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Cited by 97Fatty Acid Research and HealthAntioxidant Activity and Oxidative StressNitric Oxide and Endothelin Effects

Nitrite and nitrate-dependent generation of anti-inflammatory fatty acid nitroalkenes

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Delmastro-Greenwood, Meghan, Hughan, Kara S., Vitturi, Dario A., Salvatore, Sonia R., Grimes, George, Potti, Gopal, Shiva, Sruti, Schopfer, Francisco J. +3 more·2015·Free Radical Biology and Medicine
DOI 10.1016/j.freeradbiomed.2015.07.149PMC PMC4684780
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Abstract

A gap in our understanding of the beneficial systemic responses to dietary constituents nitrate (NO3−), nitrite (NO2−) and conjugated linoleic acid (cLA) is the identification of the downstream metabolites that mediate their actions. To examine these reactions in a clinical context, investigational drug preparations of 15N-labeled NO3− and NO2− were orally administered to healthy humans with and without cLA. Mass spectrometry analysis of plasma and urine indicated that the nitrating species nitrogen dioxide was formed and reacted with the olefinic carbons of unsaturated fatty acids to yield the electrophilic fatty acid, nitro-cLA (NO2-cLA). These species mediate the post-translational modification (PTM) of proteins via reversible Michael addition with nucleophilic amino acids. The PTM of critical target proteins by electrophilic lipids has been described as a sensing mechanism that regulates adaptive cellular responses, but little is known about the endogenous generation of fatty acid nitroalkenes and their metabolites. We report that healthy humans consuming 15N-labeled NO3− or NO2−, with and without cLA supplementation, produce 15NO2-cLA and corresponding metabolites that are detected in plasma and urine. These data support that the dietary constituents NO3−, NO2− and cLA promote the further generation of secondary electrophilic lipid products that are absorbed into the circulation at concentrations sufficient to exert systemic effects before being catabolized or excreted.

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Written from the paper’s full text.

TL;DR

In healthy humans, oral 15N-labeled nitrate or nitrite, with and without conjugated linoleic acid (cLA), generated 15NO2-cLA and related metabolites detectable in plasma and urine. These findings support that dietary nitrate, nitrite, and cLA produce absorbable electrophilic fatty acid nitroalkenes at concentrations proposed to exert systemic signaling effects.

Key findings

  • Without cLA supplementation, endogenous 14NO2-cLA was present in all volunteers and did not vary significantly over 24 h; 15NO2-cLA was not detectable after 15NO2− supplementation and was only detectable 24 h after 15NO3− consumption (0.14 nM median, range 0–25.3 nM).
  • With cLA supplementation, 15NO2-cLA became detectable at 1 h after 15NO2− + cLA (2.5 nM median, range 0–25.7 nM) and remained elevated until 6 h (3.3 nM median, range 0–11.5 nM); after 15NO3− + cLA it became detectable at 2 h and was greatest at 24 h (1.6 nM median, range 0–9.9 nM).
  • Plasma cLA isomers peaked at 3 h after 15NO2− + cLA: (9Z,11E)-cLA averaged 858 ± 138 nM (range 530–1350 nM) and (10E,12Z)-cLA averaged 430.3 ± 77.8 nM (range 271–710 nM).
  • Urinary 15NO2-cLA was greatest 6 h after 15NO2− + cLA (10.7 pmol/mg creatinine median, range 0–55.0) and 24 h after 15NO3− + cLA (12.5 pmol/mg creatinine median, range 3.8–44.3); Cys-15NO2-cLA metabolites were ~10-fold greater than Cys-14NO2-cLA at 24 h.

Methods

  • Healthy volunteers (3 males, 2 females, mean age 29 ± 7 yr) fasted overnight and were randomized to receive oral Na15NO2 (20 mg) or Na15NO3 (1 gm), then crossed over after ~7 days to the other nitrogen oxide (Trial I). In Trial II, the same 5 volunteers received cLA (3 gm) concurrently with both NOx; blood was drawn at baseline, 0.5, 1, 2, 3, 6, and 24 h, and urine was collected at baseline, 6, and 24 h for Trial II only.
  • Plasma and urine were analyzed by LC-MS/MS for 14NO2-cLA and 15NO2-cLA after lipid extraction; nitrite was measured by triiodide chemiluminescence and by diaminonaphthalene derivatization with LC-MS/MS to distinguish 14N/15N contributions; cLA was measured by PTAD derivatization LC-MS/MS.
  • Statistical analysis used samples from 5 subjects, with duplicate or triplicate analyses per subject per time point; data are reported as mean ± SEM for plasma cLA and overall 14NO2-cLA, and as median and range for 14NO2-cLA and 15NO2-cLA box plots.

Limitations

  • The paper states that detection and quantification of NO2-cLA and other electrophilic fatty acids are challenged by their facile reaction with biological nucleophiles, esterification into complex lipids, enzymatic reduction, oxidation, and mitochondrial β-oxidation; the relative signaling activities of β-oxidation products, extents of NO2-fatty acid esterification into glycerolipids, and half-lives of nitro-fatty acids in complex lipid and tissue compartments remain to be fully characterized.
  • Evident from the text: the study included only 5 healthy volunteers with normal blood pressure, so findings may not generalize to other populations; Trial I did not include urine collection; cLA was given only in Trial II; and sampling was limited to 24 h after supplementation.

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Registry

Crossref · 10.1016/j.freeradbiomed.2015.07.149

Cites

43 of 43 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 52 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    These changes corresponded with the timing of peak plasma concentrations of both (9Z,11E)-cLA and (10E,12Z)-cLA.

    “These changes corresponded with the timing of peak plasma concentrations of both (9Z,11E)-cLA and (10E,12Z)-cLA (Fig. 3A).”
    Quote in the paperNot a substantial claim
  • Findingown data

    The consumption of cLA in Trial II supported significant increases in plasma 15NO2-cLA levels from 1 to 6 h following 15NO2− + cLA administration.

    “The consumption of cLA in Trial II supported significant increases in plasma 15NO2-cLA levels from 1 to 6 h following 15NO2− + cLA administration (Fig. 2E).”
    Quote in the paperSubstantial claim
  • Findingown data

    Mass spectrometry analysis of healthy human urine and plasma at different times after oral consumption of 15N-labeled stable isotopes of NO2− and NO3− and conjugated linoleic acid (cLA) reveals substantial NO2-FA generation at concentrations capable of mediating metabolic and inflammatory signaling responses.

    “Herein, the mass spectrometry analysis of healthy human urine and plasma at different times after oral consumption of 15N-labeled stable isotopes of NO2− and NO3− and conjugated linoleic acid (cLA) reveals substantial NO2-FA generation at concentrations capable of mediating metabolic and inflammatory signaling responses.”
    Quote in the paperSubstantial claim
  • Findingown data

    Endogenous urinary mean 14NO2-cLA concentrations modestly decreased after 15NO2− + cLA or 15NO3− + cLA supplementation over the 24 h period.

    “Endogenous urinary mean 14NO2-cLA concentrations modestly decreased after 15NO2− + cLA or 15NO3− + cLA supplementation over the 24 h period (Fig. 4A).”
    Quote in the paperSubstantial claim
  • Findingown data

    The NO2− concentrations after 15NO3− supplementation in Trials I and II peaked between 2 and 3 h with a slightly longer lag time when cLA was added.

    “The NO2− concentrations after 15NO3− supplementation in Trials I and II peaked between 2 and 3 h with a slightly longer lag time when cLA was added (Fig. 3C and D).”
    Quote in the paperNot a substantial claim
  • May combine two claims. 36
  • Classified as probably not a substantial claim. 10

  • May combine two claims.

    Findingown data

    15NO2-cLA generation became detectable at 1 h after 15NO2− + cLA supplementation and remained elevated until 6 h.

    “15NO2-cLA generation became detectable at 1 h (2.5 nM median, range = 0 to 25.7 nM) after 15NO2− + cLA supplementation and remained elevated until 6 h (3.3 nM median, range = 0 to 11.5 nM).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    15NO3− + cLA mediated 15NO2-cLA generation became detectable at 2 h and was greatest at 24 h compared to baseline.

    “In contrast, 15NO3− + cLA mediated 15NO2-cLA generation became detectable at 2 h and was the greatest at 24 h (1.6 nM median, range = 0 to 9.9 nM), compared to baseline (Fig. 2E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Endogenous 14NO2-cLA was present in the plasma of all volunteers and did not vary significantly over the 24 h sampling period after both 15NO2− and 15NO3− supplementation in Trial I.

    “Endogenous 14NO2-cLA was present in the plasma of all volunteers and did not vary significantly over the 24 h sampling period after both 15NO2− and 15NO3− supplementation (15NO2−: 1.0 ± 0.3 nM and 15NO3−: 2.1 ± 0.7 nM, Fig. 2B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In Trial II, similar concentrations of endogenous 14NO2-cLA were detectable over the 24 h sampling period and were not affected by 15NOx + cLA supplementation.

    “Similar concentrations of endogenous 14NO2-cLA were detectable over the 24 h sampling period and not affected by 15NOx + cLA supplementation (15NO2− + cLA : 2.6 ± 0.8 nM and 15NO3− + cLA L 3.1 ± 0.9 nM, Fig. 2D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    15NO2-cLA was not detectable over the sampling period after 15NO2− supplementation and was only detectable 24 h after 15NO3− consumption in Trial I.

    “15NO2-cLA was not detectable over the sampling period after 15NO2− supplementation and was only detectable 24 h after 15NO3− consumption (0.14 nM median, range = 0 to 25.3 nM, Fig. 2C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    The reactions of oxides of nitrogen support substantial NO2-FA generation in humans, and the levels can be altered by dietary supplementation.

    “We now report that the reactions of these oxides of nitrogen support substantial NO2-FA generation in humans, the levels of which can be altered by dietary supplementation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The (9Z,11E)-cLA isomer remained elevated above baseline between 0.5 and 6 h before decreasing back to baseline, while the (10E,12Z)-cLA isomer, derived from the cLA supplement, was only detected from 2 to 6 h.

    “The (9Z,11E)-cLA isomer remained elevated above baseline between 0.5 and 6 h before decreasing back to baseline while the (10E,12Z)-cLA isomer, derived from the cLA supplement, was only detected from 2 to 6 h (Fig. 3A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Cysteine-NO2-cLA adducts were detected in urine, with the ion intensity of Cys-15NO2-cLA metabolites being ~10-fold greater than Cys-14NO2-cLA metabolites at 24 h.

    “Cysteine-NO2-cLA adducts were detected in urine, with the ion intensity of Cys-15NO2-cLA metabolites being ~10-fold greater than Cys-14NO2-cLA metabolites at 24 h (Supplementary Fig. S3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Urinary 15NO2-cLA levels paralleled 15NO2-cLA levels, peaking at 6 h with an equimolar contribution from 14NOx and 15NOx species.

    “This paralleled 15NO2-cLA levels, peaking at 6 h with an equimolar contribution from 14NOx and 15NOx species (Fig. 4C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Total urinary NO2− concentrations decreased after 15NO3− + cLA administration over the 24 h time period, with little contribution from 15NO3−.

    “Total urinary NO2− concentrations decreased after 15NO3− + cLA administration over the 24 h time period, with little contribution from 15NO3− (Fig. 4C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Plasma NO2− concentrations were greatest at 0.5 h post-15NO2− supplementation in both trials; total NO2− concentrations were greatest at 0.5 h in Trial I, where there was a larger contribution of 15NO2− to total NO2−.

    “Plasma NO2− concentrations were greatest at 0.5 h post-15NO2− supplementation in both trials; however, total NO2− concentrations were greatest at 0.5 h in Trial I, where there was a larger contribution of 15NO2− to total NO2− (Fig. 3C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The concentration of urinary 15NO2-cLA was greatest 6 h after 15NO2− + cLA consumption and 24 h after 15NO3− + cLA consumption.

    “The concentration of urinary 15NO2-cLA was greatest 6 h after 15NO2− + cLA consumption (10.7 pmol/mg creatinine (Cre) median, range = 0 to 55.0 pmol/mg Cre) and 24 h after 15NO3− + cLA consumption (12.5 pmol/mg Cre median, range = 3.8 to 44.3 pmol/mg Cre, Fig. 4B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    The levels of 15NO2-cLA after 15NO3− + cLA administration were lower and detectable at later time points compared to those after 15NO2− + cLA supplementation.

    “The levels of 15NO2-cLA after 15NO3− + cLA administration were lower and detectable at later time points compared to those after 15NO2− + cLA supplementation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Dietary supplementation with 15NO3− and 15NO2− caused an increase of all detectable endogenous NOx species, thereby increasing 14NO2− levels that paralleled concentrations of 15NOx species.

    “Notably, dietary supplementation with 15NO3− and 15NO2− caused an increase of all detectable endogenous NOx species, thereby increasing 14NO2− levels that paralleled concentrations of 15NOx species.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The concentration of both cLA isomers peaked at 3 h after 15NO2− + cLA administration, with (9Z,11E)-cLA averaging 858 ± 138 nM and (10E,12Z)-cLA averaging 430.3 ± 77.8 nM.

    “The concentration of both isomers peaked at 3 h after 15NO2− + cLA administration with (9Z,11E)-cLA ranging from 530 to 1350 nM with an average of 858 ± 138 nM and (10E,12Z)-cLA ranging from 271 to 710 nM, with an average of 430.3 ± 77.8 nM (Fig. 3A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    Urinary β-oxidation products and cysteine-adducts of 14NO2-cLA and 15NO2-cLA were measured, confirming the electrophilic reactivity of the parent molecules and metabolites.

    “Urinary β-oxidation products and cysteine-adducts (Supplementary Fig. S2 and S3) of 14NO2-cLA and 15NO2-cLA were also measured, confirming the electrophilic reactivity of the parent molecules and metabolites [3].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [3] Salvatore, 2013, Characterization and quantification of endogenous fatty acid nitroalkene metabolites in human urine, Journal of lipid research source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Maximum levels of plasma 15NO2− after dietary intake occurred after 30 min, thus preceding 15NO2-cLA formation.

    “Maximum levels of plasma 15NO2− after dietary intake occurred after 30 min (Fig. 3C), thus preceding 15NO2-cLA formation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the absence of cLA supplementation in combination with overnight fasting, plasma 15NO2-cLA concentrations were barely detectable and only became significantly elevated 24 h after 15NO3− consumption.

    “It was hypothesized that dietary NOx would be limiting in NO2-cLA formation; however, in the absence of cLA supplementation in combination with overnight fasting, plasma 15NO2-cLA concentrations were barely detectable and only became significantly elevated 24 h after 15NO3− consumption (Fig. 2C).”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Fatty acid nitroalkene derivatives (NO2-FA) are detected in species as diverse as plants, insects and humans.

    “Fatty acid nitroalkene derivatives (NO2-FA) are detected in species as diverse as plants, insects and humans [1–3], but little is known of how NO2-FA are endogenously generated.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [1] Piskorski, 2007, Nitroalkenes and sesquiterpene hydrocarbons from the frontal gland of three prorhinotermes termite species, Journal of chemical ecology source

      Not checked in the free run.

    • cites [2] Fazzari, 2014, Olives and olive oil are sources of electrophilic fatty acid nitroalkenes, PLoS One source

      Not checked in the free run.

    • cites [3] Salvatore, 2013, Characterization and quantification of endogenous fatty acid nitroalkene metabolites in human urine, Journal of lipid research source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    A standard curve using synthetic NO2-cLA and [13C18]-NO2-OA internal standard allowed for the quantification of endogenous products.

    “A standard curve using synthetic NO2-cLA and [13C18]-NO2-OA internal standard allowed for the quantification of endogenous products.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Healthy volunteers were randomized to receive either Na15NO2 (20 mg) or Na15NO3 (1 gm), and after a ~7 day washout period were crossed over to receive the other nitrogen oxide.

    “Healthy volunteers fasted overnight (10–12 h) and were randomized to receive either Na15NO2 (20 mg) or Na15NO3 (1 gm). Following ~7 day washout period, volunteers were crossed over to receive the other nitrogen oxide (NOx) (Trial I).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Metal center-independent reactions of ·NO and nitrite (NO2−) at neutral pH yield both symmetric and asymmetric dinitrogen trioxide (N2O3), which mediates the concerted S-nitrosation of thiols and nitration of unsaturated fatty acid conjugated dienes.

    “metal center-independent reactions of ·NO and nitrite (NO2−) at neutral pH yield both symmetric and asymmetric dinitrogen trioxide (N2O3), that in turn mediates the concerted S-nitrosation of thiols and nitration of unsaturated fatty acid conjugated dienes [10].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [10] Vitturi, 2015, Convergence of Biological Nitration and Nitrosation Reactions via Symmetrical Nitrous Anhydride (N2O3)

      Not checked in the free run.

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

    Study design and conductnone given

    The lipid extraction procedure allowed for measurement of NO2-cLA that was non-covalently bound to albumin and other proteins as well as NO2-cLA that was covalently adducted to small thiols and proteins.

    “This extraction procedure allowed for measurement of NO2-cLA that was non-covalently bound to albumin and other proteins as well as the NO2-cLA that was covalently adducted to small thiols and proteins.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    The bleed-over of 14N into the 15N transition has been accounted for in the calculations of the 15N contribution to total nitrite.

    “Additionally, the “bleed over” of 14N into the 15N transition has been accounted for in the calculations of the 15N contribution to total nitrite.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    This post-translational modification of functionally-significant hyperreactive thiols induces pleiotropic responses that include the cGMP-independent activation of transcriptional programs regulated by Nrf2 and nuclear lipid receptors, as well as inhibition of pro-inflammatory signaling by NF-kβ.

    “This post-translational modification of functionally-significant hyperreactive thiols induces pleiotropic responses that include the cGMP-independent activation of transcriptional programs regulated by nuclear factor (erythroid-derived-2)-like 2 (Nrf2) and nuclear lipid receptors, as well as inhibition of pro-inflammatory signaling by nuclear factor kappa-β (NF-kβ) [12–15].”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [12] Kansanen, 2009, Nrf2-dependent and -independent responses to nitro-fatty acids in human endothelial cells: identification of heat shock response as the major pathway activated by nitro-oleic acid, J Biol Chem source

      Not checked in the free run.

    • cites [13] Kansanen, 2011, Electrophilic Nitro-fatty Acids Activate NRF2 by a KEAP1 Cysteine 151-independent Mechanism, J Biol Chem source

      Not checked in the free run.

    • cites [14] Schopfer, 2011, Formation and signaling actions of electrophilic lipids, Chem Rev source

      Not checked in the free run.

    • cites [15] Cui, 2006, Nitrated fatty acids: Endogenous anti-inflammatory signaling mediators, J. Biol. Chem. 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 conductown data

    The quality and stability of the 15N-labeled sodium nitrite and sodium nitrate capsules were measured by stability-indicating HPLC assays, and all operations were performed in a GMP pharmaceutical manufacturing facility.

    “The quality and stability of the capsules were measured by stability indicating HPLC assays. All operations were performed in a GMP pharmaceutical manufacturing facility with chemical analysis preformed on site.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The study was performed with University of Pittsburgh IRB and FDA IND approval, and informed consent was obtained from all subjects before participation.

    “This study was performed with University of Pittsburgh IRB (PRO11120134) and FDA IND (#115926) approval. Informed consent was obtained from all subjects before participation.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    ·NO2 is formed by metalloprotein-catalyzed oxidation of NO2−, the autoxidation of ·NO, the protonation of NO2− to N2O3 followed by its disproportionation, decomposition of nitrous acid, and the homolytic scission of peroxynitrous acid and nitrosoperoxocarbonate.

    “This species is formed by metalloprotein-catalyzed oxidation of NO2−, the autoxidation of ·NO, the protonation of NO2− to N2O3 followed by its disproportionation, decomposition of nitrous acid, and the homolytic scission of two key products of ·NO and superoxide (O2 ·−) reaction, peroxynitrous acid and nitrosoperoxocarbonate [21–23].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [21] Suarez, 1989, Activated oncogenes in human skin tumors from a repair-deficient syndrome, xeroderma pigmentosum, Cancer research

      Not checked in the free run.

    • cites [22] Leach, 1987, Bacterially catalysed N-nitrosation reactions and their relative importance in the human stomach, Carcinogenesis source

      Not checked in the free run.

    • cites [23] Eiserich, 1998, Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils, Nature source

      Not checked in the free run.

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

    Premisecitation

    Dietary sources of cLA include dairy products and meat, and cLA is also a product of enterosalivary bacterial Δ9-desaturase activity.

    “Dietary sources of cLA include dairy products and meat and cLA is also a product of enterosalivary bacterial Δ9-desaturase activity [27].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] Churruca, 2009, Conjugated linoleic acid isomers: differences in metabolism and biological effects, BioFactors source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Unsaturated fatty acids having conjugated double bonds, such as cLA, are highly reactive with ·NO2, a reaction that occurs much more readily than tyrosine nitration.

    “Unsaturated fatty acids having conjugated double bonds, such as cLA, are highly reactive with ·NO2, a reaction that occurs much more readily than tyrosine nitration [24].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [24] Bonacci, 2012, Conjugated linoleic acid is a preferential substrate for fatty acid nitration, J Biol Chem source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The consumption of cLA is linked with beneficial clinical responses, including the reduction of obesity-related body mass index, improved insulin sensitivity and attenuation of airway hyperactivity.

    “The consumption of cLA is linked with beneficial clinical responses, including the reduction of obesity-related body mass index, improved insulin sensitivity and attenuation of airway hyperactivity, all of which we propose are due in part to the formation and signaling actions of NO2-cLA [29–32].”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [29] Tricon, 2004, Effects of cis-9,trans-11 and trans-10,cis-12 conjugated linoleic acid on immune cell function in healthy humans, The American journal of clinical nutrition source

      Not checked in the free run.

    • cites [30] Tricon, 2004, Opposing effects of cis-9,trans-11 and trans-10,cis-12 conjugated linoleic acid on blood lipids in healthy humans, The American journal of clinical nutrition source

      Not checked in the free run.

    • cites [31] MacRedmond, 2010, Conjugated linoleic acid improves airway hyper-reactivity in overweight mild asthmatics, Clin Exp Allergy source

      Not checked in the free run.

    • cites [32] Zulet, 2005, Inflammation and conjugated linoleic acid: mechanisms of action and implications for human health, J Physiol Biochem source

      Not checked in the free run.

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

    Premisecitation

    The predominant endogenous cLA isomer is 9-cis, 11-trans-cLA, whereas plant-derived oils contain a racemic mixture of 9-cis, 11-trans and 10-trans, 12-cis-cLA.

    “The predominant endogenous cLA isomer is 9-cis, 11-trans-cLA, whereas plant-derived oils contain a racemic mixture of 9-cis, 11-trans and 10-trans, 12-cis-cLA [28].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [28] Kitz, 2010, Omega-3 polyunsaturated fatty acids and bronchial inflammation in grass pollen allergy after allergen challenge, Respiratory medicine source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Electrophilic nitroalkenes such as NO2-cLA induce kinetically rapid posttranslational modification of nucleophilic cysteines of key transcriptional regulatory proteins and inhibit the activity of functionally-significant electrophile-sensitive enzymes.

    “Electrophilic nitroalkenes such as NO2-cLA induce kinetically rapid posttranslational modification of nucleophilic cysteines of key transcriptional regulatory proteins and inhibit the activity of functionally-significant electrophile-sensitive enzymes [11, 33, 34].”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [11] Schopfer, 2011, Formation and signaling actions of electrophilic lipids, Chem Rev source

      Not checked in the free run.

    • cites [33] Kelley, 2008, Nitro-oleic Acid, a Novel and Irreversible Inhibitor of Xanthine Oxidoreductase, J Biol Chem source

      Not checked in the free run.

    • cites [34] Bianchini, 2009, Partitioning the relative contributions of inorganic plant composition and soil characteristics to the quality of Helichrysum italicum subsp. italicum (Roth) G. Don fil. essential oil, Chemistry & biodiversity source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    NO2-cLA is found basally in the plasma and urine of healthy humans and its concentrations increase in organelles and tissues undergoing metabolic and inflammatory stress.

    “NO2-cLA is found basally in the plasma and urine of healthy humans and its concentrations increase in organelles and tissues undergoing metabolic and inflammatory stress [3, 16] (Fig. 5).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [3] Salvatore, 2013, Characterization and quantification of endogenous fatty acid nitroalkene metabolites in human urine, Journal of lipid research source

      Not checked in the free run.

    • cites [16] Trapasso, 2009, Retinoids: new use by innovative drug-delivery systems, Expert opinion on drug delivery source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Some of these transformations retain intrinsic signaling competency via preservation of their electrophilic moiety and others do not.

    “From a pharmacological perspective, some of these transformations retain intrinsic signaling competency via preservation of their electrophilic moiety and others do not.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Lipid nitroalkene derivatives represent salutary signaling molecules that both modulate inflammation and serve as sensitive biomarkers for tissue nitrative and nitrosative reactions.

    “Thus, lipid nitroalkene derivatives represent salutary signaling molecules that both modulate inflammation and serve as sensitive biomarkers for tissue nitrative and nitrosative reactions, much like 8-iso-prostaglandin F2α is a marker for oxidative reactions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Nitroalkylation directly inhibits the catalytic activity of enzymes including xanthine oxidoreductase, cyclooxygenase and soluble epoxide hydrolase, reactions that result in significant beneficial physiological responses.

    “Nitroalkylation also directly inhibits the catalytic activity of enzymes including xanthine oxidoreductase [33], cyclooxygenase [37] and soluble epoxide hydrolase (Cys521) [34], reactions that result in significant beneficial physiological responses.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [33] Kelley, 2008, Nitro-oleic Acid, a Novel and Irreversible Inhibitor of Xanthine Oxidoreductase, J Biol Chem source

      Not checked in the free run.

    • cites [37] Trostchansky, 2011, Nitroarachidonic acid, a novel peroxidase inhibitor of prostaglandin endoperoxide H synthases 1 and 2, J Biol Chem source

      Not checked in the free run.

    • cites [34] Bianchini, 2009, Partitioning the relative contributions of inorganic plant composition and soil characteristics to the quality of Helichrysum italicum subsp. italicum (Roth) G. Don fil. essential oil, Chemistry & biodiversity source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The formation of NO2-cLA and its metabolites after dietary nitrogen oxide and cLA consumption reveals that diet, digestion and metabolism yield a spectrum of absorbable electrophilic metabolites that are detectable in both plasma and urine.

    “In summary, the formation of NO2-cLA and its metabolites after dietary nitrogen oxide and cLA consumption reveals that diet, digestion and metabolism yield a spectrum of absorbable electrophilic metabolites that are detectable in both plasma and urine.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Previously reported targets of nitroalkylation include Cys285 of PPARγ, Cys273 and Cys288 of Keap1, heat shock proteins, and Cys38 of the p65 subunit of NF-κB.

    “Previously reported targets of nitroalkylation include the Cys285 of PPARγ (peroxisome proliferator activator receptor γ) [35], Cys273 and Cys288 of the Nrf2 regulatory protein Keap1 [Kelch-like ECH-associated protein 1 (Keap1)/regulator of nuclear factor (erythroid-derived-2)-like 2 (Nrf2)] [12], heat shock proteins[12], and Cys38 of the p65 subunit of nuclear factor κB (NF-κB) [36] (Fig. 5).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [35] Ferreira, 2012, Nitro-fatty acids as novel electrophilic ligands for peroxisome proliferator-activated receptors, Free radical biology & medicine source

      Not checked in the free run.

    • cites [12] Kansanen, 2009, Nrf2-dependent and -independent responses to nitro-fatty acids in human endothelial cells: identification of heat shock response as the major pathway activated by nitro-oleic acid, J Biol Chem source

      Not checked in the free run.

    • cites [36] Cui, 2006, Nitrated fatty acids: Endogenous anti-inflammatory signaling mediators, The Journal of biological chemistry source

      Not checked in the free run.

39 of 39

  • The paper relies on this without support.

    Little is known about how NO2-FA are endogenously generated.

    “Fatty acid nitroalkene derivatives (NO2-FA) are detected in species as diverse as plants, insects and humans [1–3], but little is known of how NO2-FA are endogenously generated.”

  • The paper relies on this without support.

    A gap in understanding of the beneficial systemic responses to dietary nitrate, nitrite and conjugated linoleic acid is the identification of the downstream metabolites that mediate their actions.

    “A gap in our understanding of the beneficial systemic responses to dietary constituents nitrate (NO3−), nitrite (NO2−) and conjugated linoleic acid (cLA) is the identification of the downstream metabolites that mediate their actions.”
2 of 2

Claims other papers make about this one

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Cites

Flagged first, then by citations

1 of the 44 references couldn’t be matched to a known paper.

Research works printed but not linked: 2

  1. Schopfer, 2011, Formation and signaling actions of electrophilic lipids, Chem Rev, 111, 5997, 10.1021/cr200131e · DOI · Paper page
  2. Cui, 2006, Nitrated fatty acids: Endogenous anti-inflammatory signaling mediators, The Journal of biological chemistry, 281, 35686, 10.1074/jbc.m603357200 · DOI · Paper page

Cited by

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Authors
Delmastro-Greenwood, M., Hughan, K. S., Vitturi, D. A., Salvatore, S. R., Grimes, G., Potti, G., Shiva, S., Schopfer, F. J., Gladwin, M. T., Freeman, B. A., & Gelhaus Wendell, S.
Affiliations
  • University of Pittsburgh (US)
  • National Institutes of Health (US)
ORCID
Venue
Free Radical Biology and Medicine
Publisher
Elsevier BV
Published
2015-12
Volume
89
Pages
333-341
ISSN
0891-5849 (ISSN-L)
DOI
10.1016/j.freeradbiomed.2015.07.149
PMC
PMC4684780
OpenAlex
W1743854985
Semantic Scholar
f7888ccca45b1a0ee2c17225873bd39a5a520cb6
Cited by
97
References
44
Citations by year
2026: 4 · 2025: 10 · 2024: 9 · 2023: 5 · 2022: 6 · 2021: 11 · 2020: 9 · 2019: 12 · 2018: 17 · 2017: 7 · 2016: 6 · 2015: 1
FWCI
9.82
Type
journal article
Language
en
Open access
Licence
Locations
  • Free Radical Biology and Medicine (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
NIH (R01-HL058115, R01-HL64937 (BF), P01-HL103455 (MG, BF), AT00682 (FJS), K12-HD052892 (KSH))
Topics
  • Health Sciences › Nursing › Nutrition and Dietetics › Fatty Acid Research and Health
  • Health Sciences › Medicine › Biochemistry › Antioxidant Activity and Oxidative Stress
  • Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
Keywords
nitrite, nitrate, conjugated linoleic acid, Michael addition, post-translational modification, nitric oxide, nitrate supplementation, anti-inflammatory activity, 15N-labelled nitrate
MeSH terms
Adult, Alkenes, Anti-Inflammatory Agents, Chromatography, Liquid, Female, Humans, Male, Nitrates, Nitrites, Nitro Compounds, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Dietary Supplements, Linoleic Acid

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