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Cited by 108Nitric Oxide and Endothelin EffectsHemoglobin structure and functionHeme Oxygenase-1 and Carbon Monoxide

Skeletal muscle as an endogenous nitrate reservoir

Open access
Piknova, Barbora, Park, Ji Won, Swanson, Kathryn M., Dey, Soumyadeep, Noguchi, Constance Tom, Schechter, Alan N.·2015·Nitric Oxide
DOI 10.1016/j.niox.2015.02.145PMC PMC4439352
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Abstract

The nitric oxide synthase (NOS) family of enzymes form nitric oxide (NO) from arginine in the presence of oxygen. At reduced oxygen availability NO is also generated from nitrate in a two step process by bacterial and mammalian molybdopterin proteins, and also directly from nitrite by a variety of five-coordinated ferrous hemoproteins. The mammalian NO cycle also involves direct oxidation of NO to nitrite, and both NO and nitrite to nitrate by oxy-ferrous hemoproteins. The liver and blood are considered the sites of active mammalian NO metabolism and nitrite and nitrate concentrations in the liver and blood of several mammalian species, including human, have been determined. However, the large tissue mass of skeletal muscle had not been generally considered in the analysis of the NO cycle, in spite of its long-known presence of significant levels of active neuronal NOS (nNOS or NOS1). We hypothesized that skeletal muscle participates in the NO cycle and, due to its NO oxidizing heme protein, oxymyoglobin has high concentrations of nitrate ions. We measured nitrite and nitrate concentrations in rat and mouse leg skeletal muscle and found unusually high concentrations of nitrate but similar levels of nitrite, when compared to the liver. The nitrate reservoir in muscle is easily accessible via the bloodstream and therefore nitrate is available for transport to internal organs where it can be reduced to nitrite and NO. Nitrate levels in skeletal muscle and blood in nNOS(-/-) mice were dramatically lower when compared with controls, which support further our hypothesis. Although the nitrate reductase activity of xanthine oxidoreductase in muscle is less than that of liver, the residual activity in muscle could be very important in view of its total mass and the high basal level of nitrate. We suggest that skeletal muscle participates in overall NO metabolism, serving as a nitrate reservoir, for direct formation of nitrite and NO, and for determining levels of nitrate in other organs.

AI snapshot

Written from the paper’s full text.

TL;DR

Skeletal muscle contains unusually high nitrate concentrations in rodents, exceeding blood and liver, and this pool is markedly reduced in nNOS−/− mice, suggesting muscle acts as an endogenous nitrate reservoir. Muscle has lower nitrate reductase activity than liver, but its large mass and high nitrate content may still contribute to systemic nitrite/NO homeostasis.

Key findings

  • Rat skeletal muscle nitrate was 212.4±52.1 nmol/g tissue, significantly higher than blood (76.6±2.6 nmol/g) and liver (12.7±4.6 nmol/g) (p<0.05); liver nitrate was significantly lower than blood (p<0.05).
  • In mice, skeletal muscle nitrate was 113±5.8 nmol/g in wild type vs 13.2±5 in nNOS−/−, and blood nitrate was 48.9±17.3 vs 9.7±2.3 nmol/g; both muscle and blood nitrate were significantly lower in nNOS−/− mice (p<0.05 for both).
  • Rat nitrite concentrations were comparable across tissues: liver 0.8±0.5, blood 0.5±0.2, and skeletal muscle 0.6±0.2 nmol/g, with no statistically significant differences.
  • Nitrate reductase activity was lower in skeletal muscle than liver: at 100, 300, and 500 μM nitrate, liver rates were 1.5, 4.8, and 10.9 nmol/(h×g tissue), while muscle rates were 0.4, 0.5, and 0.9 nmol/(h×g tissue); oxypurinol drastically inhibited liver nitrate reduction (p<0.05), whereas raloxifene had little effect.

Methods

  • Adult male Wistar rats (n=6, 250±50 g), adult nNOS−/− mice (n=5, C57BL/6 background), and wild-type mice (n=10, C57BL/6J) were used; blood, liver, and hind-leg skeletal muscle were collected.
  • Nitrite and nitrate were measured by chemiluminescence after tissue homogenization and protein precipitation; nitrate reductase assays in rat liver and skeletal muscle were performed at 2% oxygen, pH 7.4, 37°C with 100, 300, or 500 μM nitrate and cofactor mix, sampling at 0 min, 30 min, 1, 2, 3, 4, and 24 h.
  • Inhibitor studies used 100 μM oxypurinol (XOR inhibitor) and 50 nM raloxifene (AO inhibitor); Western blotting assessed XOR, AO, and GAPDH expression; statistical significance was tested by one-way ANOVA.

Limitations

  • Stated: nitrate reductase experiments were restricted to rat tissues because of insufficient mouse tissue samples.
  • Stated: the alternative hypothesis that high muscle nitrate results from active transport from blood cannot be completely ruled out.
  • Evident: sample sizes are small (n=5–10 per group), human total nitrate pool estimates are rough extrapolations from rat data, and some supporting data are described as not shown (e.g., pH 6 nitrate reductase results and nNOS−/− nitrite levels).

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

Integrity signals

Registry

Crossref · 10.1016/j.niox.2015.02.145

Cites

47 of 47 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 19 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Nitrite levels in all mouse tissues measured are similar between wild-type and nNOS−/− mice.

    “Nitrite levels in all mice tissues were similar in wild type and nNOS−/− mice (data not shown).”
    Quote in the paperNot a substantial claim
  • Findingown data

    In nNOS−/− mice, nitrate concentrations in skeletal muscle and blood are significantly lower than in wild-type controls.

    “Nitrate levels in skeletal muscle and blood in nNOS−/− mice were dramatically lower when compared with controls, which support further our hypothesis.”
    • Also stated in: § Results · plain
      “Nitrate concentrations of wild type and nNOS−/− mice in skeletal muscle were 113±5.8 and 13.2±5, in blood 48.9±17.3 and 9.7±2.3, and in liver and16.9±5 nmol/g, respectively. These nitrate concentrations in skeletal muscle and blood of nNOS−/− mice were significantly lower than in control mice (p<0.05 for both cases), as seen in Figure 1A.”
    Quote in the paperSubstantial claim
  • Findingown data

    In rat skeletal muscle homogenate, no significant differences in accumulated nitrite were observed at different starting nitrate concentrations.

    “No significant differences in amount of accumulated nitrite were observed at different starting conditions.”
    Quote in the paperSubstantial claim
  • Findingown data

    In liver and muscle homogenates, increased nitrite formation from nitrate was observed at pH 6 compared to pH 7.4.

    “Indeed, when nitrate reductase activity of liver and muscle homogenates were tested, increased nitrite formation from nitrate was observed at pH 6 compared to pH 7.4 (data not shown).”
    Quote in the paperSubstantial claim
  • Findingown data

    Skeletal muscle in rodents contains unusually high nitrate concentrations compared to liver and blood.

    “We measured nitrite and nitrate concentrations in rat and mouse leg skeletal muscle and found unusually high concentrations of nitrate but similar levels of nitrite, when compared to the liver.”
    • Also stated in: § Results · plain
      “Nitrate concentrations in rat liver, blood and skeletal muscle were 12.7±4.6, 76.6±2.6 and 212.4±52.1 nmol/g tissue, respectively. Thus the nitrate concentration in muscle is significantly higher than in liver and blood (p<0.05) but nitrate level in liver was significantly lower than in blood (p<0.05) (Figure 1B).”
    • Also stated in: § Discussion · plain
      “When analyzing the data in Figure 1B in terms of distributions of nitrate and nitrite ions among skeletal muscle, blood and liver, it is striking that nitrate concentrations in skeletal muscle are about 3-fold over those found in blood and about 17-fold higher than in liver.”
    Quote in the paperSubstantial claim
  • May combine two claims. 19
  • Classified as probably not a substantial claim. 11

  • May combine two claims.

    Findingown data

    Nitrite concentrations in rat skeletal muscle and liver are comparable and both are slightly higher than blood, but not statistically significantly.

    “We found that nitrite concentrations in rat liver, blood and skeletal muscle were, 0.8±0.5, 0.5±0.2 and 0.6±0.2 nmol/g tissue, respectively. As seen in Figure 1C, nitrite levels in skeletal muscle and liver were comparable and both values trended to be slightly higher (but not statistically significantly) than in blood.”
    • Also stated in: § Discussion · plain
      “This distribution is not reflected by nitrite ions, levels of which are slightly higher in muscle and liver than in blood, although the differences are not statistically significant.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In rat liver homogenate, oxypurinol drastically inhibited nitrate reduction (p<0.05), while raloxifene had little effect.

    “While oxypurinol drastically inhibited nitrate reduction (p<0.05), raloxifene had little effect, suggesting that most of the reduction was due to XOR.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There is a pronounced nitrate gradient from skeletal muscle through blood to other tissues such as liver, and a slight nitrite gradient from liver and skeletal muscle to blood.

    “Therefore, there is a well pronounced nitrate gradient from skeletal muscle through blood to other tissues, such as liver, and a slight nitrite gradient from these organs (liver and skeletal muscle) to blood.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In rat liver homogenate, nitrate reduction to nitrite increases linearly over 4 hours after nitrate addition, with rates of 1.5, 4.8, and 10.9 nmol/(h x g tissue) for 100, 300, and 500 μM nitrate, respectively.

    “In liver, after addition of the different concentrations of nitrate, we observed a linear increase of nitrite in the homogenate over the next 4 hours (Figure 2A), with rates of 1.5, 4.8 and 10.9 nmol/(h x g tissue), respectively.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In rat skeletal muscle homogenate, nitrate reduction to nitrite occurs at rates of 0.4, 0.5, and 0.9 nmol/(h x g tissue) for 100, 300, and 500 μM nitrate, respectively, which are significantly lower than in liver.

    “Figure 2B shows the results for skeletal muscle homogenate. Calculated rates of nitrate reduction to nitrite were 0.4, 0.5 and 0.9 nmol/(h x g tissue), numbers that are significantly lower than observed for liver.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    XOR is expressed at significant levels in rat skeletal muscle and liver tissues, while AO expression is very low but detectable in skeletal muscle.

    “XOR is expressed at significant levels in muscle and liver tissues while AO expression level is very low, but detectable, in skeletal muscle tissue.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    The role of myoglobin as an NO scavenger in skeletal muscle to prevent its toxicity had been proposed.

    “In fact, the role of myoglobin as an NO scavenger in skeletal muscle to prevent its toxicity had been proposed [35; 36].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [35] Brunori M Nitric oxide moves myoglobin centre stage Trends Biochem Sci 2001 26 209 10 11295538

      Not checked in the free run.

    • cites [36] Wittenberg JB Wittenberg BA Myoglobin function reassessed J Exp Biol 2003 206 2011 20 12756283 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The high nitrate content of rodent skeletal muscle is likely due to nNOSμ and nNOSβ activity, local NO production, and its oxidation by oxymyoglobin.

    “It is likely that the unexpectedly high nitrate content of rodent skeletal muscle tissue is due to the nNOSμ and nNOSβ activity of this tissue.”
    • Also stated in: § Discussion · hedged
      “We postulate that the high levels of nitrate result from local NO production and its oxidation, as skeletal muscle contains high concentrations of oxymyoglobin, which avidly reacts with NO to form nitrate and metmyoglobin.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Acidosis or hypoxia during physical activity predicts increased nitrate reduction by XOR in muscle tissue and opens the possibility of local nitrate reduction within the muscle cell, possibly to NO.

    “Further, acidosis or hypoxia during physical activity predicts increased nitrate reduction by XOR in muscle tissue and open the possibility of local nitrate reduction within the muscle cell, possibly to NO.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The nitrate pool in skeletal muscle exceeds the blood nitrate pool in both concentration and total stored amount.

    “In addition to this source, as we report here, there is also a much larger but previously unknown pool of nitrate originating from metabolic processes in the skeletal muscle that exceeds the blood nitrate pool, both in the concentration and total stored nitrate amount.”
    Quote in the paperSubstantial claim

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

    Study design and conductcitation

    The paper's data in liver and blood of control wild-type mice and rats are in good agreement with previously reported values.

    “Our data in liver and blood in control wild type animals (mice and rats) are in good agreement with these reports [32 - 34].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [32] Kleinbongard P Dejam A Lauer T Rassaf T Schindler A Picker O Scheeren T Godecke A Schrader J Schulz R Heusch G Schaub GA Bryan NS Feelisch M Kelm M Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals Free Radic Biol Med 2003 35 790 6 14583343 source

      Not checked in the free run.

    • cites [33] Hall CN Garthwaite J What is the real physiological NO concentration in vivo? Nitric Oxide 2009 21 92 103 19602444 PMC2779337 source

      Not checked in the free run.

    • cites [34] Milsom AB Fernandez BO Garcia-Saura MF Rodriguez J Feelisch M Contributions of nitric oxide synthases, dietary nitrite/nitrate, and other sources to the formation of NO signaling products Antioxid Redox Signal 2012 17 422 32 22133018 PMC3365358

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Muscle cells contain robust metmyoglobin reductase systems that reduce metmyoglobin back to myoglobin, keeping it functional as an oxygen transporter and available for further reaction with NO.

    “Muscle cells also contain robust metmyoglobin reductase systems, which reduce metmyoglobin back to myoglobin to keep it functional as an oxygen transporter within the myocyte [37; 38], and available for further reaction with NO.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [37] Taylor D Hochstein P Reduction of metmyoglobin in myocytes J Mol Cell Cardiol 1982 14 133 40 7108969

      Not checked in the free run.

    • cites [38] Echevarne C Renerre M Labas R Metmyoglobin reductase activity in bovine muscles Meat Sci 1990 27 161 72 22055228

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Although nitrate reductase activity of skeletal muscle XOR is lower than that of liver, the residual activity in muscle could be important for total nitrite and NO formation because of muscle's large mass and high nitrate levels.

    “Although the nitrate reductase activity of xanthine oxidoreductase in muscle is less than that of liver, the residual activity in muscle could be very important in view of its total mass and the high basal level of nitrate.”
    • Also stated in: § Discussion · hedged
      “However, it is worth noting that although the nitrate reductase activity of skeletal muscle tissue is low, when combined with its large total mass in body and high nitrate levels, muscle could still contribute to total nitrite and NO formation even under resting conditions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    In an adult human, using rat measured values, total nitrate available in skeletal muscle is estimated at 2.12-3.18 mmol, and total nitrite at 7-10.5 μM, while blood contains 382 μM nitrate and 2.5 μmol nitrite.

    “If we use our measured values for liver and skeletal muscle in rat, this would translate into total 2.5μmol of nitrite and 382μM of nitrate in blood and 7-10.5μM of nitrite and 2.12 – 3.18mmol of nitrate available in skeletal muscle at any given time.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Rat measured nitrate concentrations and tissue masses can be used to estimate total nitrate pools in adult humans.

  • May combine two claims.

    Interpretationnone given

    Nitrate levels in nNOS−/− mice presumably originate from dietary contribution and oxidation of NO produced by other NOS isoforms (eNOS and iNOS).

    “Nitrate levels in nNOS−/− mice presumably originate from dietary contribution and oxidation of NO produced by other NOS isoforms – eNOS (NOS3) and iNOS (NOS2).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    NO is not the only possible end product in the reaction cycle of nNOS; depending on the path by which ferric heme releases NO and returns to ferrous state, two end products are possible - NO and nitrate ions (the futile cycle).

    “NO is not the only possible end product in the reaction cycle of the nNOS enzyme. In fact, depending on the path by which ferric heme releases NO and returns to the initial ferrous oxidation state, two end products are possible – NO and nitrate ions. The details of this alternative process, termed the “futile” cycle, are described elsewhere [39 - 41].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [39] Stuehr DJ Santolini J Wang ZQ Wei CC Adak S Update on mechanism and catalytic regulation in the NO synthases J Biol Chem 2004 279 36167 70 15133020

      Not checked in the free run.

    • cites [40] Tejero J Santolini J Stuehr DJ Fast ferrous heme-NO oxidation in nitric oxide synthases FEBS J 2009 276 4505 14 19691141 PMC2737443

      Not checked in the free run.

    • cites [41] Li H Poulos TL Structure-function studies on nitric oxide synthases J Inorg Biochem 2005 99 293 305 15598508 source

      Not checked in the free run.

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

    Premisecitation

    An active nitrate transporter, sialin, has recently been described in salivary glands.

    “An active nitrate transporter, sialin, has recently had been described in salivary glands [44].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [44] Qin L Liu X Sun Q Fan Z Xia D Ding G Ong HL Adams D Gahl WA Zheng C Qi S Jin L Zhang C Gu L He J Deng D Ambudkar IS Wang S Sialin (SLC17A5) functions as a nitrate transporter in the plasma membrane Proc Natl Acad Sci U S A 2012 109 13434 9 22778404 PMC3421170 source

      Not checked in the free run.

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

    Premisecitation

    nNOS missing from the dystrophin protein complex in skeletal muscle is associated with most forms of muscular dystrophies and likely also with sarcopenia.

    “It is also known that nNOS missing from the dystrophin protein complex in the skeletal muscle is associated with most forms of muscular dystrophies and likely also with sarcopenia [23; 24].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [23] Ehmsen J Poon E Davies K The dystrophin-associated protein complex J Cell Sci 2002 115 2801 3 12082140

      Not checked in the free run.

    • cites [24] Samengo G Avik A Fedor B Whittaker D Myung KH Wehling-Henricks M Tidball JG Age-related loss of nitric oxide synthase in skeletal muscle causes reductions in calpain S-nitrosylation that increase myofibril degradation and sarcopenia Aging Cell 2012 11 1036 45 22950758 PMC3734855 source

      Not checked in the free run.

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

    Study design and conductnone given

    All animal procedures were carried out according to the NIH Guide for the Care and Use of Laboratory Animals under an approved NIDDK Animal Care and Use Committee protocol.

    “All animal procedures were carried out according to the recommendations in the Guide for the Care and Use of Laboratory Animals of NIH under NIDDK Animal Care and Use Commitee approved protocol.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    NOS activity of skeletal muscle is important even at resting conditions, which would likely make skeletal muscle the main site of production of NO and its metabolites.

    “The above values show that NOS activity of skeletal muscle is important, even at the resting conditions, which would likely make the skeletal muscle, as the largest organ in the mammalian body, the main site of production of NO and its metabolites.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Skeletal muscle is the largest organ in the mammalian body.

  • May combine two claims.

    Premisecitation

    NO is produced in skeletal muscle tissue even at rest, and its production substantially increases during contraction.

    “It is produced in muscle tissue even at rest and during contraction its production substantially increases [25].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [25] Balon TW Nadler JL Nitric oxide release is present from incubated skeletal muscle preparations J Appl Physiol (1985) 1994 77 2519 21 7896585

      Not checked in the free run.

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

    Premisecitation

    Skeletal muscle contains two active nNOS splice variants, nNOSμ and nNOSβ.

    “In fact, it was later shown that skeletal muscle contains two different active nNOS splice variants, nNOSμ an essential member of the dystrophin-associated protein complex in sarcolemma, and nNOSβ - located in the Golgi complex [22].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22] Percival JM Anderson KN Huang P Adams ME Froehner SC Golgi and sarcolemmal neuronal NOS differentially regulate contraction-induced fatigue and vasoconstriction in exercising mouse skeletal muscle J Clin Invest 2010 120 816 26 20124730 PMC2827958

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Currently, dietary nitrate is often considered to be the main source of nitrate and nitrite in the body.

    “Currently, dietary nitrate is often considered to be the main source of nitrate and nitrite in the body [43].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [43] Wagner DA Young VR Tannenbaum SR Schultz DS Deen WM Mammalian nitrate biochemistry: metabolism and endogenous synthesis IARC Sci Publ 1984 247 53 6533015

      Not checked in the free run.

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

    Study design and conductown data

    Due to insufficient mouse tissue, nitrate reductase assays were restricted to rat tissues.

    “Due to insufficient amount of tissue samples available from mouse, we were restricted to rat tissues in these experiments.”
    Quote in the paperNot a substantial claim

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

    Interpretationreasoning

    Even if active nitrate transport exists in muscle, it would not change the proposal that muscle tissue is a very important reservoir of body nitrate.

    “Even if this pathway exists in muscle, it would not change our proposal that muscle tissue is a very important reservoir of body nitrate.”
    Quote in the paperNot a substantial claim

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

    Study design and conductcitation

    The alternative hypothesis that high skeletal muscle nitrate is due to active nitrate transport from blood cannot be completely ruled out.

    “However, an alternative hypothesis, that high concentration of nitrate in the skeletal muscle is a result of its accumulation in this tissue due to the active nitrate transport from blood, cannot be completely ruled out.”
    Quote in the paperNot a substantial claim

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

    Premisereasoning

    Oxyhemoglobin in red blood cells is a well-known and efficient nitrite and NO oxidase, and blood could be a significant source of nitrate generation from nitrite and NO oxidations.

    “Also, the most abundant protein in blood, oxyhemoglobin in red blood cells, is a well-known and efficient nitrite and NO oxidase, and thus blood could also be a very significant source of nitrate generation from nitrite and NO oxidations, in addition to its role in dietary nitrate transport.”
    Quote in the paperNot a substantial claim
27 of 27

  • The paper relies on this without support.

    No prior measurements of nitrite and/or nitrate in skeletal muscle have been reported.

    “To our knowledge, values of nitrite and/or nitrate in skeletal muscle have not been reported so far.”
    “Both ions had been quantified using chemiluminescence in most of the internal organs [7; 9; 12; 19; 20], but we are not aware of any similar measurements done in skeletal muscle.”

  • The paper relies on this without support.

    Skeletal muscle is the largest organ in the mammalian body.

    “The above values show that NOS activity of skeletal muscle is important, even at the resting conditions, which would likely make the skeletal muscle, as the largest organ in the mammalian body, the main site of production of NO and its metabolites.”
    “These new results on nitrate concentrations in rodent skeletal muscle tissue suggest that this tissue, constituting one of the largest organs in the mammalian body, could play a major role in mammalian nitrogen metabolism.”

  • The paper relies on this without support.

    Rat measured nitrate concentrations and tissue masses can be used to estimate total nitrate pools in adult humans.

    “If we use our measured values for liver and skeletal muscle in rat, this would translate into total 2.5μmol of nitrite and 382μM of nitrate in blood and 7-10.5μM of nitrite and 2.12 – 3.18mmol of nitrate available in skeletal muscle at any given time.”

  • The paper relies on this without support.

    The bacterial contribution to nitrate/nitrite metabolism is dietary-dependent.

    “However, the bacterial contribution to nitrate/nitrite metabolism is also dietary-dependent and therefore can be considered only as an additional mechanism that is highly dependent on extended factors.”
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?

  • Elevations in [NO3−] and [NO2−] following NO3− supplementation have been observed in skeletal muscle and plasma.

    “Elevations in [NO3−] and [NO2−] following NO3− supplementation have been observed in skeletal muscle [88,89,90,91] and plasma [53,64,92], and are associated with positive physiological effects [41,64,74,93] that facilitate a greater capacity for muscular work [51,53,59] and/or improved muscle contractile efficiency (i.e., a lower high-energy phosphate cost of force production) [51,94].”

    From Effects of Dietary Nitrate Supplementation on Weightlifting Exercise Performance in Healthy Adults: A Systematic Review (2020) · cites this paper as [89]

Self-check

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

  • Blood nitrate amount is reported as a concentration

    Minor

    The stated 382 is approximately the total nitrate amount in 5 liters of blood, in μmol, not a concentration in μM. Dividing that amount by 5 liters gives about 76.4 μM, consistent with the reported rat blood value of 76.6 nmol/g.

    Reported 382 · recomputed 76.4

  • Muscle nitrite amount is reported as a concentration

    Minor

    The estimate of 7-10.5 is an amount across 10-15 kg of muscle, so its unit should be μmol rather than μM. Dividing its lower endpoint by 10 kg gives 0.7 μmol/kg, not a concentration of 7 μM.

    Reported 7 · recomputed 0.7

2 of 2

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Related

Authors
Piknova, B., Park, J. W., Swanson, K. M., Dey, S., Noguchi, C. T., & Schechter, A. N.
Affiliations
National Institutes of Health (US)
Corresponding author
Piknova, Barbora
ORCID
Venue
Nitric Oxide
Publisher
Elsevier BV
Published
2015-05
Volume
47
Pages
10-16
ISSN
1089-8603 (ISSN-L)
DOI
10.1016/j.niox.2015.02.145
PMC
PMC4439352
OpenAlex
W2022265373
Cited by
108
References
48
Citations by year
2026: 5 · 2025: 11 · 2024: 10 · 2023: 9 · 2022: 9 · 2021: 13 · 2020: 11 · 2019: 14 · 2018: 13 · 2017: 7 · 2016: 5 · 2015: 1
FWCI
6.39
Type
journal article
Language
en
Open access
Licence
www.elsevier.com/tdm/userlicense/1.0/ (tdm)
Locations
  • Nitric Oxide (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
National Institutes of Health (Z99 DK999999)
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 › Heme Oxygenase-1 and Carbon Monoxide
Keywords
skeletal muscle, nitrite, nitric oxide synthase, neuronal nitric oxide synthase, oxymyoglobin, xanthine oxidoreductase, nitrate reductase, nitric oxide metabolism, nitrate transport, nNOS knockout mice
MeSH terms
Animals, Male, Mice, Inbred C57BL, Nitrates, Nitric Oxide, Rats, Wistar, Mice, Knockout, Muscle, Skeletal, Nitric Oxide Synthase, Mice, Rats

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