Differential Metabolomics Reveals Ophthalmic Acid as an Oxidative Stress Biomarker Indicating Hepatic Glutathione Consumption

Metabolomics is an emerging tool that can be used to gain insights into cellular and physiological responses. Here we present a metabolome differential display method based on capillary electrophoresis time-of-flight mass spectrometry to profile liver metabolites following acetaminophen-induced hepatotoxicity. We globally detected 1,859 peaks in mouse liver extracts and highlighted multiple changes in metabolite levels, including an activation of the ophthalmate biosynthesis pathway. We confirmed that ophthalmate was synthesized from 2-aminobutyrate through consecutive reactions with γ-glutamylcysteine and glutathione synthetase. Changes in ophthalmate level in mouse serum and liver extracts were closely correlated and ophthalmate levels increased significantly in conjunction with glutathione consumption. Overall, our results provide a broad picture of hepatic metabolite changes following acetaminophen treatment. In addition, we specifically found that serum ophthalmate is a sensitive indicator of hepatic GSH depletion, and may be a new biomarker for oxidative stress. Our method can thus pinpoint specific metabolite changes and provide insights into the perturbation of metabolic pathways on a large scale and serve as a powerful new tool for discovering low molecular weight biomarkers. Metabolomics is an emerging tool that can be used to gain insights into cellular and physiological responses. Here we present a metabolome differential display method based on capillary electrophoresis time-of-flight mass spectrometry to profile liver metabolites following acetaminophen-induced hepatotoxicity. We globally detected 1,859 peaks in mouse liver extracts and highlighted multiple changes in metabolite levels, including an activation of the ophthalmate biosynthesis pathway. We confirmed that ophthalmate was synthesized from 2-aminobutyrate through consecutive reactions with γ-glutamylcysteine and glutathione synthetase. Changes in ophthalmate level in mouse serum and liver extracts were closely correlated and ophthalmate levels increased significantly in conjunction with glutathione consumption. Overall, our results provide a broad picture of hepatic metabolite changes following acetaminophen treatment. In addition, we specifically found that serum ophthalmate is a sensitive indicator of hepatic GSH depletion, and may be a new biomarker for oxidative stress. Our method can thus pinpoint specific metabolite changes and provide insights into the perturbation of metabolic pathways on a large scale and serve as a powerful new tool for discovering low molecular weight biomarkers. An excess dose of acetaminophen (AAP), 4The abbreviations used are: AAP, acetaminophen; CE-TOFMS, capillary electrophoresis time-of-flight mass spectrometry; 2AB, 2-aminobutyrate; GCS, γ-glutamylcysteine synthetase; GS, glutathione synthetase; ip, intraperitoneal; DEM, diethylmaleate; BSO, buthionine sulfoximine; MES, 2-morpholinoethanesulfonate; Oct RFV, octapole radio frequency voltage; ESI-Q-TOFMS, electrospray ionization quadrupole time-of-flight mass spectrometry; PIPES, piperazine-1,4-bis(2-ethansulfonate). the most commonly used analgesic, can lead to possibly fatal hepatitis and more than 100 such deaths occur in the United States annually. AAP is normally detoxified by sulfation or glucoronidation followed by elimination (1Vermeulen N.P. Bessems J.G. Van de Straat R. Drug Metab. Rev. 1992; 24: 367-407Crossref PubMed Scopus (310) Google Scholar). In high doses, it is metabolized by P450 cytochromes to generate the reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI) (2Dahlin D.C. Miwa G.T. Lu A.Y. Nelson S.D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1327-1331Crossref PubMed Google Scholar), which is further inactivated by conjugation with glutathione (GSH) before excretion. This results in a sudden drop in GSH levels (3Vendemiale G. Grattagliano I. Altomare E. Turturro N. Guerrieri F. Biochem. Pharmacol. 1996; 52: 1147-1154Crossref PubMed Scopus (87) Google Scholar). In absence of sufficient GSH, the reactive NAPQI can cause toxic and covalent protein modifications that lead to cell death and tissue injury (4Mitchell J.R. Jollow D.J. Potter W.Z. Davis D.C. Gillette J.R. Brodie B.B. J. Pharmacol. Exp. Ther. 1973; 187: 185-194PubMed Google Scholar, 5Gibson J.D. Pumford N.R. Samokyszyn V.M. Hinson J.A. Chem. Res. Toxicol. 1996; 9: 580-585Crossref PubMed Scopus (100) Google Scholar, 6Wallace J.L. Br. J. Pharmacol. 2004; 143: 1-2Crossref PubMed Scopus (44) Google Scholar, 7Hinson J.A. Reid A.B. McCullough S.S. James L.P. Drug Metab. Rev. 2004; 36: 805-822Crossref PubMed Scopus (247) Google Scholar). Recent transcriptomic and proteomic studies showed that AAP can cause numerous changes in gene and protein expression levels in pathways related to cellular stress response, mitochondrial function, and metabolism, as well as in cell cycle, structural, signaling, and apoptotic proteins (8Reilly T.P. Bourdi M. Brady J.N. Pise-Masison C.A. Radonovich M.F. George J.W. Pohl L.R. Biochem. Biophys. Res. Commun. 2001; 282: 321-328Crossref PubMed Scopus (115) Google Scholar, 9Ruepp S.U. Tonge R.P. Shaw J. Wallis N. Pognan F. Toxicol. Sci. 2002; 65: 135-150Crossref PubMed Scopus (260) Google Scholar). However, little is known about global changes in metabolites. Global information about when and where metabolite levels increase or decrease can reveal connections in biological networks and provide a system level understanding of the cell (10Raamsdonk L.M. Teusink B. Broadhurst D. Zhang N. Hayes A. Walsh M.C. Berden J.A. Brindle K.M. Kell D.B. Rowland J.J. Westerhoff H.V. van Dam K. Oliver S.G. Nat. Biotechnol. 2001; 19: 45-50Crossref PubMed Scopus (865) Google Scholar, 11Spinnler H.E. Ginies C. Khan J.A. Vulfson E.N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3373-3376Crossref PubMed Scopus (23) Google Scholar, 12Ideker T. Thorsson V. Ranish J.A. Christmas R. Buhler J. Eng J.K. Bumgarner R. Goodlett D.R. Aebersold R. Hood L. Science. 2001; 292: 929-934Crossref PubMed Scopus (1653) Google Scholar, 13Fraenkel D.G. Annu. Rev. Genet. 1992; 26: 159-177Crossref PubMed Scopus (37) Google Scholar, 14Fernie A.R. Trethewey R.N. Krotzky A.J. Willmitzer L. Nat. Rev. Mol. Cell. Biol. 2004; 5: 763-769Crossref PubMed Scopus (645) Google Scholar). However, unlike other functional genomic approaches, metabolome analysis methods are still under development. Current large scale metabolite analysis methods are based on gas chromatography mass spectrometry (15Fiehn O. Kopka J. Dormann P. Altmann T. Trethewey R.N. Willmitzer L. Nat. Biotechnol. 2000; 18: 1157-1161Crossref PubMed Scopus (1748) Google Scholar), liquid chromatography mass spectrometry (LC-MS) (16Plumb R. Granger J. Stumpf C. Wilson I.D. Evans J.A. Lenz E.M. Analyst. 2003; 128: 819-823Crossref PubMed Scopus (140) Google Scholar), NMR (17Reo N.V. Drug Chem. Toxicol. 2002; 25: 375-382Crossref PubMed Scopus (222) Google Scholar), Fourier transform ion cyclotron resonance mass spectrometry) (18Aharoni A. Ric de Vos C.H. Verhoeven H.A. Maliepaard C.A. Kruppa G. Bino R. Goodenowe D.B. Omics. 2002; 6: 217-234Crossref PubMed Scopus (368) Google Scholar), and capillary electrophoresis mass spectrometry (CE-MS) (19Soga T. Ohashi Y. Ueno Y. Naraoka H. Tomita M. Nishioka T. J. Proteome. Res. 2003; 2: 488-494Crossref PubMed Scopus (805) Google Scholar). Whereas these analytical tools allow global metabolite profiling, the exploration and identification of changes in compounds among the enormous amount of data generated are laborious. Here, we propose a novel strategy to analyze and differentially display metabolic profiles by coupling capillary electrophoresis with electrospray ionization time-of-flight mass spectrometry (CE-TOFMS). Using this profiling system, we determined global changes in metabolite levels in the liver and serum of AAP-treated mice, obtained insights into the perturbation of metabolic pathways related to hepatotoxicity, and identified biomarkers that can reveal acute liver injury. Animals and Drug Administration—Male C57BL6 mice, fasted overnight with free access to water, were anesthetized with an intraperitoneal (ip) injection of pentobarbital sodium (60 mg/kg). AAP or physiological saline as a vehicle was administered at 150 mg/kg. AAP was solubilized in saline prior to injection, to avoid the use of ethanol that could affect liver function. To achieve solubilization, AAP was added to warm saline kept at 42 °C followed by vortexing for 1 h. After the AAP injection, mice were allowed free access to chow diet and water. At the indicated times after AAP administration (1, 2, 4, 6, 12, and 24 h) the mice were anesthetized by urethane and were sacrificed to collect liver tissues and serum samples. The liver tissues were immediately snap-frozen in liquid nitrogen (20Shiomi M. Wakabayashi Y. Sano T. Shinoda Y. Nimura Y. Ishimura Y. Suematsu M. Hepatology. 1998; 27: 108-115Crossref PubMed Scopus (68) Google Scholar). In another set of experiments, diethylmaleate (DEM) or buthionine sulfoximine (BSO) (4 mmol/kg) was administered ip while the control mice were injected with physiological saline. At the above time points post-administration, livers and serum samples were collected for metabolome analysis. Metabolite Extraction—Frozen liver tissue (∼300 mg) was immediately plunged into methanol (1 ml) containing internal standards (300 μm each of methionine sulfone for cations, MES for anions) and homogenized for 1 min to inactivate enzymes. Then, deionized water (500 μl) was added, 300 μl of the solution were transferred to another tube, and 200 μl of chloroform were added, and the mixture thoroughly mixed. The solution was centrifuged at 12,000 × g for 15 min at 4 °C, and the 300-μl upper aqueous layer was centrifugally filtered through a Millipore 5-kDa cutoff filter to remove proteins. The filtrate was lyophilized and dissolved in 50 μl of Milli-Q water containing reference compounds (200 μm each of 3-aminopyrrolidine and trimesate) prior to CE-TOFMS analysis. For serum studies, 200-μl samples were plunged into 1.8 ml of methanol containing 55 μm each of methionine sulfone and MES and mixed well. Then 800 μl of deionized water and 2 ml of chloroform were added, and the solution was centrifuged at 2,500 × g for 5 min at 4 °C. The 800-μl upper aqueous layer was centrifugally filtered through a Millipore 5-kDa cutoff filter to remove proteins. Subsequent steps were as for liver samples. Metabolite Standards—All chemical standards were obtained from common commercial sources and dissolved in Milli-Q (Millipore, Bedford, MA) water, 0.1 n HCl or 0.1 n NaOH to obtain 10 mm or 100 mm stock solutions. Working standard mixtures were prepared by diluting stock solutions with Milli-Q water just prior to injection into the CE-TOFMS. The chemicals used were of analytical or reagent grade. Instrumentation—All CE-TOFMS experiments were performed using an Agilent CE capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany), an Agilent G3250AA LC/MSD TOF system (Agilent Technologies, Palo Alto, CA), an Agilent1100 series binary HPLC pump, and the G1603A Agilent CE-MS adapter and G1607A Agilent CE-ESI-MS sprayer kit. For system control and data acquisition we used the G2201AA Agilent ChemStation software for CE and the Analyst QS for Agilent TOFMS software. CE-MS/MS analyses for compound identification were performed on a Q-Star XL Hybrid LC-MS/MS System (Applied Biosystems, Foster City, CA) connected to an Agilent CE instrument. CE-TOFMS Conditions for Cationic Metabolite Analysis—Separations were carried out in a fused capillary μm × 100 with 1 as the T. Chem. 2000; PubMed Scopus Google Scholar). of solution were injected at 50 for and of was The capillary was at °C, and the was 5 °C. containing μm was as the liquid at 10 was in the ion and the capillary was set at V. of nitrogen gas 300 was at 10 In the and Oct were set at 50 and of each was performed using reference of reference The methanol ion and the mass for mass mass data were at a of 10 a CE-TOFMS Conditions for Metabolite capillary H. Y. N. Chem. 1998; PubMed Scopus Google was used as the capillary T. Ueno Y. Naraoka H. Ohashi Y. Tomita M. Nishioka T. Chem. 2002; PubMed Scopus Google Scholar). 50 mm solution was used as solution for CE solution was injected at 50 for and of was in containing μm and 1 μm was as the liquid at 10 was in the ion the capillary was set at V. For the and Oct were set at 100 50 and 200 of each was performed using reference of and were to used in metabolite analysis. 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