Electrophilic fatty acid nitroalkenes are systemically transported and distributed upon esterification to complex lipids
2018; Elsevier BV; Volume: 60; Issue: 2 Linguagem: Inglês
10.1194/jlr.m088815
ISSN1539-7262
AutoresMarco Fazzari, Darío A. Vitturi, Steven R. Woodcock, Sonia R. Salvatore, Bruce Α. Freeman, Francisco J. Schöpfer,
Tópico(s)Sulfur Compounds in Biology
ResumoElectrophilic 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. 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. The knowledge of absorption, distribution, metabolism, and excretion (ADME) of new drug candidates is important for safe and insightful development. The rate of drug development failures attributable to ADME deficiencies exceeds 40% and, even after new drug approval, many drugs still display ADME problems. Notably, "a chemical cannot be a drug, no matter how active nor how specific its action, unless it is also taken appropriately into the body (absorption), distributed to the right parts of the body, metabolized in a way that does not instantly remove its activity, and eliminated in a suitable manner" (Ref. 1Hodgson J. ADMET–turning chemicals into drugs.Nat. Biotechnol. 2001; 19: 722-726Crossref PubMed Scopus (302) Google Scholar; p. 722). As a covalently acting endogenous mediator and a class of new drug candidates, electrophilic nitro-fatty acids [NO2-FAs (fatty acid nitroalkenes)] have displayed beneficial effects in preclinical animal models of metabolic and inflammatory disease (2Rudolph V. Rudolph T.K. Schopfer F.J. Bonacci G. Woodcock S.R. Cole M.P. Baker P.R. Ramani R. Freeman B.A. Endogenous generation and protective effects of nitro-fatty acids in a murine model of focal cardiac ischaemia and reperfusion.Cardiovasc. 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Nitro-fatty acids occur in human plasma in the picomolar range: a targeted nitro-lipidomics GC-MS/MS study.Lipids. 2009; 44: 855-865Crossref PubMed Scopus (66) Google Scholar), (E)-10-nitro-octadec-9-enoic acid [10-nitro-oleic acid (10-NO2-OA)], is now in phase 2 clinical trials for treating focal segmental glomerulosclerosis and will soon begin trials in pulmonary arterial hypertension and obese asthmatics. NO2-FAs are generated during inflammation and digestion by reactions between unsaturated fatty acids and the nitric oxide (•NO)- and nitrite (NO2−)-derived nitrating species, nitrogen dioxide (•NO2) (10Vitturi D.A. Minarrieta L. Salvatore S.R. Postlethwait E.M. Fazzari M. Ferrer-Sueta G. Lancaster Jr., J.R. Freeman B.A. Schopfer F.J. Convergence of biological nitration and nitrosation via symmetrical nitrous anhydride.Nat. Chem. Biol. 2015; 11: 504-510Crossref PubMed Scopus (53) Google Scholar). Endogenous formation of NO2-FAs can be promoted by: a) oxidative inflammatory reactions, such as inflammatory cell activation and myocardial ischemia/reperfusion (2Rudolph V. Rudolph T.K. Schopfer F.J. Bonacci G. Woodcock S.R. Cole M.P. Baker P.R. Ramani R. Freeman B.A. Endogenous generation and protective effects of nitro-fatty acids in a murine model of focal cardiac ischaemia and reperfusion.Cardiovasc. Res. 2010; 85: 155-166Crossref PubMed Scopus (153) Google Scholar, 5Villacorta L. Minarrieta L. Salvatore S.R. Khoo N.K. Rom O. Gao Z. Berman R.C. Jobbagy S. Li L. Woodcock S.R. et al.In situ generation, metabolism and immunomodulatory signaling actions of nitro-conjugated linoleic acid in a murine model of inflammation.Redox Biol. 2018; 15: 522-531Crossref PubMed Scopus (49) Google Scholar); and b) dietary supplementation of conjugated linoleic acid and NO2− or nitrate (NO3−) (11Delmastro-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. et al.Nitrite and nitrate-dependent generation of anti-inflammatory fatty acid nitroalkenes.Free Radic. Biol. Med. 2015; 89: 333-341Crossref PubMed Scopus (67) Google Scholar). In the absence of metabolic or inflammatory stimuli, plasma and urinary NO2-FAs are normally present at ∼1–3 and ∼10 nM concentrations in humans, respectively (9Tsikas D. Zoerner A.A. Mitschke A. Gutzki F.M. Nitro-fatty acids occur in human plasma in the picomolar range: a targeted nitro-lipidomics GC-MS/MS study.Lipids. 2009; 44: 855-865Crossref PubMed Scopus (66) Google Scholar, 11Delmastro-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. et al.Nitrite and nitrate-dependent generation of anti-inflammatory fatty acid nitroalkenes.Free Radic. Biol. Med. 2015; 89: 333-341Crossref PubMed Scopus (67) Google Scholar, 12Salvatore S.R. Vitturi D.A. Baker P.R.S. Bonacci G. Koenitzer J.R. Woodcock S.R. Freeman B.A. Schopfer F.J. Characterization and quantification of endogenous fatty acid nitroalkene metabolites in human urine.J. Lipid Res. 2013; 54: 1998-2009Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 13Bonacci G. Baker P.R. Salvatore S.R. Shores D. Khoo N.K. Koenitzer J.R. Vitturi D.A. Woodcock S.R. Golin-Bisello F. Cole M.P. et al.Conjugated linoleic acid is a preferential substrate for fatty acid nitration.J. Biol. Chem. 2012; 287: 44071-44082Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Administration of NO2-FAs has shown pharmacologic benefits in animal models of atrial fibrosis (3Rudolph T.K. Ravekes T. Klinke A. Friedrichs K. Mollenhauer M. Pekarova M. Ambrozova G. Martiskova H. Kaur J.J. Matthes B. et al.Nitrated fatty acids suppress angiotensin II-mediated fibrotic remodelling and atrial fibrillation.Cardiovasc. Res. 2016; 109: 174-184Crossref PubMed Scopus (31) Google Scholar), pulmonary hypertension (6Klinke A. Moller A. Pekarova M. Ravekes T. Friedrichs K. Berlin M. Scheu K.M. Kubala L. Kolarova H. Ambrozova G. et al.Protective effects of 10-nitro-oleic acid in a hypoxia-induced murine model of pulmonary hypertension.Am. J. Respir. Cell Mol. Biol. 2014; 51: 155-162Crossref PubMed Scopus (49) Google Scholar), inflammatory bowel disease (7Borniquel S. Jansson E.A. Cole M.P. Freeman B.A. Lundberg J.O. Nitrated oleic acid up-regulates PPARgamma and attenuates experimental inflammatory bowel disease.Free Radic. Biol. Med. 2010; 48: 499-505Crossref PubMed Scopus (73) Google Scholar), adriamycin-induced nephropathy (14Liu S. Jia Z. Zhou L. Liu Y. Ling H. Zhou S.F. Zhang A. Du Y. Guan G. Yang T. Nitro-oleic acid protects against adriamycin-induced nephropathy in mice.Am. J. Physiol. Renal Physiol. 2013; 305: F1533-F1541Crossref PubMed Scopus (42) Google Scholar), cardiac ischemia/reperfusion (2Rudolph V. Rudolph T.K. Schopfer F.J. Bonacci G. Woodcock S.R. Cole M.P. Baker P.R. Ramani R. Freeman B.A. Endogenous generation and protective effects of nitro-fatty acids in a murine model of focal cardiac ischaemia and reperfusion.Cardiovasc. Res. 2010; 85: 155-166Crossref PubMed Scopus (153) Google Scholar), cutaneous inflammation (4Mathers A.R. Carey C.D. Killeen M.E. Diaz-Perez J.A. Salvatore S.R. Schopfer F.J. Freeman B.A. Falo Jr., L.D. Electrophilic nitro-fatty acids suppress allergic contact dermatitis in mice.Allergy. 2017; 72: 656-664Crossref PubMed Scopus (14) Google Scholar), and abdominal wall defect (15D'Amore A. Fazzari M. Jiang H.B. Luketich S.K. Luketich M.E. Hoff R. Jacobs D.L. Gu X. Badylak S.F. Freeman B.A. et al.Nitro-oleic acid (NO2-OA) release enhances regional angiogenesis in a rat abdominal wall defect model.Tissue Eng. Part A. 2018; 24: 889-904Crossref PubMed Scopus (13) Google Scholar), among others. The signaling actions of NO2-FAs are attributed to the reversible Michael addition reaction between the electrophilic carbon β of the vinyl nitro substituent and protein cysteine thiolates, thereby modulating adaptive gene expression and enzymatic activities (16Schopfer F.J. Cipollina C. Freeman B.A. Formation and signaling actions of electrophilic lipids.Chem. Rev. 2011; 111: 5997-6021Crossref PubMed Scopus (242) Google Scholar). For example, NO2-FAs have been shown to activate Nrf2, PPARγ, and heat shock factor-1-dependent gene expression, and inhibit NF-kB-dependent pro-inflammatory gene expression and directly inhibit enzymes, such as soluble epoxide hydrolase and xanthine oxidoreductase (17Kansanen E. Bonacci G. Schopfer F.J. Kuosmanen S.M. Tong K.I. Leinonen H. Woodcock S.R. Yamamoto M. Carlberg C. Yla-Herttuala S. et al.Electrophilic nitro-fatty acids activate NRF2 by a KEAP1 cysteine 151-independent mechanism.J. Biol. 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Yla-Herttuala S. et al.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. 2009; 284: 33233-33241Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 21Khoo N.K.H. Li L. Salvatore S.R. Schopfer F.J. Freeman B.A. Electrophilic fatty acid nitroalkenes regulate Nrf2 and NF-kappaB signaling:A medicinal chemistry investigation of structure-function relationships.Sci. Rep. 2018; 8: 2295Crossref PubMed Scopus (33) Google Scholar, 22Kelley E.E. Batthyany C.I. Hundley N.J. Woodcock S.R. Bonacci G. Del Rio J.M. Schopfer F.J. Lancaster Jr., J.R. Freeman B.A. Tarpey M.M. Nitro-oleic acid, a novel and irreversible inhibitor of xanthine oxidoreductase.J. Biol. Chem. 2008; 283: 36176-36184Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Despite advances in understanding NO2-FA pharmacology and encouraging preclinical and clinical responses, a complete pharmacokinetic (PK) profile is still missing, due to challenges associated with the chemical reactivity and complex metabolic pathways of these mediators. For example, the quantitative analysis and characterization of NO2-FA metabolism are complicated by reversible reactions with low molecular weight and protein thiols (23Baker L.M. Baker P.R. Golin-Bisello F. Schopfer F.J. Fink M. Woodcock S.R. Branchaud B.P. Radi R. Freeman B.A. Nitro-fatty acid reaction with glutathione and cysteine. Kinetic analysis of thiol alkylation by a Michael addition reaction.J. Biol. Chem. 2007; 282: 31085-31093Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 24Batthyany C. Schopfer F.J. Baker P.R. Duran R. Baker L.M. Huang Y. Cervenansky C. Branchaud B.P. Freeman B.A. Reversible post-translational modification of proteins by nitrated fatty acids in vivo.J. Biol. Chem. 2006; 281: 20450-20463Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar), esterification into complex lipids (25Fazzari M. Khoo N. Woodcock S.R. Li L. Freeman B.A. Schopfer F.J. Generation and esterification of electrophilic fatty acid nitroalkenes in triacylglycerides.Free Radic. Biol. Med. 2015; 87: 113-124Crossref PubMed Scopus (26) Google Scholar, 26Melo T. Domingues P. Ferreira R. Milic I. Fedorova M. Santos S.M. Segundo M.A. Domingues M.R. Recent advances on mass spectrometry analysis of nitrated phospholipids.Anal. Chem. 2016; 88: 2622-2629Crossref PubMed Scopus (21) Google Scholar), and inherent instability of the nitroalkene under conditions of enzymatic and base hydrolysis, greatly limiting previous PK evaluations. Recent studies have shed new light on the ADME of NO2-FAs. Radiotracer analysis of orally administered [14C]10-NO2-OA in rats has shown a long-lasting accumulation of radioactivity in adipose tissue (27Salvatore S.R. Vitturi D.A. Fazzari M. Jorkasky D.K. Schopfer F.J. Evaluation of 10-nitro oleic acid bio-elimination in rats and humans.Sci. Rep. 2017; 7: 39900Crossref PubMed Scopus (22) Google Scholar, 28Fazzari M. Khoo N.K. Woodcock S.R. Jorkasky D.K. Li L. Schopfer F.J. Freeman B.A. Nitro-fatty acid pharmacokinetics in the adipose tissue compartment.J. Lipid Res. 2017; 58: 375-385Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). In addition, NO2-FA-containing triacylglycerides (NO2-FA-TAGs) have been characterized in adipocytes and rat plasma after 10-NO2-OA administration (25Fazzari M. Khoo N. Woodcock S.R. Li L. Freeman B.A. Schopfer F.J. Generation and esterification of electrophilic fatty acid nitroalkenes in triacylglycerides.Free Radic. Biol. Med. 2015; 87: 113-124Crossref PubMed Scopus (26) Google Scholar). A better understanding of NO2-FA distribution and metabolism in supplemented adipocytes and rat adipose tissue was provided by the quantification of both the free acid and esterified 10-NO2-OA (28Fazzari M. Khoo N.K. Woodcock S.R. Jorkasky D.K. Li L. Schopfer F.J. Freeman B.A. Nitro-fatty acid pharmacokinetics in the adipose tissue compartment.J. Lipid Res. 2017; 58: 375-385Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). These studies confirmed that adipose tissue differentially stores electrophilic NO2-FAs and their nonelectrophilic metabolites into complex lipids, a pool that is mobilized by lipase hydrolysis to be distributed to remote organs. Dietary fatty acids are normally absorbed by intestinal enterocytes, esterified into TAGs, packaged into chylomicrons, and secreted into the mesenteric lymph duct, to then reach the systemic circulation and the peripheral tissues (29Iqbal J. Hussain M.M. Intestinal lipid absorption.Am. J. Physiol. Endocrinol. Metab. 2009; 296: E1183-E1194Crossref PubMed Scopus (538) Google Scholar). The incorporation of lipophilic drugs into chylomicron TAGs and their lymphatic distribution protects from first-pass hepatic metabolism and can increase oral bioavailability (30Trevaskis N.L. Kaminskas L.M. Porter C.J. From sewer to saviour - targeting the lymphatic system to promote drug exposure and activity.Nat. Rev. Drug Discov. 2015; 14: 781-803Crossref PubMed Scopus (388) Google Scholar). In this regard, a clear understanding of the systemic distribution of NO2-FAs is lacking and relative plasma free versus esterified NO2-FA levels after oral supplementation have yet to be defined. Herein, we provide new insight into the PK of an exemplary lipid electrophile, 10-NO2-OA. We reveal that the systemic distribution and targeted delivery of orally administered NO2-FA occur as complex lipid esterified species, following esterification and stabilization in triglycerides (TAGs). Synthesis and spectrophotometric quantitation of (E)-10-nitro-octadec-9-enoic acid (10-NO2-OA), (Z)-10-nitro-octadec-9-enoic acid [(Z)-10-NO2-OA], and the internal standard, (E)-10-nitro[15N]octadec-9-enoic-15,15,16,16-[d4] acid ([15N]O2-[d4]OA), were performed as previously (27Salvatore S.R. Vitturi D.A. Fazzari M. Jorkasky D.K. Schopfer F.J. Evaluation of 10-nitro oleic acid bio-elimination in rats and humans.Sci. Rep. 2017; 7: 39900Crossref PubMed Scopus (22) Google Scholar, 31Woodcock S.R. Bonacci G. Gelhaus S.L. Schopfer F.J. Nitrated fatty acids: synthesis and measurement.Free Radic. Biol. Med. 2013; 59: 14-26Crossref PubMed Scopus (49) Google Scholar, 32Woodcock S.R. Marwitz A.J. Bruno P. Branchaud B.P. Synthesis of nitrolipids. All four possible diastereomers of nitrooleic acids: (E)- and (Z)-, 9- and 10-nitro-octadec-9-enoic acids.Org. Lett. 2006; 8: 3931-3934Crossref PubMed Scopus (48) Google Scholar). Synthesis of the labeled internal standard, (E)-10-nitro[15N]octadecanoic-15,15,16,16-[d4] acid ([15N]O2-[d4]SA) was performed by reducing [15N]O2-[d4]OA with sodium borohydride. Recombinant human FLAG-tagged prostaglandin reductase-1 (PtGR-1) was expressed in HEK-293T cells and purified as described previously (33Vitturi D.A. Chen C.S. Woodcock S.R. Salvatore S.R. Bonacci G. Koenitzer J.R. Stewart N.A. Wakabayashi N. Kensler T.W. Freeman B.A. et al.Modulation of nitro-fatty acid signaling: prostaglandin reductase-1 is a nitroalkene reductase.J. Biol. Chem. 2013; 288: 25626-25637Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated. Solvents used for extractions and mass spectrometric analyses were from Burdick and Jackson (Muskegon, MI). Male beagle dogs (7 months old, n = 5 per group) were orally administered with 31.25 mg/kg 10-NO2-OA (dissolved in sesame oil) twice a day, about 6 h apart, or with vehicle (sesame oil) for 14 days. Blood samples were collected at 0, 1, 4, 7, 10, 16, and 24 h at 1 and 14 days, centrifuged, and the resultant plasma was stored at −80°C until used. Animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (National Institutes of Health Publication No.85-23, revised 1996). Synthesis of 10-nitro-octadec-trans-8-enoic acid [10-NO2-(t8,9)-18:1] was carried out by charging a 25 ml round-bottom flask with 9-acetoxy-10-nitrooctadecanoic ester (193 mg) (32Woodcock S.R. Marwitz A.J. Bruno P. Branchaud B.P. Synthesis of nitrolipids. All four possible diastereomers of nitrooleic acids: (E)- and (Z)-, 9- and 10-nitro-octadec-9-enoic acids.Org. Lett. 2006; 8: 3931-3934Crossref PubMed Scopus (48) Google Scholar), cesium carbonate (73 mg, one-half equivalent), and benzene (10–15 ml). The solution was refluxed under nitrogen with vigorous stirring under a Dean-Stark trap for azeotropic removal of water for 24 h at 80–90°C. After completion, the solution was cooled to room temperature, partitioned with 5–10 ml of 1 M hydrochloric acid and 10 ml of diethyl ether. After stirring, the mixture was separated and the aqueous fraction extracted three times with 10 ml aliquots of diethyl ether. The organic layers were combined and washed once with water and once with saturated aqueous sodium chloride, and then dried over sodium sulfate. The solution was filtered through a plug of silica gel/Celite and the solvent removed under reduced pressure. The crude product was redissolved in 5 ml phosphate buffer/5 ml methanol with 393 mg l-cysteine (10-fold excess) and stirred for 2 h to remove nitroalkene-cysteine adducts, then quenched with a few drops of acetic acid and extracted three times with ethyl acetate. The organic layers were combined and washed twice with water and twice with saturated aqueous sodium chloride, and dried over sodium sulfate. The final solution was evaporated under reduced pressure and then applied to a flash chromatography column (ethyl acetate-hexanes 0–5%) to yield 96 mg of 10-NO2-(t8,9)-18:1 after de-esterification by standard techniques. Characteristic 1H-NMR signals were observed at δ 4.78 (q, J = 7.7 Hz), 5.54 (dd, J = 15.3, 9.0 Hz), and 5.77 (dt, J = 15.3, 6.7 Hz) ppm. Synthesis of NO2-FA-TAGs was performed by esterification of the appropriate NO2-FA with 1,2-dipalmitin. In a typical reaction, 66 mg of 10-NO2-OA (0.2 mmol) were charged to a vial along with 50 mg N,N′-dicyclohexylcarbodiimide (0.24 mmol) and a catalytic amount (5 mg) of 4-dimethylaminopyridine, and then dissolved in dichloromethane. The solution was stirred briefly before 114 mg of 1,2-dipalmitin (0.2 mmol) were added and the solvent volume adjusted to 10 ml. The vial was sealed under nitrogen and stirred overnight at room temperature. The final TAG was obtained by filtering off the solids, removing the solvent by rotary evaporation, and purifying the crude product via column chromatography (82 mg, 48% yield). Products were analyzed by 1H-NMR and HPLC-high-resolution (HR)-MS/MS for structural confirmation as previously (25Fazzari M. Khoo N. Woodcock S.R. Li L. Freeman B.A. Schopfer F.J. Generation and esterification of electrophilic fatty acid nitroalkenes in triacylglycerides.Free Radic. Biol. Med. 2015; 87: 113-124Crossref PubMed Scopus (26) Google Scholar). Analysis of nonesterified 10-NO2-OA and metabolites was performed by adding 250 μl of acetonitrile to 50 μl of plasma in the presence of 20 pmol of [15N]O2-[d4]OA and [15N]O2-[d4]SA. Samples were vortexed, centrifuged at 20,000 g for 10 min at 4°C, and the supernatant was analyzed. TAGs were extracted from 20 μl of plasma using the Bligh and Dyer method (34Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42828) Google Scholar), dried under a stream of nitrogen, and reconstituted in 100 μl of ethyl acetate. To confirm the electrophilic character of NO2-OA-derived metabolites, acetonitrile-precipitated plasma samples (50 μl) were dried and reacted with β-mercaptoethanol (BME) by adding 100 μl of phosphate buffer (50 mM, pH 7.4)/ acetonitrile (9:1, v/v) in the presence of 500 mM of BME followed by a 90 min incubation at 37°C. Also, 10-NO2-OA, 10-NO2-(t8,9)-18:1, (Z)-10-NO2-OA, and 10-nitro-stearic acid (10-NO2-SA) standards (20 pmol) were spiked with a 10 pmol [15N]O2-[d4]OA and [15N]O2-[d4]SA mix of internal standards, and reaction with BME was performed as above, but with an incubation time of 15 min. Samples were analyzed by HPLC-ESI-MS/MS. Plasma (20 μl) was spiked with 10 pmol of [15N]O2-[d4]OA and [15N]O2-[d4]SA, and acid hydrolysis was performed with minor modifications as previously (28Fazzari M. Khoo N.K. Woodcock S.R. Jorkasky D.K. Li L. Schopfer F.J. Freeman B.A. Nitro-fatty acid pharmacokinetics in the adipose tissue compartment.J. Lipid Res. 2017; 58: 375-385Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Samples were incubated with 1 ml of acetonitrile/HCl (9:1, v/v) at room temperature to obtain the free acid levels of NO2-FAs (before hydrolysis condition) and 90°C for 1 h for the total levels of NO2-FAs (after hydrolysis condition). After incubation, 1 ml of 0.5 M phosphate buffer was added and extracted with 2 ml of hexane. Then, samples were vortexed, centrifuged at 1,000 g for 5 min at 4°C, and the hexane phase recovered and dried under a stream of nitrogen. Finally, NO2-FAs were reconstituted in methanol for HPLC-MS/MS analysis. The esterified levels of NO2-FAs were obtained by subtracting the free acid levels (acetonitrile extracts, before hydrolysis) from the total levels (after hydrolysis condition). Stability and recovery of synthetic 10-NO2-OA, 10-NO2-SA, 10-NO2-(t8,9)-18:1, (Z)-10-NO2-OA, 10-NO2-OA-containing TAG (10-NO2-OA-TAG), and 10-NO2-SA-containing TAG (10-NO2-SA-TAG) standards (30 pmol) were assessed in the presence of 0.5 mg of triolein under the same acidic hydrolysis conditions as above, with the only difference that the internal standard mixture was added before the extraction with hexane. The relative levels before and after hydrolysis conditions were reported as area ratio with the internal standards [15N]O2-[d4]OA and [15N]O2-[d4]SA, respectively (supplemental Fig. S3A–E). The extent of hydrolysis of the NO2-FA-containing TAG standards (10-NO2-OA-TAG and 10-NO2-SA-TAG) was further confirmed by TLC and iodine staining. The concentrations of (E)-10-NO2-OA and (Z)-10-NO2-OA were determined by UV-Vis spectrometry by using the same extinction coefficient of 7.5 mM−1cm−1 at λ = 257 nm in methanol. The concentration of 10-NO2-(t8,9)-18:1 was adjusted by LC-MS/MS analysis using (E)-10-NO2-OA and (Z)-10-NO2-OA standards as reference values. The NADPH concentration in phosphate buffer was calculated using an extinction coefficient (ε340) of 6.22 mM−1cm−1. Then, (E)-10-NO2-OA, (Z)-10-NO2-OA, and 10-NO2-(t8,9)-18:1 (500 nM) were incubated at room temperature with 30 ng of PtGR-1 with 250 μM of NADPH in 10 mM of sodium phosphate buffer (pH 7.0) supplemented with 100 μM of DTPA and 100 nM of internal standard [15N]O2-[d4]SA. Aliquots were collected at 0, 1, 5, 15, 30, and 60 min, and reactions were stopped by 10-fold dilution in methanol, and 10-NO2-SA formation and 10-NO2-OA isomer consumption were quantified by HPLC-ESI-MS/MS. Lipids were extracted from 100 μl of plasma using the Bligh and Dyer method, and the chloroform phase was dried under a stream of nitrogen and dissolved in 0.5 ml of hexane/methyl tert-butyl ether/acetic acid (100:3:0.3 v/v/v). Lipid classes were chromatographically separated using solid phase extraction Strata NH2 columns (100 mg/1 ml), preconditioned with 2 ml of acetone/water (7:1, v/v) and equilibrated with 2 ml of hexane. The lipid extracts solubilized in hexane/methyl tert-butyl ether/acetic acid were loaded on the columns and cholesterol ester (CE), TAG, monoglyceride+diglyceride (MAG+DAG), FFA, and phospholipid (PL) fractions were sequentially eluted with 1 ml of hexane, hexane/chloroform/ethyl acetate (100:5:5, v/v/v), chloroform/2-propanol (2:1, v/v), diethyl ether/2% acetic acid, and methanol, respectively. For analysis, the TAG and MAG+DAG fractions were pooled together because some NO2-FA-TAG eluted in the latter fraction. The lipid fractions were dried under a flow of N2, dissolved in 1.8 ml of acetonitrile spiked with a 40 pmol mix of labeled [15N]O2-[d4]OA and [15N]O2-[d4]SA, and vortexed. Then, 0.9
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