CoChat
Sign Up
Cited by 148Fatty Acid Research and HealthAntioxidant Activity and Oxidative StressNitric Oxide and Endothelin Effects

Conjugated linoleic acid is a preferential substrate for fatty acid nitration

Open access
Bonacci, Gustavo, Baker, Paul R.S., Salvatore, Sonia R., Shores, Darla, Khoo, Nicholas K.H., Koenitzer, Jeffrey R., Vitturi, Dario A., Woodcock, Steven R. +7 more·2012·Journal of Biological Chemistry
DOI 10.1074/jbc.m112.401356PMC PMC3531723
Open this paper in CoChat — free accountOpen PDF

Abstract

The oxidation and nitration of unsaturated fatty acids by oxides of nitrogen yield electrophilic derivatives that can modulate protein function via post-translational protein modifications. The biological mechanisms accounting for fatty acid nitration and the specific structural characteristics of products remain to be defined. Herein, conjugated linoleic acid (CLA) is identified as the primary endogenous substrate for fatty acid nitration in vitro and in vivo, yielding up to 10(5) greater extent of nitration products as compared with bis-allylic linoleic acid. Multiple enzymatic and cellular mechanisms account for CLA nitration, including reactions catalyzed by mitochondria, activated macrophages, and gastric acidification. Nitroalkene derivatives of CLA and their metabolites are detected in the plasma of healthy humans and are increased in tissues undergoing episodes of ischemia reperfusion. Dietary CLA and nitrite supplementation in rodents elevates NO(2)-CLA levels in plasma, urine, and tissues, which in turn induces heme oxygenase-1 (HO-1) expression in the colonic epithelium. These results affirm that metabolic and inflammatory reactions yield electrophilic products that can modulate adaptive cell signaling mechanisms.

AI snapshot

Written from the paper’s full text.

TL;DR

Conjugated linoleic acid (CLA) is identified as the preferential unsaturated fatty acid substrate for nitration, producing nitro-CLA (NO2-CLA) under inflammatory, mitochondrial, and gastric acidic conditions. NO2-CLA is detectable in healthy human plasma, and dietary CLA plus nitrite increases NO2-CLA in rodents and induces colonic HO-1 expression.

Key findings

  • CLA nitration yielded up to 10^5 greater nitration products than bis-allylic linoleic acid (LA); with pure NO2 gas, 1.5 µM NO2-CLA versus 20 pM NO2-LA formed. Mitochondrial supplementation with CLA induced ~26-fold increase in NO2-CLA, whereas oleic acid and LA did not increase nitrated products.
  • NO2-CLA was detected in healthy human plasma at 0.3–1.3 nM (average 0.72 nM, median 0.62 nM, n = 7), increased in cardiac tissue after focal ischemia-reperfusion, and was generated by activated macrophages, MPO/H2O2/NO2−, ONOO−, mitochondria, and gastric acidification. L-NAME decreased macrophage-dependent CLA nitration by 30% and media nitrite by 50%.
  • Dietary CLA plus Na[15N]O2 in rodents elevated NO2-CLA in stomach, intestine, colon, plasma, liver, and urine. In mice given 100 nmol each, stomach NO2-CLA peaked at 0.9 ± 0.3 nmol/g tissue at 2 h; higher doses gave 33 ± 22 nmol/g stomach, with small intestine 1.5 ± 1.1 and colon 1.2 ± 1.7 nmol/g.
  • CLA plus nitrite or synthetic NO2-CLA induced HO-1 expression in rat colonic epithelium; [15N]O2-CLA induced dose-dependent HO-1 in RAW 264.7 macrophages comparable to NO2-LA and 9-NO2-CLA.

Methods

  • In vitro reactions used isolated rat liver/heart mitochondria, RAW 264.7 macrophages activated with LPS/INFγ, MPO/H2O2/NO2−, ONOO−, and pure NO2 gas; products were quantified and structurally characterized by HPLC-ESI-MS/MS and high-resolution MS, with β-ME reactivity and Dess-Martin oxidation used to assess electrophilicity and hydroxy groups.
  • In vivo/clinical work used male Sprague-Dawley rats (200–250 g) and C57Bl6 mice, gavage with CLA and Na[15N]O2, archived murine cardiac ischemia-reperfusion tissue, and plasma from seven healthy human subjects; HO-1 was assessed by immunocytochemistry in rat colon and by Western blot in RAW 264.7 cells.
  • Data were analyzed by one-way analysis of variance with post hoc Tukey test.

Limitations

  • The paper states that the biological mechanisms accounting for fatty acid nitration and the structural characteristics of products require additional characterization; the fate of the acyl chain radical formed upon NO2 addition is unclear, and no evidence for stable dinitro or nitro-nitrito CLA intermediates was apparent under the mild acidic conditions used.
  • Evident from the text: the human plasma measurement is from only n = 7 healthy subjects, so broader population prevalence and disease-associated ranges are not established.
  • Evident from the text: most mechanistic evidence comes from in vitro systems or rodent models, and HO-1 induction is shown in colon epithelium and macrophages without establishing full downstream signaling or clinical outcomes.

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

Integrity signals

Registry

Crossref · 10.1074/jbc.m112.401356

Cites

57 of 57 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 46 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Higher doses of CLA and Na15NO2 in nonfasting mice gave 33 ± 22 nmol of NO2-CLA/g of tissue in the stomach.

    “When higher doses of CLA and Na[ 15 N]O 2 were given to nonfasting mice (100 and 10 mol, respectively), 33 Ϯ 22 nmol of NO 2 -CLA/g of tissue was detected in the stomach.”
    Quote in the paperSubstantial claim
  • Findingown data

    Mitochondrial generation of nitrated fatty acids is dependent on pH and NO2- concentrations, with a 10-fold increase in fatty acid nitration at pH 6.0 compared with pH 7.0.

    “The mitochondrial generation of these species was dependent on both pH and NO 2 Ϫ concentrations, with a 10-fold increase in fatty acid nitration at pH 6.0 (1.1 pg of NO 2 -FA/mg of mitochondrial protein), compared with pH 7.0 (0.1 pg of NO 2 -FA/mg of mitochondrial protein).”
    Quote in the paperSubstantial claim
  • Findingown data

    Nitroalkene derivatives of CLA and their metabolites are detected in the plasma of healthy humans.

    “Nitroalkene derivatives of CLA and their metabolites are detected in the plasma of healthy humans”
    • Also stated in: § Fatty Acid Nitration in Vitro and in Vivo · plain
      “Analysis by HPLC-MS/MS revealed NO 2 -CLA in healthy human plasma”
    Quote in the paperSubstantial claim
  • Findingown data

    Nitroalkene derivatives of CLA are increased in tissues undergoing episodes of ischemia reperfusion.

    “and are increased in tissues undergoing episodes of ischemia reperfusion.”
    Quote in the paperSubstantial claim
  • Findingown data

    No additional nitration products were detected upon LA supplementation in macrophages.

    “No additional nitration products were detected upon LA supplementation, further affirming the susceptibility of CLA to nitration.”
    Quote in the paperSubstantial claim
  • May combine two claims. 33
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 20

  • May combine two claims.

    Findingown data

    Supplementation with oleic acid and linoleic acid did not impact the extent of fatty acid nitration and did not increase yields of nitrated products.

    “The addition of both oleic acid (OA) and LA (1 M each) did not impact the extent of fatty acid nitration and did not increase yields of nitrated products.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The reaction of LA with ONOO- gave no detectable nitrated species, whereas ONOO- induced a dose-dependent nitration of (9Z,11E)-CLA.

    “The reaction of LA (1 M) with ONOO Ϫ (0 -20 M) also gave no detectable nitrated species, whereas ONOO Ϫ induced a dose-dependent nitration of (9Z,11E)-CLA (Fig. 3g).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Conjugated linoleic acid (CLA) is the primary endogenous substrate for fatty acid nitration in vitro and in vivo, yielding up to 10^5 greater extent of nitration products as compared with bis-allylic linoleic acid.

    “Herein, conjugated linoleic acid (CLA) is identified as the primary endogenous substrate for fatty acid nitration in vitro and in vivo, yielding up to 10 5 greater extent of nitration products as compared with bis-allylic linoleic acid.”
    • Also stated in: § Abstract · plain
      “CLA is the preferential endogenous substrate for the fatty acid nitration reactions promoted by ⅐ NO and NO 2 Ϫ .”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Addition of the NOS inhibitor L-NAME decreased macrophage-dependent CLA nitration by 30%, in concert with a 50% reduction in media NO2- levels, revealing a contribution of NO-derived species to CLA nitration.

    “Addition of the NOS inhibitor L-NAME decreased macrophage-dependent CLA nitration by 30%, in concert with a 50% reduction in media NO 2 Ϫ levels, revealing a contribution of ⅐ NO-derived species to CLA nitration (Fig. 3d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The reaction of LA with MPO, NO2-, and H2O2 yielded oxidation products, with nitrated products undetectable.

    “The reaction of LA with MPO, NO 2 Ϫ , and H 2 O 2 yielded oxidation products, with nitrated products undetectable under these conditions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    All treated animals showed detectable NO2-CLA levels throughout the gastrointestinal tract, which was absent in control animals and animals gavaged with CLA alone.

    “All treated animals showed detectable NO 2 -CLA levels throughout the gastrointestinal tract, which was absent in control animals and animals gavaged with CLA alone.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    NO2-CLA formation in mitochondria is time-, pH-, and [NO2-]-dependent and correlates with decreased mitochondrial levels of free CLA.

    “NO 2 -CLA formation was time-, pH-, and [NO 2 Ϫ ]-dependent and correlates with decreased mitochondrial levels of free CLA (Fig. 3b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    NO2-CLA reacted rapidly with β-ME, yielding products detected by the neutral loss of β-ME.

    “NO 2 -CLA reacted rapidly, yielding products detected by the neutral loss of ␤-ME (78 atomic mass units) upon CID fragmentation (reverse Michael addition) to give NO 2 -CLA (MRM 402.4/324.2),”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    Multiple enzymatic and cellular mechanisms account for CLA nitration, including reactions catalyzed by mitochondria, activated macrophages, and gastric acidification.

    “Multiple enzymatic and cellular mechanisms account for CLA nitration, including reactions catalyzed by mitochondria, activated macrophages, and gastric acidification.”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    The NO2 group of mitochondrial nitroalkenes is placed at positions C-9 or C-12, with unsaturations at C-9 and C-11, consistent with nitration of conjugated linoleic acid rather than bis-allylic LA.

    “The differences in the elution time between synthetic standard and mitochondrially derived fatty acid nitration products, along with the appearance of the 14 atomic mass units shift in product ion fragments upon CID (m/z 219.1 versus 205.1), indicated that the NO 2 group of mito- chondrial nitroalkenes is placed at positions C-9 or C-12, with unsaturations at C-9 and C-11, respectively”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CLA is present in mitochondrial membranes and is a preferential substrate for nitration reactions.

    “This analysis revealed that CLA is present in mitochondrial membranes and is a preferential substrate for nitration reactions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Up to a 400-fold molar excess of tyrosine over CLA does not compete for nitration of CLA by MPO/H2O2/NO2− and actually promotes CLA nitration.

    “Herein, up to a 400-fold M excess of tyrosine over CLA did not compete for the nitration of CLA by MPO/H 2 O 2 /NO 2 Ϫ , and actually promoted CLA nitration.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    Addition at the C-9 and C-12 positions prevails over additions at C-10 and C-11 and gives a delocalized allylic radical.

    “Thus, addition at the C-9 and C-12 positions prevails over additions at C-10 and C-11 and gives a delocalized allylic radical (42).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [42] Claridge, 1998, The reactions of nitrogen dioxide with dienes, J. Chem. Soc.

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    NO2-OH-OA (m/z 342.2) and NO2-oxo-OA (m/z 340.3) were detected.

    “The detection of NO 2 -OH-OA (m/z 342.2) and NO 2 -oxo-OA (m/z 340.3) further expands the potential product profiles of fatty acids nitrated by ⅐ NO 2 .”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CLA is nitrated by mitochondria, activated RAW 264.7 macrophages, MPO, and ONOO−.

    “CLA nitration by mitochondria, activated RAW 264.7 macrophages, MPO, and ONOO ؊ .”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Nitrogen dioxide mediates nitration of CLA and NO2-CLA is electrophilic.

    “Nitrogen dioxide-mediated nitration of CLA and electrophilic reactivity of NO 2 -CLA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Gastric formation of [15N]O2-CLA modulates HO-1 expression in gut epithelium and levels in plasma, tissue, and urine.

    “Gastric formation of [ 15 N]O 2 -CLA modulates HO-1 expression in gut epithelium and levels in plasma, tissue, and urine.”
    Quote in the paperSubstantial claim

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

    Findingnone given

    These fatty acid derivatives and their metabolites are detected in the plasma of healthy humans and are generated during digestion, metabolic stress, and inflammation.

    “These fatty acid derivatives and their metabolites are detected in the plasma of healthy humans and are generated during digestion, metabolic stress, and inflammation.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Nitric oxide does not directly nitrate protein or lipids; its oxidation to the proximal nitrating species NO2 is essential.

    “Nitric oxide does not directly nitrate protein or lipids, rather its oxidation to the proximal nitrating species ⅐ NO 2 is essential.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [16] Lundberg, 2011, Roles of dietary inorganic nitrate in cardiovascular health and disease, Cardiovasc. Res. source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    CLA is distinct from LA by having a conjugated rather than methylene-interrupted diene.

    “Notably, CLA is distinct from octadeca-(9Z,12Z)-dienoic acid (linoleic acid, LA) by having a conjugated rather than methylene-interrupted diene.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [6] Turpeinen, 2002, Bioconversion of vaccenic acid to conjugated linoleic acid in humans, Am. J. Clin. Nutr. source

      Not checked in the free run.

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

    Study design and conductnone given

    All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh.

    “all procedures were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh (protocol numbers 0911852, 0901770B-4, and 0905750B-7).”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Nitrated fatty acids have been detected both in animal tissues subjected to inflammatory conditions and endogenously in healthy human plasma and urine.

    “Nitrated fatty acids have been detected both in animal tissues subjected to inflammatory conditions and endogenously in healthy human plasma and urine (25,38).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [25] Baker, 2004, Red cell membrane and plasma linoleic acid nitration products. Synthesis, clinical identification, and quantitation, Proc. Natl. Acad. Sci. U.S.A. source

      Not checked in the free run.

    • cites [38] Lima, 2002, Characterization of linoleic acid nitration in human blood plasma by mass spectrometry, Biochemistry source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Nitration of CLA generates electrophilic nitroalkenes and α,β-unsaturated ketone, while NO2-OH-OA requires further oxidation to gain electrophilic reactivity.

    “Thus, although nitration of CLA generates electrophilic nitroalkenes and ␣-␤-unsaturated ketone, NO 2 -OH-OA re- quires further oxidation to gain electrophilic reactivity.”
    Quote in the paperSubstantial claim

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

    Premisenone given

    The biological mechanisms accounting for fatty acid nitration and the specific structural characteristics of products remain to be defined.

    “The biological mechanisms accounting for fatty acid nitration and the specific structural characteristics of products remain to be defined.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [17] Rudolph, 2010, Endogenous generation and protective effects of nitro-fatty acids in a murine model of focal cardiac ischaemia and reperfusion, Cardiovasc. Res. source

      Not checked in the free run.

    • cites [18] Baker, 2005, Fatty acid transduction of nitric oxide signaling. Multiple nitrated unsaturated fatty acid derivatives exist in human blood and urine and serve as endogenous peroxisome proliferator-activated receptor ligands, J. Biol. Chem. source

      Not checked in the free run.

    • cites [19] Ferreira, 2009, Macrophage activation induces formation of the anti-inflammatory lipid cholesteryl-nitrolinoleate, Biochem. J. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Electrophilic derivatives of unsaturated fatty acids (nitroalkene and α,β-unsaturated carbonyl species) are critical proximal mediators of signaling actions, because electrophilic fatty acids are orders of magnitude more potent than native unsaturated fatty acids in modulating tissue-protective and adaptive signaling mechanisms.

    “Notably, electrophilic derivatives of these unsaturated fatty acids (i.e.nitroalkene and ␣,␤-unsaturated carbonyl species) are critical proximal mediators of these signaling actions, because electrophilic fatty acids are orders of magnitude more potent than native unsaturated fatty acids in modulating these and other key tissue-protective and adaptive signaling mechanisms (2,3,11).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [2] Schopfer, 2011, Formation and signaling actions of electrophilic lipids, Chem. Rev. source

      Not checked in the free run.

    • cites [3] Groeger, 2010, Cyclooxygenase-2 generates anti-inflammatory mediators from ω-3 fatty acids, Nat. Chem. Biol. source

      Not checked in the free run.

    • cites [11] Bates, 2011, Nitroalkene fatty acids mediate activation of Nrf2/ARE-dependent and PPARγ-dependent transcription by distinct signaling pathways and with significantly different potencies, Biochemistry source

      Not checked in the free run.

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

    Study design and conductnone given

    All settings (laser power, photomultiplier tube voltage, and offset) were kept constant for the collection of all images.

    “All settings (laser power, photomultiplier tube voltage, and offset) were kept constant for the collection of all images.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Human plasma was obtained with University of Pittsburgh IRB approval PRO07110032.

    “Lipids from human plasma (1.5 ml) (University of Pittsburgh IRB approval PRO07110032) were extracted”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Artifactual nitration during extraction procedures was controlled in parallel by adding a large excess of Na15NO2 and 1,3-hexadiene to plasma and following the formation of [15N]O2-CLA.

    “Artifactual nitration during extraction procedures was controlled in parallel by adding a large excess (100 M) of Na[ 15 N]O 2 and 1 mM 1,3-hexadiene to plasma and fol- lowing the formation of [ 15 N]O 2 -CLA in the presence of [ 13 C 18 ]NO 2 -LA as an internal standard.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    NO2- supplemented diets are associated with a variety of beneficial anti-inflammatory and metabolic actions, including regulation of mitochondrial function, adipogenesis, oxygen delivery to tissues, and blood pressure.

    “Importantly, NO 2 Ϫ -supplemented diets are associated with a variety of beneficial anti-inflammatory and metabolic actions, including the regulation of mitochondrial function, adipogenesis, oxygen delivery to tissues, and blood pressure (20).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [20] Dezfulian, 2009, Nitrite therapy after cardiac arrest reduces reactive oxygen species generation, improves cardiac and neurological function, and enhances survival via reversible inhibition of mitochondrial complex I, Circulation source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Increased rates of generation of reactive inflammatory mediators (partially reduced oxygen species and various oxides of nitrogen), metabolic acidosis, and low oxygen tensions all contribute to unsaturated lipid nitration.

    “Biochemical reaction systems reported herein reinforce that the increased rates of generation of reactive inflammatory mediators (partially reduced oxygen species and various oxides of nitrogen), metabolic acidosis, and low oxygen tensions all contribute to unsaturated lipid nitration.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The in vitro biochemical reaction systems used in this study are representative of in vivo oxidative inflammatory conditions.

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

    Premisecitation

    Multiple mechanisms can account for endogenous NO2 generation, including oxidation of NO by O2, reaction of NO with superoxide to yield ONOO-, one-electron oxidation of NO2- by heme peroxidases, and acid-catalyzed protonation of NO2- to HNO2.

    “Multiple mechanisms can account for endogenous ⅐ NO 2 generation, including the following: (a) oxidation of ⅐ NO by oxygen (O 2 ); (b) reaction of ⅐ NO with superoxide (O 2 .) to yield ONOO Ϫ ; (c) the oneelectron oxidation of NO 2 Ϫ by heme peroxidases (i.e.myeloperoxidase (MPO) and eosinophil peroxidase); and (d) the acidcatalyzed protonation of NO 2 Ϫ to nitrous acid (HNO 2 ).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] Lundberg, 2011, Roles of dietary inorganic nitrate in cardiovascular health and disease, Cardiovasc. Res. source

      Not checked in the free run.

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

    Premisecitation

    Electrophilic fatty acid derivatives react with nucleophilic amino acids of proteins via Michael addition, thereby altering protein structure and function.

    “Electrophilic fatty acid derivatives react with nucleophilic amino acids of proteins via Michael addition, thereby altering protein structure and function (2).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [2] Schopfer, 2011, Formation and signaling actions of electrophilic lipids, Chem. Rev. source

      Not checked in the free run.

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

    Premisecitation

    Nitrite is the product of nitrate reduction by commensal bacterial nitrate reductases in saliva and the gut and is a product of NO oxidation.

    “It is also the product of nitrate (NO 3 Ϫ ) reduction by commensal bacterial nitrate reductases in saliva and the gut (53) and is a product of ⅐ NO oxidation.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [53] Lundberg, 2008, The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics, Nat. Rev. Drug. Discov. source

      Not checked in the free run.

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

    Premisecitation

    Mechanisms giving rise to NO2 include exposure to NO2 gas, O2-derived inflammatory by-products ONOOH and nitrosoperoxocarbonate (ONOOCO2), and heme protein-catalyzed oxidation of NO2− to NO2.

    “These include exposure to ⅐ NO 2 gas, the O 2 . -derived inflammatory by-products ONOOH and nitrosoperoxocarbonate (ONOOCO 2 ) (45), and heme protein-catalyzed oxidation of NO 2 Ϫ to ⅐ NO 2 (46).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [45] Denicola, 1996, Peroxynitrite reaction with carbon dioxide/bicarbonate. Kinetics and influence on peroxynitrite-mediated oxidations, Arch. Biochem. Biophys. source

      Not checked in the free run.

    • cites [46] Eiserich, 1998, Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils, Nature source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    There is a convergence of actions attributed to NO2−, CLA, and fatty acid nitroalkenes, as these species induce positive metabolic responses and anti-inflammatory actions.

    “There is a provocative convergence of the actions attributed to both the precursors (NO 2 Ϫ and CLA) and the products (fatty acid nitroalkenes), as these species induce positive metabolic responses and anti-inflammatory actions.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Nitrite serves as an in vivo reservoir for NO generation, alters mitochondrial function, and manifests a broad array of anti-inflammatory actions in model systems.

    “Nitrite serves as an in vivo reservoir for ⅐ NO generation, alters mitochondrial function (54), and manifests a broad array of anti-inflammatory actions in model systems (22).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [54] Shiva, 2009, Nitrite mediates cytoprotection after ischemia/reperfusion by modulating mitochondrial function, Basic Res. Cardiol. source

      Not checked in the free run.

    • cites [22] Lundberg, 2009, Nitrate and nitrite in biology, nutrition, and therapeutics, Nat. Chem. Biol. source

      Not checked in the free run.

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

    Premisecitation

    The biochemical mechanisms by which fatty acid nitration occurs are those that give rise to NO2.

    “The biochemical mechanisms by which fatty acid nitration occurs are those that give rise to ⅐ NO 2 (44).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [44] O'Donnell, 1999, Nitration of unsaturated fatty acids by nitric oxide-derived reactive nitrogen species peroxynitrite, nitrous acid, nitrogen dioxide, and nitronium ion, Chem. Res. Toxicol. source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    Different electrophilic reactivities displayed by nitrated CLA products influence downstream signaling actions.

    “This in turn will influence downstream signaling actions due to the different electrophilic reactivities displayed by these compounds.”
    Quote in the paperSubstantial claim

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

    Interpretationreasoning

    Inhibition of fatty acid nitration by NOS inhibitors could be a consequence of suppressing formation of NO2− or limiting the reaction of O2 with NO and subsequent ONOO−-dependent NO2 generation.

    “Because NO 2 Ϫ is a product of ⅐ NO autoxidation, the inhibition of fatty acid nitration by NOS inhibitors could be a consequence of the following: (a) suppressing the formation of NO 2 Ϫ or (b) limiting the reaction of O 2 .with ⅐ NO and subsequent ONOO Ϫ -dependent ⅐ NO 2 generation.”
    Quote in the paperSubstantial claim

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

    Interpretationnone given

    Hydrogen abstraction of the NO2-CLA radical intermediate by NO2 can generate HNO2 and reform the corresponding vinyl nitro product.

    “Alternatively, hydrogen abstraction of the NO 2 -CLA radical intermediate by ⅐ NO 2 can generate HNO 2 and reform the corresponding vinyl nitro product.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Nitrito functional groups undergo rapid hydrolysis to yield the corresponding alcohol, with further elimination of water, resulting in a vinyl nitro product.

    “Nitrito functional groups would undergo rapid hydrolysis to yield the corresponding alcohol, with further elimination of water, resulting in a vinyl nitro product (43).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [43] Napolitano, 2000, Acid-promoted reactions of ethyl linoleate with nitrite ions. Formation and structural characterization of isomeric nitroalkane, nitrohydroxy, and novel 3-nitro-1,5-hexadiene and 1,5-dinitro-1,3-pentadiene products, J. Org. Chem. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Conjugated dienes are a preferential target of nitration reactions during oxidative inflammatory conditions and digestion, leading to the formation of electrophilic products that act as potent transcriptional regulatory mediators.

    “In summary, conjugated dienes are a preferential target of nitration reactions during oxidative inflammatory conditions and digestion, leading to the formation of electrophilic products that act as potent transcriptional regulatory”
    Quote in the paperSubstantial claim
42 of 42

  • The paper relies on this without support.

    NO2-CLA detected in healthy human plasma is endogenously formed rather than derived from dietary sources.

    “Endogenous levels of NO 2 -CLA in plasma ranged from 0.3 to 1.3 nM”

  • The paper relies on this without support.

    Very low concentrations or rates of generation of reversibly reactive electrophiles mediate transient and functionally significant reactions with susceptible protein targets, eliciting a broad range of responses.

    “These modifications have typically been viewed as toxic, but recent data affirm that very low concentrations or rates of generation of reversibly reactive electrophiles mediate transient and functionally significant reactions with susceptible protein targets, eliciting a broad range of responses.”

  • The paper relies on this without support.

    Electrophilic NO2-FA potently limit inflammation via multiple mechanisms, including PPARγ activation, inhibition of expression of pro-inflammatory NF-κB-regulated genes, and up-regulation of HO-1 and other phase 2 genes regulated by Keap1/Nrf2.

    “The consideration of the endogenous generation, metabolism, and reactions of electrophilic NO 2 -FA is of relevance because these species potently limit inflammation via multiple mechanisms, including PPAR␥ activation, the inhibition of expression of pro-inflammatory NF-B-regulated genes, and up-regulation of HO-1 and other phase 2 genes regulated by Keap1/Nrf2.”

  • The paper relies on this without support.

    The in vitro biochemical reaction systems used in this study are representative of in vivo oxidative inflammatory conditions.

    “Biochemical reaction systems reported herein reinforce that the increased rates of generation of reactive inflammatory mediators (partially reduced oxygen species and various oxides of nitrogen), metabolic acidosis, and low oxygen tensions all contribute to unsaturated lipid nitration.”

  • The paper relies on this without support.

    The gas-phase and solution reactivity of conjugated dienes extrapolates to biological conditions, explaining preferential nitration of CLA.

    “This preferential reactivity of ⅐ NO 2 toward CLA originates from the conjugated diene moiety, which in the gas phase is more reactive than simple monoalkenes by a factor of 10 3 -10 4 (39).”
5 of 5

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?

  • Under low pH, non-enzymatic reduction of nitrite to NO produces an intermediate nitrogen dioxide radical that can react with dietary, unsaturated, electrophilic fatty acids such as oleic and linoleic acid, to form nitro-conjugated fatty acids and S-nitrosothiol derivatives.

    “Under low pH, non-enzymatic reduction of nitrite to NO produces an intermediate nitrogen dioxide radical that can react with dietary, unsaturated, electrophilic fatty acids such as the essential FAs, oleic and linoleic acid, to form nitro-conjugated fatty acids and S-nitrosothiol derivatives[93,384].”

    From Enterosalivary nitrate metabolism and the microbiome: Intersection of microbial metabolism, nitric oxide and diet in cardiac and pulmonary vascular health (2017) · cites this paper as [384]

  • 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].”

    From Nitrite and nitrate-dependent generation of anti-inflammatory fatty acid nitroalkenes (2015) · cites this paper as [24]

  • Electrophilic nitroalkenes exert signaling actions via the modulation of the expression and activity of both anti-inflammatory and pro-inflammatory enzymes.

    “Electrophilic nitroalkenes exert signaling actions via the modulation of the expression and activity of both anti-inflammatory [32] and pro-inflammatory enzymes [23].”

    From Olives and olive oil are sources of electrophilic fatty acid nitroalkenes (2014) · cites this paper as [32]

  • The presence of cLA in the oils from the three olive cultivars studied herein was confirmed by HPLC-MS/MS detection of a Diels Alder reaction of conjugated dienes with PTAD.

    “The presence of cLA in the oils from the three olive cultivars studied herein was confirmed by HPLC-MS/MS detection of a Diels Alder reaction of conjugated dienes with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD, m/z 454) (Figure S2 [32]).”

    From Olives and olive oil are sources of electrophilic fatty acid nitroalkenes (2014) · cites this paper as [32]

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.

  • MPO experiment reports two CLA concentrations

    Major

    The MPO nitration procedure specifies 1 M fatty acid, but the caption for the MPO experiment specifies 10 M CLA.

  • Colon nuclei stain identified as both Hoechst and DAPI

    Minor

    The immunocytochemistry procedure says the nuclei were stained with Hoechst dye, while the caption identifies the nuclear staining as DAPI.

  • Peroxynitrite experiment reports two LA concentrations

    Minor

    The procedure specifies 10 M CLA or LA for the peroxynitrite reaction, while the results state that LA was used at 1 M.

3 of 3

Cites

Flagged first, then by citations

Cited by

Sorted by citations

Related

Authors
Bonacci, G., Baker, P. R. S., Salvatore, S. R., Shores, D., Khoo, N. K. H., Koenitzer, J. R., Vitturi, D. A., Woodcock, S. R., Golin-Bisello, F., Cole, M. P., Watkins, S., St. Croix, C., Batthyany, C. I., Freeman, B. A., & Schopfer, F. J.
Affiliations
University of Pittsburgh (US)
Corresponding author
Schopfer, Francisco J.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2012-12
Volume
287
Issue
53
Pages
44071-44082
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m112.401356
PMC
PMC3531723
OpenAlex
W1991345823
Semantic Scholar
b4ab20590eff2af6a413f858db19f6201009b52c
Cited by
147
References
56
Citations by year
2026: 2 · 2025: 6 · 2024: 11 · 2023: 11 · 2022: 8 · 2021: 9 · 2020: 11 · 2019: 16 · 2018: 19 · 2017: 8 · 2016: 13 · 2015: 15 · 2014: 9 · 2013: 9
FWCI
9.86
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • HAL (submittedVersion)
  • Conicet (submittedVersion, cc-by-nc-nd)
Funders
  • National Heart, Lung, and Blood Institute (R01HL64937, R01HL064937, R01HL058115, P01HL103455, K99HL095769, R00HL095769, R01HL132550)
  • National Institute of General Medical Sciences (U54GM103529)
  • National Institute of Diabetes and Digestive and Kidney Diseases (P30DK072506)
  • National Center for Complementary and Integrative Health (R01AT006822)
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
conjugated linoleic acid, nitroalkenes, nitric oxide, heme oxygenase-1, post-translational protein modification, electrophilic metabolites, ischemia-reperfusion injury, nitrite supplementation, macrophages, colonic epithelium, dietary linoleic acid
MeSH terms
Animals, Cell Line, Fatty Acids, Humans, Mice, Inbred C57BL, Mitochondria, Nitrates, Nitric Oxide, Nitrites, Signal Transduction, Linoleic Acid, Mice

About these checks

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.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review