Nitro-fatty acids are formed in response to virus infection and are potent inhibitors of STING palmitoylation and signaling.

The adaptor molecule stimulator of IFN genes (STING) is central to production of type I IFNs in response to infection with DNA viruses and to presence of host DNA in the cytosol. Excessive release of type I IFNs through STING-dependent mechanisms has emerged as a central driver of several interferonopathies, including systemic lupus erythematosus (SLE), Aicardi-Goutières syndrome (AGS), and stimulator of IFN genes-associated vasculopathy with onset in infancy (SAVI). The involvement of STING in these diseases points to an unmet need for the development of agents that inhibit STING signaling. Here, we report that endogenously formed nitro-fatty acids can covalently modify STING by nitro-alkylation. These nitro-alkylations inhibit STING palmitoylation, STING signaling, and subsequently, the release of type I IFN in both human and murine cells. Furthermore, treatment with nitro-fatty acids was sufficient to inhibit production of type I IFN in fibroblasts derived from SAVI patients with a gain-of-function mutation in STING. In conclusion, we have identified nitro-fatty acids as endogenously formed inhibitors of STING signaling and propose for these lipids to be considered in the treatment of STING-dependent inflammatory diseases.

Electrophilic fatty acid nitroalkenes are systemically transported and distributed upon esterification to complex lipids.

Electrophilic nitro-fatty acids [NO2-FAs (fatty acid nitroalkenes)] showed beneficial signaling actions in preclinical studies and safety in phase 1 clinical trials. A detailed description of the pharmacokinetics (PK) of NO2-FAs is complicated by the capability of electrophilic fatty acids to alkylate thiols reversibly and become esterified in various complex lipids, and the instability of the nitroalkene moiety during enzymatic and base hydrolysis. Herein, we report the mechanism and kinetics of absorption, metabolism, and distribution of the endogenously detectable and prototypical NO2-FA, 10-nitro-oleic acid (10-NO2-OA), in dogs after oral administration. Supported by HPLC-high-resolution-MS/MS analysis of synthetic and plasma-derived 10-NO2-OA-containing triacylglycerides (TAGs), we show that a key mechanism of NO2-FA distribution is an initial esterification into complex lipids. Quantitative analysis of plasma free and esterified lipid fractions confirmed time-dependent preferential incorporation of 10-NO2-OA into TAGs when compared with its principal metabolite, 10-nitro-stearic acid. Finally, new isomers of 10-NO2-OA were identified in vivo, and their electrophilic reactivity and metabolism characterized. Overall, we reveal that NO2-FAs display unique PK, with the principal mechanism of tissue distribution involving complex lipid esterification, which serves to shield the electrophilic character of this mediator from plasma and hepatic inactivation and thus permits efficient distribution to target organs.

Nitro-fatty acid inhibition of triple-negative breast cancer cell viability, migration, invasion, and tumor growth.

Triple-negative breast cancer (TNBC) comprises ∼20% of all breast cancers and is the most aggressive mammary cancer subtype. Devoid of the estrogen and progesterone receptors, along with the receptor tyrosine kinase ERB2 (HER2), that define most mammary cancers, there are no targeted therapies for patients with TNBC. This, combined with a high metastatic rate and a lower 5-year survival rate than for other breast cancer phenotypes, means there is significant unmet need for new therapeutic strategies. Herein, the anti-neoplastic effects of the electrophilic fatty acid nitroalkene derivative, 10-nitro-octadec-9-enoic acid (nitro-oleic acid, NO2-OA), were investigated in multiple preclinical models of TNBC. NO2-OA reduced TNBC cell growth and viability in vitro, attenuated TNFα-induced TNBC cell migration and invasion, and inhibited the tumor growth of MDA-MB-231 TNBC cell xenografts in the mammary fat pads of female nude mice. The up-regulation of these aggressive tumor cell growth, migration, and invasion phenotypes is mediated in part by the constitutive activation of pro-inflammatory nuclear factor κB (NF-κB) signaling in TNBC. NO2-OA inhibited TNFα-induced NF-κB transcriptional activity in human TNBC cells and suppressed downstream NF-κB target gene expression, including the metastasis-related proteins intercellular adhesion molecule-1 and urokinase-type plasminogen activator. The mechanisms accounting for NF-κB signaling inhibition by NO2-OA in TNBC cells were multifaceted, as NO2-OA (a) inhibited the inhibitor of NF-κB subunit kinase ß phosphorylation and downstream inhibitor of NF-κB degradation, (b) alkylated the NF-κB RelA protein to prevent DNA binding, and (c) promoted RelA polyubiquitination and proteasomal degradation. Comparisons with non-tumorigenic human breast epithelial MCF-10A and MCF7 cells revealed that NO2-OA more selectively inhibited TNBC function. This was attributed to more facile mechanisms for maintaining redox homeostasis in normal breast epithelium, including a more favorable thiol/disulfide balance, greater extents of multidrug resistance protein-1 (MRP1) expression, and greater MRP1-mediated efflux of NO2-OA-glutathione conjugates. These observations reveal that electrophilic fatty acid nitroalkenes react with more alkylation-sensitive targets in TNBC cells to inhibit growth and viability.

The Chemical Basis of Thiol Addition to Nitro-conjugated Linoleic Acid, a Protective Cell-signaling Lipid.

Nitroalkene fatty acids are formed in vivo and exert protective and anti-inflammatory effects via reversible Michael addition to thiol-containing proteins in key signaling pathways. Nitro-conjugated linoleic acid (NO2-CLA) is preferentially formed, constitutes the most abundant nitrated fatty acid in humans, and contains two carbons that could potentially react with thiols, modulating signaling actions and levels. In this work, we examined the reactions of NO2-CLA with low molecular weight thiols (glutathione, cysteine, homocysteine, cysteinylglycine, and ß-mercaptoethanol) and human serum albumin. Reactions followed reversible biphasic kinetics, consistent with the presence of two electrophilic centers in NO2-CLA located on the ß- and δ-carbons with respect to the nitro group. The differential reactivity was confirmed by computational modeling of the electronic structure. The rates (kon and koff) and equilibrium constants for both reactions were determined for different thiols. LC-UV-Visible and LC-MS analyses showed that the fast reaction corresponds to ß-adduct formation (the kinetic product), while the slow reaction corresponds to the formation of the δ-adduct (the thermodynamic product). The pH dependence of the rate constants, the correlation between intrinsic reactivity and thiol pKa, and the absence of deuterium solvent kinetic isotope effects suggested stepwise mechanisms with thiolate attack on NO2-CLA as rate-controlling step. Computational modeling supported the mechanism and revealed additional features of the transition states, anionic intermediates, and final neutral products. Importantly, the detection of cysteine-δ-adducts in human urine provided evidence for the biological relevance of this reaction. Finally, human serum albumin was found to bind NO2-CLA both non-covalently and to form covalent adducts at Cys-34, suggesting potential modes for systemic distribution. These results provide new insights into the chemical basis of NO2-CLA signaling actions.

Nitro-fatty acid formation and metabolism.

Nitro-fatty acids (NO2-FA) are pleiotropic modulators of redox signaling pathways. Their effects on inflammatory signaling have been studied in great detail in cell, animal and clinical models primarily using exogenously administered nitro-oleic acid. While we know a considerable amount regarding NO2-FA signaling, endogenous formation and metabolism is relatively unexplored. This review will cover what is currently known regarding the proposed mechanisms of NO2-FA formation, dietary modulation of endogenous NO2-FA levels, pathways of NO2-FA metabolism and the detection of NO2-FA and corresponding metabolites.

Convergence of biological nitration and nitrosation via symmetrical nitrous anhydride.

The current perspective holds that the generation of secondary signaling mediators from nitrite (NO2(-)) requires acidification to nitrous acid (HNO2) or metal catalysis. Herein, the use of stable isotope-labeled NO2(-) and LC-MS/MS analysis of products reveals that NO2(-) also participates in fatty acid nitration and thiol S-nitrosation at neutral pH. These reactions occur in the absence of metal centers and are stimulated by autoxidation of nitric oxide ((•)NO) via the formation of symmetrical dinitrogen trioxide (nitrous anhydride, symN2O3). Although theoretical models have predicted physiological symN2O3 formation, its generation is now demonstrated in aqueous reaction systems, cell models and in vivo, with the concerted reactions of (•)NO and NO2(-) shown to be critical for symN2O3 formation. These results reveal new mechanisms underlying the NO2(-) propagation of (•)NO signaling and the regulation of both biomolecule function and signaling network activity via NO2(-)-dependent nitrosation and nitration reactions.

Synaptic Zn2+ inhibits neurotransmitter release by promoting endocannabinoid synthesis.

Although it is well established that many glutamatergic neurons sequester Zn(2+) within their synaptic vesicles, the physiological significance of synaptic Zn(2+) remains poorly understood. In experiments performed in a Zn(2+)-enriched auditory brainstem nucleus--the dorsal cochlear nucleus--we discovered that synaptic Zn(2+) and GPR39, a putative metabotropic Zn(2+)-sensing receptor (mZnR), are necessary for triggering the synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). The postsynaptic production of 2-AG, in turn, inhibits presynaptic probability of neurotransmitter release, thus shaping synaptic strength and short-term synaptic plasticity. Zn(2+)-induced inhibition of transmitter release is absent in mutant mice that lack either vesicular Zn(2+) or the mZnR. Moreover, mass spectrometry measurements of 2-AG levels reveal that Zn(2+)-mediated initiation of 2-AG synthesis is absent in mice lacking the mZnR. We reveal a previously unknown action of synaptic Zn(2+): synaptic Zn(2+) inhibits glutamate release by promoting 2-AG synthesis.

Modulation of nitro-fatty acid signaling: prostaglandin reductase-1 is a nitroalkene reductase.

Inflammation, characterized by the activation of both resident and infiltrated immune cells, is accompanied by increased production of oxidizing and nitrating species. Nitrogen dioxide, the proximal nitrating species formed under these conditions, reacts with unsaturated fatty acids to yield nitroalkene derivatives. These electrophilic products modulate protein function via post-translational modification of susceptible nucleophilic amino acids. Nitroalkenes react with Keap1 to instigate Nrf2 signaling, activate heat shock response gene expression, and inhibit NF-κB-mediated signaling, inducing net anti-inflammatory and tissue-protective metabolic responses. We report the purification and characterization of a NADPH-dependent liver enzyme that reduces the nitroalkene moiety of nitro-oleic acid, yielding the inactive product nitro-stearic acid. Prostaglandin reductase-1 (PtGR-1) was identified as a nitroalkene reductase by protein purification and proteomic studies. Kinetic measurements, inhibition studies, immunological and molecular biology approaches as well as clinical analyses confirmed this identification. Overexpression of PtGR-1 in HEK293T cells promoted nitroalkene metabolism to inactive nitroalkanes, an effect that abrogated the Nrf2-dependent induction of heme oxygenase-1 expression by nitro-oleic acid. These results situate PtGR-1 as a critical modulator of both the steady state levels and signaling activities of fatty acid nitroalkenes in vivo.

Biomimetic nitration of conjugated linoleic acid: formation and characterization of naturally occurring conjugated nitrodienes.

Nitro-conjugated linoleic acids (NO2-cLA), endogenous nitrodiene lipids which act as inflammatory signaling mediators, were isolated and single isomers purified from the biomimetic acidic nitration products of conjugated linoleic acid (CLA). Structures were elucidated by means of detailed NMR and HPLC-MS/MS spectroscopic analysis and the relative double bond configurations assigned. Additional synthetic methods produced useful quantities and similar isomeric distributions of these unusual and reactive compounds for biological studies and isotopic standards, and the potential conversion of nitro-linoleic to nitro-conjugated linoleic acids was explored via a facile base-catalyzed isomerization. This represents one of the few descriptions of naturally occurring conjugated nitro dienes (in particular, 1-nitro 1,3-diene), an unusual and highly reactive motif with few biological examples extant.

Digestive interaction between dietary nitrite and dairy products generates novel nitrated linolenic acid products.

Nitrated fatty acids are important anti-inflammatory and protective lipids formed in the gastric compartment, with conjugated linoleic acid (rumenic acid, RA, 9Z,11E-18:2) being the primary substrate for lipid nitration. The recently reported identification of nitrated rumelenic acid (NO2-RLA) in human urine has led to hypothesize that rumelenic acid (RLA, 9Z,11E,15Z-18:3) from dairy fat is responsible for the formation of NO2-RLA. To evaluate the source and mechanism of NO2-RLA formation, 15N labeled standards of NO2-RLA were synthesized and characterized. Afterward, milk fat with different RA and RLA levels was administered to mice in the presence of nitrite, and the appearance of nitrated fatty acids in plasma and urine followed. We confirmed the formation of NO2-RLA and defined the main metabolites in plasma, urine, and tissues. In conclusion, RLA obtained from dairy products is the main substrate for forming this novel electrophilic lipid reported to be present in human urine.

Development of nitroalkene-based inhibitors to target STING-dependent inflammation.

Stimulator of Interferon Genes (STING) is essential for the inflammatory response to cytosolic DNA. Despite that aberrant activation of STING is linked to an increasing number of inflammatory diseases, the development of inhibitors has been challenging, with no compounds in the pipeline beyond the preclinical stage. We previously identified endogenous nitrated fatty acids as novel reversible STING inhibitors. With the aim of improving the specificity and efficacy of these compounds, we developed and tested a library of nitroalkene-based compounds for in vitro and in vivo STING inhibition. The structure-activity relationship study revealed a robustly improved electrophilicity and reduced degrees of freedom of nitroalkenes by conjugation with an aromatic moiety. The lead compounds CP-36 and CP-45, featuring a ß-nitrostyrene moiety, potently inhibited STING activity in vitro and relieved STING-dependent inflammation in vivo. This validates the potential for nitroalkene compounds as drug candidates for STING modulation to treat STING-driven inflammatory diseases, providing new robust leads for preclinical development.

Characterization and quantification of endogenous fatty acid nitroalkene metabolites in human urine.

The oxidation and nitration of unsaturated fatty acids transforms cell membrane and lipoprotein constituents into mediators that regulate signal transduction. The formation of 9-NO2-octadeca-9,11-dienoic acid and 12-NO2-octadeca-9,11-dienoic acid stems from peroxynitrite- and myeloperoxidase-derived nitrogen dioxide reactions as well as secondary to nitrite disproportionation under the acidic conditions of digestion. Broad anti-inflammatory and tissue-protective responses are mediated by nitro-fatty acids. It is now shown that electrophilic fatty acid nitroalkenes are present in the urine of healthy human volunteers (9.9 ± 4.0 pmol/mg creatinine); along with electrophilic 16- and 14-carbon nitroalkenyl ß-oxidation metabolites. High resolution mass determinations and coelution with isotopically-labeled metabolites support renal excretion of cysteine-nitroalkene conjugates. These products of Michael addition are in equilibrium with the free nitroalkene pool in urine and are displaced by thiol reaction with mercury chloride. This reaction increases the level of free nitroalkene fraction >10-fold and displays a K(D) of 7.5 × 10(-6) M. In aggregate, the data indicates that formation of Michael adducts by electrophilic fatty acids is favored under biological conditions and that reversal of these addition reactions is critical for detecting both parent nitroalkenes and their metabolites. The measurement of this class of mediators can constitute a sensitive noninvasive index of metabolic and inflammatory status.

Conjugated linoleic acid is a preferential substrate for fatty acid nitration.

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.

CYB5R3 in type II alveolar epithelial cells protects against lung fibrosis by suppressing TGF-ß1 signaling.

Type II alveolar epithelial cell (AECII) redox imbalance contributes to the pathogenesis of idiopathic pulmonary fibrosis (IPF), a deadly disease with limited treatment options. Here, we show that expression of membrane-bound cytochrome B5 reductase 3 (CYB5R3), an enzyme critical for maintaining cellular redox homeostasis and soluble guanylate cyclase (sGC) heme iron redox state, is diminished in IPF AECIIs. Deficiency of CYB5R3 in AECIIs led to sustained activation of the pro-fibrotic factor TGF-ß1 and increased susceptibility to lung fibrosis. We further show that CYB5R3 is a critical regulator of ERK1/2 phosphorylation and the sGC/cGMP/protein kinase G axis that modulates activation of the TGF-ß1 signaling pathway. We demonstrate that sGC agonists (BAY 41-8543 and BAY 54-6544) are effective in reducing the pulmonary fibrotic outcomes of in vivo deficiency of CYB5R3 in AECIIs. Taken together, these results show that CYB5R3 in AECIIs is required to maintain resilience after lung injury and fibrosis and that therapeutic manipulation of the sGC redox state could provide a basis for treating fibrotic conditions in the lung and beyond.

Small molecule nitroalkenes inhibit RAD51-mediated homologous recombination and amplify triple-negative breast cancer cell killing by DNA-directed therapies.

Nitro fatty acids (NO 2 -FAs) are endogenously generated lipid signaling mediators from metabolic and inflammatory reactions between conjugated diene fatty acids and nitric oxide or nitrite-derived reactive species. NO 2 -FAs undergo reversible Michael addition with hyperreactive protein cysteine thiolates to induce posttranslational protein modifications that can impact protein function. Herein, we report a novel mechanism of action of natural and non-natural nitroalkenes structurally similar to ( E ) 10-nitro-octadec-9-enoic acid (CP-6), recently de-risked by preclinical Investigational New Drug-enabling studies and Phase 1 and Phase 2 clinical trials and found to induce DNA damage in a TNBC xenograft by inhibiting homologous-recombination (HR)-mediated repair of DNA double-strand breaks (DSB). CP-6 specifically targets Cys319, essential in RAD51-controlled HR-mediated DNA DSB repair in cells. A nitroalkene library screen identified two structurally different nitroalkenes, a non-natural fatty acid [( E ) 8-nitro- nonadec-7-enoic acid (CP-8)] and a dicarboxylate ester [dimethyl ( E )nitro-oct-4-enedioate (CP- 23)] superior to CP-6 in TNBC cells killing, synergism with three different inhibitors of the poly ADP-ribose polymerase (PARP) and γ-IR. CP-8 and CP-23 effectively inhibited γ-IR-induced RAD51 foci formation and HR in a GFP-reported assay but did not affect benign human epithelial cells or cell cycle phases. In vivo, CP-8 and CP-23's efficacies diverged as only CP-8 showed promising anticancer activities alone and combined with the PARP inhibitor talazoparib in an HR-proficient TNBC mouse model. As preliminary preclinical toxicology analysis also suggests CP-8 as safe, our data endorse CP-8 as a novel anticancer molecule for treating cancers sensitive to homologous recombination-mediated DNA repair inhibitors.

Small molecule nitroalkenes inhibit RAD51-mediated homologous recombination and amplify triple-negative breast cancer cell killing by DNA-directed therapies.

Nitro fatty acids (NO2-FAs) are endogenously generated lipid signaling mediators from metabolic and inflammatory reactions between conjugated diene fatty acids and nitric oxide or nitrite-derived reactive species. NO2-FAs undergo reversible Michael addition with hyperreactive protein cysteine thiolates to induce posttranslational protein modifications that can impact protein function. Herein, we report a novel mechanism of action of natural and non-natural nitroalkenes structurally similar to (E) 10-nitro-octadec-9-enoic acid (CP-6), recently de-risked by preclinical Investigational New Drug-enabling studies and Phase 1 and Phase 2 clinical trials and found to induce DNA damage in a TNBC xenograft by inhibiting homologous-recombination (HR)-mediated repair of DNA double-strand breaks (DSB). CP-6 specifically targets Cys319, essential in RAD51-controlled HR-mediated DNA DSB repair in cells. A nitroalkene library screen identified two structurally different nitroalkenes, a non-natural fatty acid [(E) 8-nitro-nonadec-7-enoic acid (CP-8)] and a dicarboxylate ester [dimethyl (E)nitro-oct-4-enedioate (CP-23)] superior to CP-6 in TNBC cells killing, synergism with three different inhibitors of the poly ADP-ribose polymerase (PARP) and γ-IR. CP-8 and CP-23 effectively inhibited γ-IR-induced RAD51 foci formation and HR in a GFP-reported assay but did not affect benign human epithelial cells or cell cycle phases. In vivo, CP-8 and CP-23's efficacies diverged as only CP-8 showed promising anticancer activities alone and combined with the PARP inhibitor talazoparib in an HR-proficient TNBC mouse model. As preliminary preclinical toxicology analysis also suggests CP-8 as safe, our data endorse CP-8 as a novel anticancer molecule for treating cancers sensitive to homologous recombination-mediated DNA repair inhibitors.

Protein-metabolite interactomics of carbohydrate metabolism reveal regulation of lactate dehydrogenase.

Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates, and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl-coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment.

Obese female mice do not exhibit overt hyperuricemia despite hepatic steatosis and impaired glucose tolerance.

Recent reports have clearly demonstrated a tight correlation between obesity and elevated circulating uric acid levels (hyperuricemia). However, nearly all preclinical work in this area has been completed with male mice, leaving the field with a considerable gap in knowledge regarding female responses to obesity and hyperuricemia. This deficiency in sex as a biological variable extends beyond unknowns regarding uric acid (UA) to several important comorbidities associated with obesity including nonalcoholic fatty liver disease (NAFLD). To attempt to address this issue, herein we describe both phenotypic and metabolic responses to diet-induced obesity (DIO) in female mice. Six-week-old female C57BL/6J mice were fed a high-fat diet (60% calories derived from fat) for 32 weeks. The DIO female mice had significant weight gain over the course of the study, higher fasting blood glucose, impaired glucose tolerance, and elevated plasma insulin levels compared to age-matched on normal chow. While these classic indices of DIO and NAFLD were observed such as increased circulating levels of ALT and AST, there was no difference in circulating UA levels. Obese female mice also demonstrated increased hepatic triglyceride (TG), cholesterol, and cholesteryl ester. In addition, several markers of hepatic inflammation were significantly increased. Also, alterations in the expression of redox-related enzymes were observed in obese mice compared to lean controls including increases in extracellular superoxide dismutase (Sod3), heme oxygenase (Ho)-1, and xanthine dehydrogenase (Xdh). Interestingly, hepatic UA levels were significantly elevated (~2-fold) in obese mice compared to their lean counterparts. These data demonstrate female mice assume a similar metabolic profile to that reported in several male models of obesity in the context of alterations in glucose tolerance, hepatic steatosis, and elevated transaminases (ALT and AST) in the absence of hyperuricemia affirming the need for further study.

Immunomodulatory actions of a kynurenine-derived endogenous electrophile.

The up-regulation of kynurenine metabolism induces immunomodulatory responses via incompletely understood mechanisms. We report that increases in cellular and systemic kynurenine levels yield the electrophilic derivative kynurenine-carboxyketoalkene (Kyn-CKA), as evidenced by the accumulation of thiol conjugates and saturated metabolites. Kyn-CKA induces NFE2 like bZIP transcription factor 2- and aryl hydrocarbon receptor-regulated genes and inhibits nuclear factor κB- and NLR family pyrin domain containing 3-dependent proinflammatory signaling. Sickle cell disease (SCD) is a hereditary hemolytic condition characterized by basal inflammation and recurrent vaso-occlusive crises. Both transgenic SCD mice and patients with SCD exhibit increased kynurenine and Kyn-CKA metabolite levels. Plasma hemin and kynurenine concentrations are positively correlated, indicating that Kyn-CKA synthesis in SCD is up-regulated during pathogenic vascular stress. Administration of Kyn-CKA abrogated pulmonary microvasculature occlusion in SCD mice, an important factor in lung injury development. These findings demonstrate that the up-regulation of kynurenine synthesis and its metabolism to Kyn-CKA is an adaptive response that attenuates inflammation and protects tissues.

Mass spectrometry-based study defines the human urine nitrolipidome.

Nitrated fatty acids (NO2-FA) are an endogenous class of signaling mediators formed mainly during digestion and inflammation. The signaling actions of NO2-FA have been extensively studied, but their detection and characterization lagged. Several different nitrated fatty acid species have been reported in animals and humans, but their formation remains controversial, and a systemic approach to define the endogenous pool of NO2-FA is needed. Herein, we screened for endogenous NO2-FA in urine from healthy human volunteers as this is the main excretion route for NO2-FA and its metabolites, and it provides an excellent matrix for evaluation. Only isomers of two fatty acids, conjugated linoleic and linolenic acid were found to be nitrated. Several, previously unknown, nitrated species were identified and confirmed using high-resolution mass spectrometry, fragmentation analysis, and compared to synthetic nitrated standards, the main group corresponding to nitrated conjugated linolenic acid (NO2-CLnA). In contrast, we were unable to confirm the presence of previously reported nitrated omega-3's, oleic acid, arachidonic acid and α- and γ-linolenic acid, suggesting that their biological formation and presence in humans should be re-evaluated. Metabolite analysis of NO2-CLnA in human urine identified cysteine adducts and ß-oxidation products, which were compared to the metabolic products of nitrated standards obtained using primary mouse hepatocytes. Importantly, NO2-CLnA isomers belong to two defined groups, are electrophilic, participate in Michael addition reactions and account for 39% of total urinary NO2-FA, highlighting their relative abundance and possible role in cell signaling.