Nitro-fatty acid pharmacokinetics in the adipose tissue compartment
2016; Elsevier BV; Volume: 58; Issue: 2 Linguagem: Inglês
10.1194/jlr.m072058
ISSN1539-7262
AutoresMarco Fazzari, N. Khoo, Steven R. Woodcock, Diane K. Jorkasky, Lihua Li, Francisco J. Schöpfer, Bruce Α. Freeman,
Tópico(s)Metabolomics and Mass Spectrometry Studies
ResumoElectrophilic nitro-FAs (NO2-FAs) promote adaptive and anti-inflammatory cell signaling responses as a result of an electrophilic character that supports posttranslational protein modifications. A unique pharmacokinetic profile is expected for NO2-FAs because of an ability to undergo reversible reactions including Michael addition with cysteine-containing proteins and esterification into complex lipids. Herein, we report via quantitative whole-body autoradiography analysis of rats gavaged with radiolabeled 10-nitro-[14C]oleic acid, preferential accumulation in adipose tissue over 2 weeks. To better define the metabolism and incorporation of NO2-FAs and their metabolites in adipose tissue lipids, adipocyte cultures were supplemented with 10-nitro-oleic acid (10-NO2-OA), nitro-stearic acid, nitro-conjugated linoleic acid, and nitro-linolenic acid. Then, quantitative HPLC-MS/MS analysis was performed on adipocyte neutral and polar lipid fractions, both before and after acid hydrolysis of esterified FAs. NO2-FAs preferentially incorporated in monoacyl- and diacylglycerides, while reduced metabolites were highly enriched in triacylglycerides. This differential distribution profile was confirmed in vivo in the adipose tissue of NO2-OA-treated mice. This pattern of NO2-FA deposition lends new insight into the unique pharmacokinetics and pharmacologic actions that could be expected for this chemically-reactive class of endogenous signaling mediators and synthetic drug candidates. Electrophilic nitro-FAs (NO2-FAs) promote adaptive and anti-inflammatory cell signaling responses as a result of an electrophilic character that supports posttranslational protein modifications. A unique pharmacokinetic profile is expected for NO2-FAs because of an ability to undergo reversible reactions including Michael addition with cysteine-containing proteins and esterification into complex lipids. Herein, we report via quantitative whole-body autoradiography analysis of rats gavaged with radiolabeled 10-nitro-[14C]oleic acid, preferential accumulation in adipose tissue over 2 weeks. To better define the metabolism and incorporation of NO2-FAs and their metabolites in adipose tissue lipids, adipocyte cultures were supplemented with 10-nitro-oleic acid (10-NO2-OA), nitro-stearic acid, nitro-conjugated linoleic acid, and nitro-linolenic acid. Then, quantitative HPLC-MS/MS analysis was performed on adipocyte neutral and polar lipid fractions, both before and after acid hydrolysis of esterified FAs. NO2-FAs preferentially incorporated in monoacyl- and diacylglycerides, while reduced metabolites were highly enriched in triacylglycerides. This differential distribution profile was confirmed in vivo in the adipose tissue of NO2-OA-treated mice. This pattern of NO2-FA deposition lends new insight into the unique pharmacokinetics and pharmacologic actions that could be expected for this chemically-reactive class of endogenous signaling mediators and synthetic drug candidates. Unsaturated FAs of cellular membranes, lipoproteins, and dietary fats are susceptible to oxidation and nitration by reactive oxygen species and nitrogen oxides during inflammatory and metabolic stress (1.Guillén M.D. Goicoechea E. Toxic oxygenated alpha,beta-unsaturated aldehydes and their study in foods: a review.Crit. Rev. Food Sci. Nutr. 2008; 48: 119-136Crossref PubMed Scopus (149) Google Scholar, 2.O'Donnell V.B. Eiserich J.P. Chumley P.H. Jablonsky M.J. Krishna N.R. Kirk M. Barnes S. Darley-Usmar V.M. 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Chem. 2014; 79: 25-33Crossref PubMed Scopus (21) Google Scholar). For example, NO2-FAs are detected at micromolar concentrations in rodent cardiac mitochondria subjected to an episode of ischemia-reperfusion and at nanomolar concentrations in healthy human plasma and urine (12.Rudolph 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 (151) Google Scholar, 13.Lima E.S. Di Mascio P. Rubbo H. Abdalla D.S. Characterization of linoleic acid nitration in human blood plasma by mass spectrometry.Biochemistry. 2002; 41: 10717-10722Crossref PubMed Scopus (91) Google Scholar, 14.Salvatore 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). Additionally, NO2-FAs have been detected in Arabidopsis, fresh olives, and extra virgin olive oil (15.Mata-Pérez C. Sánchez-Calvo B. Padilla M.N. Begara-Morales J.C. Luque F. Melguizo M. Jiménez-Ruiz J. Fierro-Risco J. Peñas-Sanjuán A. Valderrama R. et al.Nitro-fatty acids in plant signaling: nitro-linolenic acid induces the molecular chaperone network in Arabidopsis.Plant Physiol. 2016; 170: 686-701Crossref PubMed Scopus (79) Google Scholar, 16.Fazzari M. Trostchansky A. Schopfer F.J. Salvatore S.R. Sanchez-Calvo B. Vitturi D. Valderrama R. Barroso J.B. Radi R. Freeman B.A. et al.Olives and olive oil are sources of electrophilic fatty acid nitroalkenes.PLoS One. 2014; 9: e84884Crossref PubMed Scopus (79) Google Scholar). Notably, plasma and urinary NO2-FA concentrations in humans are increased following oral supplementation with NO2−, nitrate (NO3−), and conjugated linoleic acid (CLA) (17.Delmastro-Greenwood M. Hughan K.S. Vitturi D.A. Salvatore S.R. Grimes G. Potti G. Shiva S. Schopfer F.J. Gladwin M.T. Freeman B.A. 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, 18.Bonacci G. Schopfer F. Salvatore S. Rudolph T.K. Rudolph V. Khoo K. Koenitzer J. Golin-Bisello F. Baker P.R. Shores D. et al.Conjugated linoleic acid a preferential substrate for fatty acid nitration.J. Biol. Chem. 2012; 287: 44071-44082Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Recently, NO2-FA-containing phospholipids have been identified in cardiac mitochondria isolated from an animal model of type 1 diabetes (10.Melo 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 NO2-FA-containing triacylglycerides (TAGs) have been detected in nitro-oleic acid (NO2-OA)-supplemented adipocytes and plasma of rats after gavage with NO2-OA (3.Fazzari 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). The analytical advances permitting the detection of FA nitro-alkene derivatives in complex lipids provide new opportunities for better understanding NO2-FA pharmacokinetics, metabolism, and potential toxicology. To date, the identification and quantitation of NO2-FAs in complex lipids has been limited by: 1) the instability of NO2-FAs during enzymatic and basic hydrolysis; 2) the diversity of potential structures wherein NO2-FAs can be incorporated (e.g., sterols, phospholipids, glycolipids, glycerolipids); 3) challenges of the analysis NO2-FA-containing complex lipids and their relative low abundance in cells and tissues; and 4) the lack of synthetic standards (19.Manini P. Capelli L. Reale S. Arzillo M. Crescenzi O. Napolitano A. Barone V. d'Ischia M. Chemistry of nitrated lipids: remarkable instability of 9-nitrolinoleic acid in neutral aqueous medium and a novel nitronitrate ester product by concurrent autoxidation/nitric oxide-release pathways.J. Org. Chem. 2008; 73: 7517-7525Crossref PubMed Scopus (22) Google Scholar, 20.Schopfer F.J. Baker P.R. Giles G. Chumley P. Batthyany C. Crawford J. Patel R.P. Hogg N. Branchaud B.P. Lancaster Jr., J.R. et al.Fatty acid transduction of nitric oxide signaling. Nitrolinoleic acid is a hydrophobically stabilized nitric oxide donor.J. Biol. Chem. 2005; 280: 19289-19297Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Herein, we show the preferential distribution of orally administered 10-nitro-[14C]oleic acid radiolabeled at carbon 10 (10-NO2-[14C]OA) in adipose tissue of rats over a 2 week period via quantitative whole-body autoradiography (QWBA). Then, after lipid class fractionation, we report the quantitative analysis of the differential incorporation of NO2-FA and metabolites into cultured adipocytes before and after acid hydrolysis, using HPLC-MS/MS. We observed the preferential incorporation of electrophilic versus nonelectrophilic NO2-FAs in adipocyte mono- and di-acylglycerides (MAG+DAGs), a phenomenon confirmed in adipose tissue obtained from NO2-OA-treated mice. These findings reveal tissue-specific pharmacokinetics and the preferential role of adipose tissue in distribution and metabolism of NO2-FAs in vivo. Oleic acid (OA), CLA (catalog number UC-60A), and α-linolenic acid were from Nu-Check Prep, Inc. (Elysian, MN). The [13C18]labeled OA was obtained from Spectra Stable Isotopes (Columbia, MD). The [15N]labeled sodium nitrate was purchased from Cambridge Isotope Laboratories (Tewksbury, MA). Deuterium-labeled 1-bromononane-6,6,7,7-d4 was obtained from CDN Isotopes (Quebec, Canada). The 10-NO2-[14C]OA (labeled at carbon 10) was synthesized by ABC Laboratories, Inc. (Columbia, MO). Nitro-linolenic acid (NO2-LnA), nonspecific 9- and 10-nitro-octadec-9-enoic acid (NO2-OA), and isotopically labeled analog nitro-[13C18]octadec-9-enoic acid (NO2-[13C18]OA) were synthesized by direct alkene nitroselenation of the corresponding native FAs, as previously described (21.Woodcock 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 (48) Google Scholar). Nitro-CLA (NO2-CLA) was synthesized according to a previously described method for conjugated diene nitration (11.Woodcock S.R. Salvatore S.R. Bonacci G. Schopfer F.J. Freeman B.A. Biomimetic nitration of conjugated linoleic acid: formation and characterization of naturally occurring conjugated nitrodienes.J. Org. Chem. 2014; 79: 25-33Crossref PubMed Scopus (21) Google Scholar). The [15N]nitro-[D4]octadecenoic acid (NO2-[15N/D4]OA) was synthesized from the corresponding isotopically labeled precursor, 1-[15N]nitrononane-6,6,7,7-d4 (obtained by nucleophilic substitution of [15N]labeled sodium nitrate and 1-bromononane-6,6,7,7-d4) and a nine-carbon aldehyde-ester via a previously described nitroaldol procedure (21.Woodcock 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 (48) Google Scholar, 22.Woodcock 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). Nitro-stearic acid (NO2-SA) and its isotopically labeled analog, [15N]nitro-[D4]octadecanoic acid (NO2-[15N/D4]SA), were synthesized by selective reduction with sodium borohydride of the nitro-alkene double bond in NO2-OA and NO2-[15N/D4]OA, respectively. Nitro-alkene standards of various lengths (C16, C15, C14, C13, and C12) were synthesized by nitroselenation (21.Woodcock 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 (48) Google Scholar) of the corresponding native FAs and used as calibrators to normalize for MS responses. Before each experiment, NO2-OA, NO2-CLA, and NO2-LnA concentrations were measured spectrophotometrically in methanol using the following extinction coefficients: ε312 = 11,200 M−1cm−1 for NO2-CLA, ε257 = 7,000 M−1cm−1 for NO2-OA , and ε305 = 7,000 M−1cm−1 for NO2-LnA (21.Woodcock 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 (48) Google Scholar). DMEM, FBS, HBSS, and antibiotic-antimycotic solutions were from Corning Cellgro (Herndon, VA). Strata NH2 solid phase extraction columns (55 μm, 70 A) were from Phenomenex (Torrance, CA). Solvents were LC-MS grade from Burdick and Jackson (Muskegon, MI). Chemicals were of analytical grade and purchased from Sigma (St. Louis, MO) unless otherwise stated. The 3T3-L1 preadipocytes were maintained and differentiated into adipocytes as previously (23.Khoo N.K. Mo L. Zharikov S. Kamga-Pride C. Quesnelle K. Golin-Bisello F. Li L. Wang Y. Shiva S. Nitrite augments glucose uptake in adipocytes through the protein kinase A-dependent stimulation of mitochondrial fusion.Free Radic. Biol. Med. 2014; 70: 45-53Crossref PubMed Scopus (19) Google Scholar). Fully differentiated adipocytes were then treated with 100 μM CLA or 5 μM OA, NO2-SA, 10-nitro-oleic acid (10-NO2-OA), NO2-CLA, NO2-LnA in HBSS. Aliquots of cellular media were obtained at 1, 2, 4, 8, and 24 h, spiked with 20 pmol NO2-[13C18]OA and NO2-[15N/D4]SA as internal standards, extracted using the Bligh and Dyer method (24.Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42694) Google Scholar), dried under a stream of nitrogen and reconstituted in 0.2 ml methanol for HPLC-MS/MS analysis. At the end of the treatment, adipocytes were rinsed with cold PBS, scraped, and lipids extracted. The 10-NO2-[14C]OA (labeled at carbon 10) was administered by a single oral gavage as a solution in sesame oil to male Sprague-Dawley rats (8–10 weeks old, n = 8) at a dose level of 30 mg/4 MBq/2 ml sesame oil per kilogram body weight. Rats were euthanized at 1, 6, 24, 48, 72, 120, 168, and 336 h after dose administration and QWBA was then carried out on the carcass of n = 1 animal for each time point. A frozen carcass was set in a block of 2% (w/v) aqueous carboxymethyl cellulose at −80°C. Samples of whole blood reference standards containing six different concentrations of radioactivity were placed into holes drilled into the block to facilitate signal calibration. The block was mounted onto the stage of a microtome in a cryostat maintained at −20°C. Sagittal sections (∼30 μm) were obtained at six different levels through the carcass of each animal: 1) kidney; 2) intra-orbital lacrimal gland; 3) harderian gland; 4) adrenal gland; 5) half brain and thyroid; and 6) brain and spinal cord. The sections, mounted on sectioning tape, were freeze-dried using a Lyolab B freeze-drier. One section from each level was exposed to imaging plates and an adjacent freeze-dried section at each level was mounted and used for reference purposes when evaluating the images. After exposure in a refrigerated lead-lined exposure box for 3 days, imaging plates were scanned using a FLA5000 radioluminography system. The electronic images were analyzed using an image analysis package (Seescan Densitometry software, version 2.0). The limits of quantification for the procedure corresponded to the lowest and highest calibration standards (0.12 to 528 μg equivalents of 10-NO2-OA per gram). Wherever possible, the maximum area for each tissue within a single autoradiogram was defined for measurement. These radiolabeling experiments were conducted at Huntingdon Life Sciences (Cambridgeshire, UK) with approval by the Huntingdon Life Sciences Ethical Review Process Committee. Plasma and blood cell radioactivity was measured by liquid scintillation analysis using Wallac 1409 automatic liquid scintillation counters. Radioactivity in amounts less than twice that of the background concentration in the samples was considered to be below the limit of accurate quantification (BLQ). All murine studies were conducted with the approval of the University of Pittsburgh Institutional Animal Care and Use Committee (25.Kelley E.E. Baust J. Bonacci G. Golin-Bisello F. Devlin J.E. St Croix C.M. Watkins S.C. Gor S. Cantu-Medellin N. Weidert E.R. et al.Fatty acid nitroalkenes ameliorate glucose intolerance and pulmonary hypertension in high-fat diet-induced obesity.Cardiovasc. Res. 2014; 101: 352-363Crossref PubMed Scopus (63) Google Scholar). In brief, 6–8 week old male C57Bl/6j mice were subjected to high-fat diet (HFD) purchased from Research Diets Inc. (D12492; New Brunswick, NJ) for 20 weeks. Age-matched controls were maintained on a standard rodent chow diet (Pro Lab RHM 3000 rodent diet; PMI Feeds, Inc., St. Louis, MO). Mice were fed ad libitum for 20 weeks and given free access to water. At week 13.5 of the HFD study, mice were anesthetized with isoflurane before Alzet osmotic pumps (Cupertino, CA) containing vehicle (polyethylene glycol/ethanol, 92:8) or 9- and 10-NO2-OA were implanted subcutaneously in the back region. The osmotic mini pump was set to deliver 8 mg NO2-OA /kg/day. At the end of the 20th week, mice were euthanized and epididymal fat pads were quickly removed (n = 9 per treatment group), snap-frozen, and stored at −80°C. Sections of adipose tissues (∼100 mg) were homogenized in a bullet blender for 5 min in 0.8 ml phosphate buffer 50 mM pH 7.4, and lipids were extracted. Adipocyte and adipose tissue lipids were extracted according to Bligh and Dyer (24.Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42694) Google Scholar), dried under a stream of nitrogen, and dissolved in 0.5 ml hexane/methyl tert-butyl ether/acetic acid (100:3:0.3 v/v/v). Lipid classes were further resolved chromatographically using solid phase extraction Strata NH2 columns loaded with 500 mg lipid per 6 ml bed volume (26.Zou W. Separation of lipids by solid phase extraction (SPE)..http://www.nature.com/protocolexchange/protocols/2156Date: 2011Google Scholar, 27.Pietsch A. Lorenz R.L. Rapid separation of the major phospholipid classes on a single aminopropyl cartridge.Lipids. 1993; 28: 945-947Crossref PubMed Scopus (57) Google Scholar). Columns were preconditioned by washing twice with 2 ml acetone/water (7:1, v/v) and twice with 2 ml hexane. The samples solubilized in hexane/methyl tert-butyl ether/acetic acid were loaded on the columns and cholesterol esters (CEs), TAGs, MAG+DAGs, FFAs, phosphatidylcholines (PCs), phosphatidylethanolamines, phosphatidylserines, and phosphatidylinositols were sequentially eluted with 12 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, acetonitrile/1-propanol (2:1, v/v), methanol, isopropanol/methanolic HCl (4:1, v/v), and methanol/methanolic HCl (9:1, v/v), respectively. The solvents were evaporated under a stream of nitrogen and then CEs, TAGs, and MAG+DAGs were dissolved in 0.2 ml ethyl acetate, while FFAs and phospholipid fractions were solvated in 0.2 ml methanol. Direct injection MS analysis of each fraction confirmed lipid class abundance and composition. Fractionated lipid classes were hydrolyzed by a modification of a previously reported protocol (28.Aveldaño M.I. Horrocks L.A. Quantitative release of fatty acids from lipids by a simple hydrolysis procedure.J. Lipid Res. 1983; 24: 1101-1105Abstract Full Text PDF PubMed Google Scholar). Fractions (10 μl) were spiked with 20 pmol NO2-[13C18]OA/NO2-[15N/D4]SA mix internal standards. To limit artifactual acid-catalyzed nitration reactions, 50 μl methanolic sulfanilamide (1 g/10 ml) was added to scavenge adventitious NO2−. Then, the mixture was dried and incubated with 500 μl acetonitrile/HCl (9:1, v/v) at 100°C for 1 h. To assess whether NO2-FAs were present before hydrolysis, samples were also incubated with 500 μl acetonitrile/water (9:1, v/v). Then, 95 μl water or ammonium hydroxide was added to samples before and after hydrolysis, respectively. Samples were vortexed, centrifuged at 18,000 g for 10 min at 4°C, and NO2-FAs analyzed by HPLC-MS/MS. The full hydrolysis of TAG and phospholipid standards was assessed by TLC and iodine staining. Analysis of NO2-FAs was performed by HPLC-MS/MS using an analytical C18 Luna column (2 × 100 mm, 5 μm; Phenomenex) at a 0.6 ml/min flow rate, with a gradient solvent system consisting of water containing 0.1% acetic acid (solvent A) and acetonitrile containing 0.1% acetic acid (solvent B). Samples were chromatographically resolved using the following gradient program: 45–100% solvent B (0–8 min); 100% solvent B (8–10 min) followed by 2 min re-equilibration to initial conditions. NO2-FAs were detected using an API4000 Q-trap triple quadrupole mass spectrometer (AB Sciex, San Jose, CA) equipped with an ESI source in negative mode. The following parameters were used: declustering potential, –75 V; collision energy, –35 eV; and a desolvation temperature of 650°C. NO2-FAs and their corresponding metabolites were detected using the multiple reaction monitoring (MRM) transitions shown in supplemental Table S1. Quantification of NO2-FAs in cell media over 24 h in adipocytes and adipose tissue was performed by stable isotopic dilution analysis using NO2-OA and NO2-SA calibration curves in the presence of NO2-[13C18]OA (MRM 344.3/46) and NO2-[15N/D4]SA (MRM 333.3/47) internal standards. Nitro-FAs of various lengths (C16, C15, C14, C13, and C12 followed as MRMs 298.3/46, 284.3/46, 270.3/46, 256.3/46, and 242.3/46) were used as external calibrants to normalize the effect of nitro-FA chain length on MS response intensity. Coefficient responses were obtained by plotting ion counts versus carbon chain length at fixed concentrations (supplemental Fig. S1). QWBA revealed the tissue distribution of NO2-OA over time. After oral administration of a single dose of 10-NO2-[14C]OA (30 mg/kg) to rats, radioactivity was readily absorbed from the gastrointestinal tract and widely distributed throughout the animal body. The vast majority of tissues reached maximum radiolabel distribution by 6 h after dosing (Table 1, supplemental Table S2), with radioactivity concentrations declining in most tissues by 24 h. Notably, brown and abdominal white adipose tissue displayed the highest levels of radioactivity 72 h postdosing in comparison with other organs, affirming that NO2-FA and potential metabolites preferentially accumulate in adipose tissue (Fig. 1).TABLE 1.Time-dependent distribution of radioactivity in rat tissues after oral administration of 10-NO2-[14C]OATissue/OrganMicrogram Equivalents 10-NO2-OA per Gram1 h6 h24 h48 h72 h120 h168 h336 hPlasmaaDetermined by direct liquid scintillation analysis.11.219.61.480.6250.3380.2070.122BLQBrain0.3581.720.2150.3730.2700.2020.163BLQKidney medulla17.328.82.080.9310.6820.4410.3241.12Liver16.728.54.481.971.190.7410.5090.299Lungs4.1617.21.521.620.7830.4500.3510.275Heart (myocardium)11.319.01.771.711.191.200.7102.21Fat (abdominal)bTissue corrected for quenching.1.7711.14.573.105.607.813.875.49Fat (brown)8.5243.141.384.011.910.63.753.03Muscle (skeletal)0.9311.640.3560.2910.3070.2110.2330.202Stomach wall (non-glandular)38.3555cValue should be treated as an estimate as above the upper limit of quantification.19.67.041.350.8270.3610.189Radioactivity levels in selected rat tissues were determined by QWBA following a single oral administration of 30 mg/kg 10-NO2-[14C]OA (labeled at carbon 10) (n = 1 for each time point). Values in bold represent maximum tissue concentrations (Cmax).a Determined by direct liquid scintillation analysis.b Tissue corrected for quenching.c Value should be treated as an estimate as above the upper limit of quantification. Open table in a new tab Radioactivity levels in selected rat tissues were determined by QWBA following a single oral administration of 30 mg/kg 10-NO2-[14C]OA (labeled at carbon 10) (n = 1 for each time point). Values in bold represent maximum tissue concentrations (Cmax). To better characterize the distribution of NO2-FA and metabolites in cellular lipid fractions, cultured 3T3-L1 adipocytes were supplemented with the biologically relevant mono- and poly-unsaturated nitro-alkenes, 10-NO2-OA, NO2-CLA, and NO2-LnA, and the saturated nitro-alkane, NO2-SA. The quantitation of NO2-FAs before and after acid hydrolysis of adipocyte lipids revealed specific patterns of incorporation, intracellular distribution, and metabolism in each lipid fraction. As expected, free acid nitrated species were only detectable in the FFA fraction before hydrolysis. Neither elongation products nor metabolites shorter than C12 were observed for all NO2-FA treatments. NO2-OA was principally reduced to NO2-SA and metabolized to its corresponding dinor, tetranor and hexanor β-oxidation products (3.Fazzari 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, 29.Rudolph V. Schopfer F.J. Khoo N.K. Rudolph T.K. Cole M.P. Woodcock S.R. Bonacci G. Groeger A.L. Golin-Bisello F. Chen C.S. et al.Nitro-fatty acid metabolome: saturation, desaturation, beta-oxidation, and protein adduction.J. Biol. Chem. 2009; 284: 1461-1473Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Esterified NO2-OA was ∼18 times more abundant in MAG+DAG than in TAG (Fig. 2A), while the nonelectrophilic NO2-SA and its β-oxidation metabolites showed the opposite distribution. The NO2-SA metabolite and its dinor, tetranor, and hexanor β-oxidation products were ∼11-fold (Fig. 2B), ∼13-fold (Fig. 2C), ∼11-fold (Fig. 2D), and ∼3.7-fold (Fig. 2E) more abundant in TAG than in MAG+DAG, respectively. This preferential incorporation of nitro-alkane metabolites was also confirmed in NO2-SA-supplemented adipocytes, with NO2-SA, dinor, tetranor, and hex
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