The simultaneous quantification of cytochrome P450 dependent linoleate and arachidonate metabolites in urine by HPLC-MS/MS
2002; Elsevier BV; Volume: 43; Issue: 9 Linguagem: Inglês
10.1194/jlr.d200018-jlr200
ISSN1539-7262
AutoresJohn W. Newman, Takaho Watanabe, Bruce D. Hammock,
Tópico(s)Metabolomics and Mass Spectrometry Studies
ResumoA method for the simultaneous quantification of urinary linoleic and arachidonic acid derived epoxides and diols, as well as the arachidonate omega hydroxylated product has been developed. The method employs negative mode electrospray ionization and HPLC with tandem mass spectroscopy for quantification. Odd chain length saturated epoxy and dihydroxy fatty acids are used as analytical surrogates resulting in linear calibrations (r 2 ⩾ 0.9995). Standard addition analyses showed that matrix effects do not prevent these surrogates from yielding reliable quantitative results. Using 4 ml urine aliquots at a final extract volume of 100 μl and injecting 10 μl, method detection limits and limits of quantification were ⩽0.5 and 1.5 nM, respectively. The sensitivity for dihydroxy lipids was from 3- to 10-fold greater than the corresponding epoxy fatty acid. Shot to shot run times of 31 min were achieved.Rodent and human urine analyses indicated the method sensitivity is sufficient for general research applications. In addition, diurnal fluctuations in linoleate and arachidonate derived metabolites were observed in human subjects. A method for the simultaneous quantification of urinary linoleic and arachidonic acid derived epoxides and diols, as well as the arachidonate omega hydroxylated product has been developed. The method employs negative mode electrospray ionization and HPLC with tandem mass spectroscopy for quantification. Odd chain length saturated epoxy and dihydroxy fatty acids are used as analytical surrogates resulting in linear calibrations (r 2 ⩾ 0.9995). Standard addition analyses showed that matrix effects do not prevent these surrogates from yielding reliable quantitative results. Using 4 ml urine aliquots at a final extract volume of 100 μl and injecting 10 μl, method detection limits and limits of quantification were ⩽0.5 and 1.5 nM, respectively. The sensitivity for dihydroxy lipids was from 3- to 10-fold greater than the corresponding epoxy fatty acid. Shot to shot run times of 31 min were achieved. Rodent and human urine analyses indicated the method sensitivity is sufficient for general research applications. In addition, diurnal fluctuations in linoleate and arachidonate derived metabolites were observed in human subjects. Cytochrome P450s (CYPs) produce a number of oxidized fatty acids, including epoxy and monohydroxy metabolites (1Capdevila J.H. Falck J.R. Harris R.C. Cytochrome P450 and arachidonic acid bioactivation: molecular and functional properties of the arachidonate monooxygenase.J. Lipid Res. 2000; 41: 163-181Google Scholar, 2Roman R.J. P-450 metabolites of arachidonic acid in the control of cardivascular function.Physiol. Rev. 2001; 82: 131-185Google Scholar, 3Zhang Y. Oltman C.L. Lu T. Lee H.C. Dellsperger K.C. VanRollins M. EET homologs potently dilate coronary microvessels and activate BK(Ca) channels.Am. J. Physiol. Heart Circ. Physiol. 2001; 280: H2430-H2440Google Scholar, 4Draper A.J. Hammock B.D. Identification of CYP2C9 as a human liver microsomal linoleic acid epoxygenase.Arch. Biochem. Biophys. 2000; 376: 199-205Google Scholar). Polyunsaturated epoxides can also be formed by the rearrangement of lipid hydroperoxides (5Sevanian A. Mead J.F. Stein R.A. Epoxides as products of lipid auto-oxidation in rat lungs.Lipids. 1978; 14: 634-643Google Scholar, 6Iwase H. Takatori T. Niijima H. Nagao M. Amano T. Iwadate K. Matsuda Y. Nakajima M. Kobayashi M. Formation of leukotoxin (9,10-epoxy-12-octadecenoic acid) during the autoxidation of phospholipids promoted by hemoproteins.Biochim. Biophys. Acta. 1997; 1345: 27-34Google Scholar, 7Möllenberg A. Spiteller G. Transformations of 12,13-epoxy-11-hydroxy-9-octadecanoic acid and 4,5-N-acetylsphingosine by incubation with liver homogenate and liver microsomes.Z. Naturforsch. 2000; 55c: 981-986Google Scholar). These chemically stable epoxides are further metabolized to their corresponding vicinal diols by the soluble epoxide hydrolase (8Greene J.F. Newman J.W. Williamson K.C. Hammock B.D. Toxicity of epoxy fatty acids and related compounds to cells expressing human soluble epoxide hydrolase.Chem. Res. Toxicol. 2000; 13: 217-226Google Scholar, 9Weintraub N.L. Fang X. Kaduce T.L. VanRollins M. Chatterjee P. Spector A.A. Epoxide hydrolases regulate epoxyeicosatrienoic acid incorporation into coronary endothelial phospholipids.Am. J. Physiol. 1999; 277: H2098-H2108Google Scholar). The hydroxylated lipids can be further transformed into glucuronides (10Street J.M. Evens J.E. Natowicz M.R. Glucuronic acid-conjugated dihydroxy fatty acids in the urine of patients with generalized peroxisomal disorders.J. Biol. Chem. 1996; 271: 3507-3516Google Scholar, 11Watzer B. Reinalter S. Seyberth H.W. Schweer H. Determination of free and glucuronide conjugated 20-hydroxyarachidonic acid (20-HETE) in urine by gas chromatography/negative ion chemical ionization mass spectrometry.Prostaglandins Leukot. Essent. Fatty Acids. 2000; 62: 175-181Google Scholar), and possibly other conjugates, prior to excretion. Many lipid oxidation products, including products of CYP metabolism, have been reported in mammalian urine (10Street J.M. Evens J.E. Natowicz M.R. Glucuronic acid-conjugated dihydroxy fatty acids in the urine of patients with generalized peroxisomal disorders.J. Biol. Chem. 1996; 271: 3507-3516Google Scholar, 11Watzer B. Reinalter S. Seyberth H.W. Schweer H. Determination of free and glucuronide conjugated 20-hydroxyarachidonic acid (20-HETE) in urine by gas chromatography/negative ion chemical ionization mass spectrometry.Prostaglandins Leukot. Essent. Fatty Acids. 2000; 62: 175-181Google Scholar, 12Toto R. Siddhanta A. Manna S. Pramanik B. Falck J.R. Capdevila J. Arachidonic acid epoxygenase: Detection of epoxyeicosatrienoic acids in human urine.Biochim. Biophys. Acta. 1987; 919: 132-139Google Scholar, 13Catella F. Lawson J.A. Fitzgerald D.J. FitzGerald G.A. Endogenous biosynthesis of arachidonic acid epoxides in humans: increased formation in pregnancy-induced hypertension.Proc. Natl. Acad. Sci. USA. 1990; 87: 5893-5897Google Scholar, 14Maier K.G. Henderson L. Narayanan J. Alonso-Galicia M. Falck J.R. Roman R.J. Fluorescent HPLC assay for 20-HETE and other P-450 metabolites of arachidonic acid.Am. J. Physiol. Heart Circ. Physiol. 2000; 279: H863-H871Google Scholar). As has been the case for prostanoids and thromboxanes (15Tsikas D. Application of gas chromatography-mass spectrometry and gas chromatography-tandem mass spectrometry to assess in vivo synthesis of prostaglandins, thromboxane, leukotrienes, isoprostanes and related compounds in humans.J. Chromatogr. B.Biomed. Appl. 1998; 71: 201-245Google Scholar, 16Schwedhelm E. Tsikas D. Durand T. Gutzki F-M. Guy A. Rossi J-C. Froelich J.C. Tandem mass spectrometric quantification of 8-iso-prostaglandin F2alpha and its metabolite 2,3-dinor-5,6-dihydro-8-iso-prostaglandin F2alpha in human urine.J. Chromatogr. B. Biomed. Appl. 2000; 744: 99-112Google Scholar, 17Tsikas D. Gutzki F-M. Boehme M. Fuchs I. Froelich J.C. Solid- and liquid-phase extraction for the gas chromatographic-tandem mass spectrometric quantification of 2,3-dinor-thromboxane B2 and 2,3-dinor-6-oxo-prostaglandin F1alpha in human urine.J. Chromatogr. A. 2000; 885: 351-359Google Scholar), the quantification of CYP derived oxylipids in urine will likely provide insight into the activity of this biochemical cascade, as well as the physiological state of the subject. The enzymatic oxidation of arachidonic acid is a key factor in the blood pressure regulatory cascade (2Roman R.J. P-450 metabolites of arachidonic acid in the control of cardivascular function.Physiol. Rev. 2001; 82: 131-185Google Scholar, 18Rahman M. Wright J.T. Douglas J.G. The role of the cytochrome p450-dependent metabolites of arachidonic acid in blood pressure regulation and renal function: A review.Am. J. Hypertens. 1997; 10: 356-365Google Scholar). Hydroxylation of the ω-carbon of the arachidonic acid chain yields 20-hydroxy eicosatetraenoic acid (20-HETE), a potent vasoconstrictor (2Roman R.J. P-450 metabolites of arachidonic acid in the control of cardivascular function.Physiol. Rev. 2001; 82: 131-185Google Scholar). The 20-HETE inhibits large and medium conductance calcium dependent potassium (K+Ca) channels (19Zou A.P. Fleming J.T. Falck J.R. Jacobs E.R. Gebremedhin D. Harder D.R. Roman R.J. 20-HETE is an endogenous inhibitor of the large-conductance Ca(2+)-activated K+ channel in renal arterioles.Am. J. Physiol. 1996; 270: R228-R237Google Scholar), preventing cellular hyperpolarization. This action of 20-HETE is opposed by the epoxides of arachidonic acid, i.e. epoxy eicosatrienoic acids (EETs), which increase the open state probability of K+Ca channels (3Zhang Y. Oltman C.L. Lu T. Lee H.C. Dellsperger K.C. VanRollins M. EET homologs potently dilate coronary microvessels and activate BK(Ca) channels.Am. J. Physiol. Heart Circ. Physiol. 2001; 280: H2430-H2440Google Scholar, 20Campbell W.B. Gebremedhin D. Pratt P.F. Harder D.R. Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors.Circ. Res. 1996; 78: 415-423Google Scholar, 21Dumoulin M. Salvail D. Gaudreault S.B. Cadieux A. Rousseau E. Epoxyeicosatrienoic acids relax airway smooth muscles and directly activate reconstituted KCa channels.Am. J. Physiol. 1998; 275: L423-L431Google Scholar). In particular, the 11(12)-EET has been suggested as an endothelial derived hyperpolarization factor (20Campbell W.B. Gebremedhin D. Pratt P.F. Harder D.R. Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors.Circ. Res. 1996; 78: 415-423Google Scholar, 22Fisslthaler B. Popp R. Michaelis U.R. Kiss L. Fleming I. Busse R. Cyclic stretch enhances the expression and activity of coronary endothelium-derived hyperpolarizing factor synthase.Hypertension. 2001; 38: 1427-1432Google Scholar). The EET regioisomers also affect mitogenesis and hormone secretion (1Capdevila J.H. Falck J.R. Harris R.C. Cytochrome P450 and arachidonic acid bioactivation: molecular and functional properties of the arachidonate monooxygenase.J. Lipid Res. 2000; 41: 163-181Google Scholar). Complicating matters, the specificity of regioisomeric activity varies between cell type and vascular bed (2Roman R.J. P-450 metabolites of arachidonic acid in the control of cardivascular function.Physiol. Rev. 2001; 82: 131-185Google Scholar). The 14,15-, 11,12-, and 8,9-EETs are excellent substrates for the soluble epoxide hydrolase (sEH). The sEH transforms the epoxides into their corresponding dihydroxy eicosatrienoic acids or dihydroxyeicosatrienoic acids (DiHETs) (23Chacos N. Capdevila J. Falck J.R. Martin-Wixtrom C. Gill S.S. Hammock B.D. Estabrook R.A. The reaction of arachidonic acid epoxides (epoxyeicosatrienoic acids) with cytosolic epoxide hydrolase.Arch. Biochem. Biophys. 1983; 233: 639-648Google Scholar). The 5,6-EET is hydrated slowly by sEH (23Chacos N. Capdevila J. Falck J.R. Martin-Wixtrom C. Gill S.S. Hammock B.D. Estabrook R.A. The reaction of arachidonic acid epoxides (epoxyeicosatrienoic acids) with cytosolic epoxide hydrolase.Arch. Biochem. Biophys. 1983; 233: 639-648Google Scholar). While the DiHETs have vasodilatory actions in canine coronary arterioles (24Oltman C.L. Weintraub N.L. VanRollins M. Dellsperger K.C. Epoxyeicosatrienoic acids and dihydroxyeicosatrienoic acids are potent vasodilators in the canine coronary microcirculation.Circ. Res. 1998; 83: 932-939Google Scholar), the introduction of free hydroxyl moieties inhibits their incorporation into membranes (25VanRollins M. Kaduce T.L. Knapp H.R. Spector A.A. 14,15-Epoxyeicosatrienoic acid metabolism in endothelial cells.J. Lipid Res. 1993; 34: 1931-1942Google Scholar) and promotes their elimination as glucuronide conjugates in humans (11Watzer B. Reinalter S. Seyberth H.W. Schweer H. Determination of free and glucuronide conjugated 20-hydroxyarachidonic acid (20-HETE) in urine by gas chromatography/negative ion chemical ionization mass spectrometry.Prostaglandins Leukot. Essent. Fatty Acids. 2000; 62: 175-181Google Scholar, 26Jude A.R. Little J.M. Freeman J.P. Evans J.E. Radominska-Pandya A. Grant D.F. Linoleic acid diols are novel substrates for human UDP-glucuronosyltransferases.Arch. Biochem. Biophys. 2000; 380: 294-302Google Scholar, 27Sacerdoti D. Balazy M. Angeli P. Gatta A. McGiff J.C. Eicosanoid excretion in hepatic cirrhosis. Predominance of 20-HETE.J. Clin. Invest. 1997; 100: 1264-1270Google Scholar). In contrast, hydroxy lipid glucuronidation appears to be deficient in rats, and the hydroxyl lipids are eliminated directly (28Wang M.H. Zand B.A. Nasjletti A. Laniado-Schwartzman M. Renal 20-hydroxyeicosatetraenoic acid synthesis during pregnancy.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2002; 282: R383-R389Google Scholar). While receiving less attention, linoleate derived dihydroxy lipids are also bioactive. These octadecanoid diols or dihydroxyoctadecenoic acids (DiHOMEs) inhibit mitochondrial respiration (8Greene J.F. Newman J.W. Williamson K.C. Hammock B.D. Toxicity of epoxy fatty acids and related compounds to cells expressing human soluble epoxide hydrolase.Chem. Res. Toxicol. 2000; 13: 217-226Google Scholar, 29Moran J.H. Mon T. Hendrickson T.L. Mitchell L.A. Grant D.F. Defining mechanisms of toxicity for linoleic acid monoepoxides and diols in Sf-21 cells.Chem. Res. Toxicol. 2001; 14: 431-437Google Scholar, 30Sisemore M.F. Zheng J. Yang J.C. Thompson D.A. Plopper C.G. Cortopassi G.A. Hammock B.D. Cellular characterization of leukotoxin diol-induced mitochondrial dysfunction.Arch. Biochem. Biophys. 2001; 392: 32-37Google Scholar), increase vascular permeability (31Slim R. Hammock B.D. Toborek M. Robertson L.W. Newman J.W. Morisseau C.H. Watkins B.A. Saraswathi V. Hennig B. The role of methyl-linoleic acid epoxide and diol metabolites in the amplified toxicity of linoleic acid and polychlorinated biphenyls to vascular endothelial cells.Toxicol. Appl. Pharmacol. 2001; 171: 184-193Google Scholar), and have been identified as their glucuronide conjugates in the urine of children with generalized peroxisomal disorders (10Street J.M. Evens J.E. Natowicz M.R. Glucuronic acid-conjugated dihydroxy fatty acids in the urine of patients with generalized peroxisomal disorders.J. Biol. Chem. 1996; 271: 3507-3516Google Scholar). Therefore, the simultaneous determination of both linoleate and arachidonate derived oxidation products in urine may provide unique insights into the associations between these compounds under various physiological conditions, including disease. The following study was conducted with two primary goals. First, to establish a simple and sensitive analytical method for the simultaneous detection of the EETs, DiHETs, and 20-HETE, along with the linoleate derived epoxides and diols in urine. The second goal was to provide a preliminary assessment of baseline excretion profiles of these oxidized lipids in apparently healthy humans. The International Union of Pure and Applied Chemistry (IUPAC) has adopted the abbreviations for oxidized fatty acids following the recommendations of Smith et al. (32Smith D.L. Willis A.L. A suggested shorthand nomenclature for the eicosanoids.Lipids. 1987; 22: 983-986Google Scholar, 33Smith W.L. Gorgeat P. Hamberg M. Roberts II, L.J. Willis A.L. Yamamoto S. Ramwell P.W. Rokach J. Samuelsson B. Corey E.J. Pace-Asciak C.R. Nomenclature.in: Fitzpatrick F.A. Methods in Enzymology. Academic Press, Inc., San Diego, CA1990: 1-9Google Scholar) as shown in Table 1 and Table 2. In short, for unsaturated epoxy fatty acids, abbreviations indicate epoxide position, chain length, and degrees of unsaturation. Therefore, 14(15)-epoxyeicostri-(5Z,8Z,11Z)-enoic acid is reduced to 14(15)-EpETrE while 9(10)-epoxyoctadec-(12Z)-enoic acid becomes 9(10)-epoxyoctadecenoic acid (EpOME). Dihydroxy lipids are named similarly, such that 14,15-dihydroxyeicostri-(5Z,8Z,11Z)-enoic acid becomes 14,15-DiHETrE, while 20-hydroxyeicosatetra-(5Z,8Z,11Z,14Z)-enoic acid is 20-HETE. The EpETrEs and DiHETrEs are more commonly known as EETs and DiHETs, respectively. The common abbreviations are used throughout the text to improve the readability of the manuscript. Unsaturated lipids were purchased from NuChek Prep (Elysian, MN). Purified DiHET regioisomers synthesized in the laboratory of Dr. J. R. “Camille” Falck were a kind gift from Dr. Darryl C. Zeldin at the National Institute of Environmental Health Sciences (Research Triangle Park, NC). The 20-HETE and 5(6)-EET were purchased from Cayman Chemical (Ann Arbor, MI). The 20-hydroxy eicosanoic acid (20-HE) was purchased from Larodan Fine Chemicals (Malmö, Sweden). Hexane, ethyl acetate, chloroform, and glacial acetic acid of HPLC Grade or better were purchased from Fisher Scientific (Pittsburgh, PA). OmniSolv™ acetonitrile and methanol purchased from EM Science (Gibbstown, NJ) was used for all reverse phase HPLC analyses. All other chemical reagents were purchase from either Sigma (St. Louis, MO) or Aldrich Chemical Co (Milwaukee, WI) unless indicated. Purification of epoxy lipids was accomplished on an HP1100 series HPLC with a HP1050 diode array detector (Agilent Technologies; San Jose, CA). Preparative scale isolations used a 22.5 × 250 mm, 5 μm ODS(2) Sphereclone column (Phenomenex, Torrence, CA). Analytical scale analysis with diode array detection was performed on a 4.6 × 250 mm, 5 μm ODS(2) PhaseSep analytical HPLC column (Waters, Milford, MA). GC/mass spectroscopy (MS) based confirmation was accomplished using a HP6890 gas chromatograph equipped with a 30 m × 0.25 mm i.d., 0.25 μm DB-17HT (Agilent Technologies) interfaced with an HP5973 mass spectral detector. The quantification of lipid oxidation products was performed by negative mode electrospray ionization with tandem quadrupole mass spectroscopy (MS/MS). All HPLC/MS analyses were performed with a Waters 2790 separation module equipped with a 2.0 × 150mm, 5μm Luna C18(2) column (Phenomenex) held at 20°C. The sample chamber was held at 10°C. A 75 cm segment of 0.005 inch i.d. PEEK tubing interfaced the HPLC to the electrospray ionization probe of a Quattro Ultima tandem-quadrupole mass spectrometer (Micromass, Manchester, UK). Solvent flow rates were fixed at 350 μl/min with a cone gas flow of 125 l/h, desolvation gas flow of 650 l/h, a source temperature of 125°C, and a desolvation temperature of 400°C. Electrospray ionization was accomplished in the negative mode with a capillary voltage fixed at −3.0 kV. For MS/MS experiments, argon was used as the collision gas at a pressure of 2.3 × 10−3 Torr while quadrupole mass resolution settings were fixed at 12.0 (i.e., ∼1.5 Da resolution). The photo multiplier voltage was 650 V. Optimal cone voltage and collision voltages were established experimentally. Ion dwell times of 0.45 s were used. A single solvent system was used for all preparative to analytical scale HPLC analyses described in this manuscript. The applied solvents were modified from Kiss et al. (34Kiss L. Schutte H. Mayer K. Grimm H. Padberg W. Seeger W. Grimminger F. Synthesis of arachidonic acid-derived lipoxygenase and cytochrome P450 products in the intact human lung vasculature.Am. J. Respir. Crit. Care Med. 2000; 161: 1917-1923Google Scholar): Solvent A = 51:40:9 acetonitrile-water-methanol (v/v/v) with 0.1% glacial acetic acid; Solvent B = 85:15 acetonitrile-methanol (v/v) with 0.1% glacial acetic acid. Sprague-Dawley rats (SDs) and 13-week-old spontaneously hypertensive rats (SHRs) were obtained from Charles River Laboratories (Wilmington, MA) and housed in metabolic chambers with free access to food and water. All animal handling was performed in accordance with approved animal use protocols. Urine from SD rats was provided by Dr. John Imig at the Medical College of Georgia (Augusta, GA). Epoxy fatty acid methyl esters were synthesized using dimethyl dioxirane (mono-unsaturated free acids) or meta-chloro perbenzoic acid (polyunsaturated methyl esters) and purified as previously described (8Greene J.F. Newman J.W. Williamson K.C. Hammock B.D. Toxicity of epoxy fatty acids and related compounds to cells expressing human soluble epoxide hydrolase.Chem. Res. Toxicol. 2000; 13: 217-226Google Scholar, 35Gunstone F.D. Schuler H.R. Fatty acids, part 45. Epoxyoctadecenoates, dihydroxyoctadecenoates, and diepoxyoctadacanoates: preparation, chromatographic properties, and reactions with boron trifluoride etherate.Chem. Phys. Lipids. 1975; 15: 174-188Google Scholar, 36Murray R.W. Singh M. Synthesis of epoxides using dimethyldioxirane: trans-stilbene oxide.in: Shinkai I. Organic Synthesis. John Wiley & Sons, New York1997: 91-100Google Scholar, 37Newman J.W. Hammock B.D. Optimized thiol derivatizing reagent for the mass spectral analysis of disubstituted epoxy fatty acids.J. Chromatogr. A. 2001; 925: 223-240Google Scholar). Purified epoxides were chemically hydrolyzed to dihydroxy lipid methyl esters using 1:1 acetonitrile-aqueous 5% perchloric acid (v/v) (8Greene J.F. Newman J.W. Williamson K.C. Hammock B.D. Toxicity of epoxy fatty acids and related compounds to cells expressing human soluble epoxide hydrolase.Chem. Res. Toxicol. 2000; 13: 217-226Google Scholar, 35Gunstone F.D. Schuler H.R. Fatty acids, part 45. Epoxyoctadecenoates, dihydroxyoctadecenoates, and diepoxyoctadacanoates: preparation, chromatographic properties, and reactions with boron trifluoride etherate.Chem. Phys. Lipids. 1975; 15: 174-188Google Scholar, 36Murray R.W. Singh M. Synthesis of epoxides using dimethyldioxirane: trans-stilbene oxide.in: Shinkai I. Organic Synthesis. John Wiley & Sons, New York1997: 91-100Google Scholar, 37Newman J.W. Hammock B.D. Optimized thiol derivatizing reagent for the mass spectral analysis of disubstituted epoxy fatty acids.J. Chromatogr. A. 2001; 925: 223-240Google Scholar). Methyl esters were transformed to free fatty acids using published procedures (38Falck J.R. Yadagiri P. Capdevila J. Synthesis of Epoxyeicosatrienoic Acids and Heteroatom Analogs.in: Fitzpatrick F.A. Methods in Enzymology: Arachidonate related lipid mediators. Academic Press, Inc., San Diego, CA1990: 357-364Google Scholar). Briefly, methyl esters were dissolved at 10 mg/ml in methanol and chilled to 0°C. While stirring, the chilled solution was enriched by the dropwise addition of 335 μl of 0.33 N sodium hydroxide per ml of ester solution. The reaction was maintained at room temperature for 12–20 h. Upon completion, the reaction was diluted with 750 μl of water per ml of reaction. The mixture was brought to pH 4.25 with 220 μl 0.25 M oxalic acid (per ml of reaction solution) and extracted four times with ethyl acetate. The extract was washed with water until the pH of the wash was unchanged. The organic fraction was dried with ∼1 g of hexane washed anhydrous sodium sulfate and the residual solvent was removed by rotary evaporation. The progress of the reactions was screened by spotting ∼10 μl of the mixture on a 10 cm silica gel TLC plate (EM Science). The TLC plate was developed in 80:20 hexane-ethyl acetate (v/v) and visualized with heat after spraying with 4% phosphomolybdic acid in 20% ethanolic water. In the case of the EETs, 100 mg of the methyl ester was hydrolyzed in 16 h. The resulting straw colored oil was purified by loading onto 2 g of silica gel (Baker 60/40 mesh) in a 15 ml sintered glass funnel and washing with 50 ml of pentane, 20 ml of 10% ethyl acetate in pentane and 20 ml of ethyl acetate. The final ethyl acetate fraction yielded 70 mg of colorless oil (197 μmol, 70% yield). Similar yields were achieved with EpOMEs and DiHOMEs. The isomeric abundance of the linoleate derived epoxide mixture was quantitatively characterized. A 10 mg aliquot of the silica gel purified reaction mixture was dissolved in 1 ml of methanol. A 10 μl aliquot of this solution was diluted to 500 pg/μl, spiked with the internal standards described below, methylated, thiolated, silylated, and analyzed by GC/MS as previously described (37Newman J.W. Hammock B.D. Optimized thiol derivatizing reagent for the mass spectral analysis of disubstituted epoxy fatty acids.J. Chromatogr. A. 2001; 925: 223-240Google Scholar). An aliquot of the epoxide mixture was hydrolyzed as described above to produce diols. The resulting diols were purified by silica gel column chromatography and quantified as trimethyl silyl-ether/methyl esters by GC/MS (37Newman J.W. Hammock B.D. Optimized thiol derivatizing reagent for the mass spectral analysis of disubstituted epoxy fatty acids.J. Chromatogr. A. 2001; 925: 223-240Google Scholar). Epoxyeicosanoid regioisomers were purified using reverse phase HPLC. A total of 70 mg of the epoxy fatty acid mixture (∼10 mg/injection) were introduced to a preparative column (see Instrumentation) and separated at a flow rate of 10 mL/min (0–35 min, 70% Solvent A; 45 to 55 min, 100% Solvent B), while recording absorbance at 210 and 232 nm. Traces of 232 nm dense compounds, presumably peroxides, were observed eluting before 10 min. Epoxide isomer identity was initially assigned based on relative retention times reported in the literature (34Kiss L. Schutte H. Mayer K. Grimm H. Padberg W. Seeger W. Grimminger F. Synthesis of arachidonic acid-derived lipoxygenase and cytochrome P450 products in the intact human lung vasculature.Am. J. Respir. Crit. Care Med. 2000; 161: 1917-1923Google Scholar): 14(15)-EET = 25.6 min, 11(12)-EET = 28.8 min, 8(9)-EET = 30.3 min, 5(6)-EET = 34.5 min). Peak heights showed 14(15) > 11(12) > 8(9) >> 5(6)-EET. The 5(6)-EET fatty acid purification was not attempted. The 14(15)-EET appeared fully resolved and was collected directly. The 11(12)- and 8(9)-isomers showed ∼60% baseline resolution. Therefore, these peaks were collected from leading edge to 50% of the height after the apex and from the apex to the trailing edge, respectively. The collected central fractions were combined, re-purified and appropriate fractions combined. The combined HPLC fractions were extracted three times with an equal volume of pentane and the solvent was removed by vacuum rotary evaporation. The residues were re-dissolved in chloroform, transferred to storage vials, and reduced to a constant mass under a gentle stream of nitrogen at 40°C. Each fraction was then diluted and ∼50 μg was reanalyzed with an isocratic solvent flow of 96% Solvent A and 4% Solvent B at 2 ml/min on the described analytical column (see Instrumentation). This solvent system gave optimal EET isomer resolution: peak widths = 0.35 min, 14(15)-EET (12.5 min), 11(12)-EET (14.3 min), and 8(9)-EET (14.7 min). The 11(12)-EET isolated under these conditions was pure (20 mg, >98% UV, 205 nm Abs). However, the 14(15)-isomer showed 13% of a late eluting unknown impurity and the 8(9)-EET was contaminated with ∼10% 11(12)-EET. The impure fractions were re-purified using the preparative system but injected masses were kept below 5 mg, resulting in the isolation of 14(15)-EET (18 mg) and 8(9)-EET (6 mg) with a final purity of >98% as determined by UV absorbance at 205 nm. Full scan (195–600 nm) diode array spectra revealed no detectable secondary peaks. Methylation, thiolation, and silylation of the purified EETs followed by GC/MS analysis (37Newman J.W. Hammock B.D. Optimized thiol derivatizing reagent for the mass spectral analysis of disubstituted epoxy fatty acids.J. Chromatogr. A. 2001; 925: 223-240Google Scholar) confirmed isomer identification and indicated that the purified materials contained less than 0.5% DiHETs. The final purified fractions were concentrated and transferred as described above. The lipids were stored under nitrogen at –32°C until use. These materials were used for the generation of all EET calibration solutions. Odd chain length monounsaturated fatty acids were used to prepare epoxide and diol extraction surrogates. Specifically, 10(11)-epoxyheptadecanoic acid [10(11)-EpHep] and 10,11-dihydroxynonadecanoic acid (10,11-DiHN) were synthesized as described above. Eight independent calibration solutions containing ∼1 pmol/μl of each internal standard were prepared in acetonitrile. The six lowest concentrations ranged from ∼5–1,500 fmol/μl (nM). These standards contained all EpOME, DiHOME, EET, and DiHET isomers along with 20-HETE. The two additional standards extended the calibration for the EpOMEs and DiHOMEs to 25 μM and the EETs, except for the 5(6)-isomer, to 10 μM. These calibration solutions were sub-aliquoted into Wheaton pre-scored gold-band amber ampoules (Fisher Scientific), sealed under nitrogen and stored at ⩽−32°C. The intensity of the deprotonated molecular ion was evaluated at cone voltages between −40 V and −120 V. Initially, calibration solutions were introduced directly into the mass spectrometer with a 1:1 mixture of Solvent A and Solvent B. A secondary analyses was performed with chromatographic resolution with cone voltages of −40 V, −50 V, −60 V, and −120 V. This experiment indicated that the loop injections accurately reflected the ionization behavior in all solvent proportions appearing during the gradient analysis and that the regioisomers showed identical ionization behavior (data not shown). To maximize analytical sensitivity using multi-reaction monitoring, the collision cell voltages must be optimized to produce the highest abundance transition ion, i.e. a characteristic ion from the collision induced dissociation of the molecular ion selected in the first quadrupole. To identify these energies, analytes were separated using a rapid HPLC gradient (0 min = 60% Solvent A, 15 min = 100% Solvent B, flow rate = 350 μl/min) eluting all analytes within 8 min. Product ion spectra were collected for each molecular ion using collision voltages of −8 V, −10 V, −13 V, −17 V, −20 V, −23 V, −26 V, and −30 V. The presence of target analytes in available urine samples required the use of a non-urine matrix for spike enrich
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