cardiology 2007 43 4 437 444 17765919 PMC2735077 source

      Not checked in the free run.

    • cites [127] Tripatara P Nitrite-derived nitric oxide protects the rat kidney against ischemia/reperfusion injury in vivo: Role for xanthine oxidoreductase Journal of the American Society of Nephrology 2007 18 2 570 580 17202421 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Neuroglobin falls short of this production rate but may be much faster if factors favor the heme pentacoordinate state.

    “Neuroglobin falls short of this production rate but may be much faster if factors also favor the heme pentacoordinate state.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    Cytochrome c can produce NO this fast when in the pentacoordinate heme state.

    “Cytochrome c can produce NO this fast when in the pentacoordinate heme state.”
    Quote in the paperNot a substantial claim
80 of 80

  • The authors state this as an assumption.

    The rate of NO production from cytochrome c oxidase is roughly linear down to a nitrite concentration of 10 μM.

    “assuming the rate of production is roughly linear down to a nitrite concentration of 10 μM”

  • The authors state this as an assumption.

    The pK of nitrite is 3.15 for calculating the percentage of nitrite in anion form.

    “assuming a pK of 3.15”

  • The paper relies on this without support.

    XOR produces negligible NO from nitrite in normal volunteer red blood cells at physiological pH values.

    “However, XOR produces negligible NO from nitrite in normal volunteer red blood cells at physiological pH values.”

  • The paper relies on this without support.

    NO reduces platelet activation, probably through binding to soluble guanylyl cyclase within platelets.

    “NO is known to reduce platelet activation, probably through binding to soluble guanylyl cyclase within platelets”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Carbonic anhydrase NO-production estimate exceeds its stated maximum rate

    Major

    At 40 μM enzyme and a turnover rate of 2 × 10−4 s−1, the maximum production rate is 0.008 μM/s, even before accounting for the stated 1 μM nitrite concentration. The reported 0.2 μM/s is 25 times that maximum.

    Reported 0.2 · recomputed 0.008

  • Distal-histidine binding has opposite stated effects on nitrite reduction

    Minor

    The paper says increased affinity for the distal histidine increases nitrite reduction, but also says histidine binding limits that rate.

2 of 2

The citation lists haven’t been fetched yet

References 133 · Cited by 157

Research works printed but not linked: 2

  1. Webb, 2005, Xanthine oxidase activity provides an alternative source of no during ischaemia from inorganic nitrite in rat blood vessels, Br. J. Clin. Pharmacol., 59, 631
  2. Srihirun, 2012, Platelet Inhibition by Nitrite Is Dependent on Erythrocytes and Deoxygenation, PLoS One, 7, e30380, 10.1371/journal.pone.0030380 · DOI · Paper page

Authors
Kim-Shapiro, D. B., & Gladwin, M. T.
Affiliations
University of Pittsburgh (US)
Corresponding author
Gladwin, Mark T.
ORCID
Gladwin, Mark T.
Venue
Nitric Oxide
Publisher
Elsevier BV
Published
2014-04
Volume
38
Pages
58-68
ISSN
1089-8603 (ISSN-L)
DOI
10.1016/j.niox.2013.11.002
PMC
PMC3999231
OpenAlex
W2027419908
Semantic Scholar
e370178c4711c430a17e096202a35024a95c7f3b
Cited by
157
References
133
Citations by year
2026: 3 · 2025: 9 · 2024: 6 · 2023: 7 · 2022: 9 · 2021: 11 · 2020: 12 · 2019: 13 · 2018: 17 · 2017: 22 · 2016: 14 · 2015: 22 · 2014: 10
FWCI
1.69
Type
journal article
Language
en
Open access
Locations
  • Nitric Oxide (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Institutes of Health
  • National Heart, Lung, and Blood Institute (R29HL058091, HL058091, HL098032, R37HL058091, R01HL058091, R01HL098032)
Topics
  • Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Hemoglobin structure and function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
Keywords
hypoxic vasodilation, blood pressure regulation, redox signaling, nitrite reduction, hemoglobin, hypoxia, acidosis, enzyme kinetics, allosteric regulation
MeSH terms
Molybdenum Cofactors, Animals, Coenzymes, Hemeproteins, Humans, Kinetics, Metalloproteins, Nitrites, Pteridines

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-09.

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