LC‐MS/MS Quantitation of Mercapturic Acid Conjugates of Lipid Peroxidation Products as Markers of Oxidative Stress

Heather C. Kuiper1, Jan F. Stevens1

1 Linus Pauling Institute and the Department of Pharmaceutical Sciences, Oregon State University, Corvallis, Oregon
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 17.14
DOI:  10.1002/0471140856.tx1714s45
Online Posting Date:  August, 2010
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Abstract

Oxidative stress–induced lipid peroxidation (LPO) leads to the formation of cytotoxic and genotoxic 2‐alkenals. LPO products such as 4‐hydroxy‐2(E)‐nonenal (HNE) and 4‐oxo‐2(E)‐nonenal (ONE) have been the subject of many studies due to their association with the development of cardiovascular and neurodegenerative diseases, as well as cancer. LPO products are excreted in the urine after conjugation with glutathione (GSH) and subsequent metabolism to mercapturic acid (MA) conjugates. Urinary LPO‐MA metabolites are stable end‐product metabolites and have gained interest as non‐invasive in vivo biomarkers of oxidative stress. This protocol describes a method for the quantitative analysis of LPO‐MA metabolites in urine using isotope‐dilution liquid chromatography coupled with electrospray tandem mass spectrometry (LC‐MS/MS). Included are protocols for preparation of labeled LPO‐MA conjugates from unlabeled LPO products and deuterium labeled MA. Curr. Protoc. Toxicol. 45:17.14.1‐17.14.17. © 2010 by John Wiley & Sons, Inc.

Keywords: lipid peroxidation; mercapturic acid; mass spectrometry; oxidative stress

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: LC‐MS/MS Analysis and Quantitation of LPO‐MA Conjugates
  • Support Protocol 1: Synthesis of ONA
  • Support Protocol 2: Synthesis of HNA
  • Support Protocol 3: Synthesis of ONO
  • Support Protocol 4: Synthesis of MAd3
  • Support Protocol 5: Synthesis of LPO‐MA and LPO‐MAd3 Conjugates
  • Support Protocol 6: Constructing a Calibration Curve
  • Support Protocol 7: Quantitation of Urinary Creatinine
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: LC‐MS/MS Analysis and Quantitation of LPO‐MA Conjugates

  Materials
  • Urine sample
  • 1 N HCl
  • Internal standard solutions (see recipe)
  • Ethyl acetate
  • Mobile phase solutions A and B (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • Vortex mixer
  • Pasteur pipets
  • 13 × 100–mm disposable borosilicate tubes
  • Zymark TurboVap LV nitrogen evaporation system (Caliper Life Sciences) or equivalent equipment
  • Small‐volume HPLC injection vials with caps (e.g., 12 × 32–mm glass vials with fused glass inserts, 300 µl capacity, and silicone/PTFE septa; MicroSolv Technology Corporation)
  • High‐performance liquid chromatography (HPLC) system (Shimadzu Prominence HPLC system or equivalent equipment), including a gradient pump system, degasser, and autosampler
  • HPLC column: 250 × 2–mm Synergi Max RP C 12 column (Phenomenex)
  • Triple quadrupole or triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems MDS Sciex 4000 QTrap, equipped with a TurboV electrospray source and Analyst 1.4.1 data acquisition software, or equivalent equipment)

Support Protocol 1: Synthesis of ONA

  Materials
  • t‐Butanol
  • 2‐pentylfuran
  • KH 2PO 4
  • NaClO 2
  • Chloroform
  • Brine
  • MgSO 4 (anhydrous)
  • Tetrahydrofuran
  • Acetone
  • Pyridine
  • Ethyl ether
  • 1.3 M NaOH
  • 1 N HCl
  • 25‐ml round‐bottom flasks
  • Stir plate
  • Separatory funnel
  • Filter apparatus
  • Rotary evaporator
  • NMR

Support Protocol 2: Synthesis of HNA

  Materials
  • ONA ( protocol 2)
  • Ethanol
  • Sodium borohydride
  • 1 N HCl
  • Ethyl ether
  • MgSO 4
  • 25‐ml round‐bottom flasks
  • Stir plate
  • Separatory funnel
  • Filter apparatus
  • Rotary evaporator
  • NMR
  • Additional reagents and equipment for preparing a solution of ONA ( protocol 2)

Support Protocol 3: Synthesis of ONO

  Materials
  • LPO product ONE (Cayman Chemical)
  • Methanol
  • Sodium phosphate buffer, 0.1 M, pH 3 (see recipe)
  • 50 mM sodium cyanoborohydride in 1 N NaOH
  • Ethyl acetate
  • 50‐ml round‐bottom flasks
  • Stir plate
  • Separatory funnel
  • Filter apparatus
  • Rotary evaporator
  • NMR

Support Protocol 4: Synthesis of MAd3

  Materials
  • 1.5 M NaOH
  • Cystine
  • [2H 6]Acetic anhydride
  • 1,4‐Dithiothreitol
  • Ethyl ether
  • Liquid nitrogen
  • Methanol
  • 1 N HCl
  • Ethyl acetate
  • Round‐bottom flasks (25 ml)
  • Ice bath
  • Rotary evaporator
  • Lyophilizer
  • 52 × 2.5–cm Sephadex LH‐20 column
  • Separatory funnel
  • High‐performance liquid chromatography (HPLC) system (Shimadzu Prominence HPLC system or equivalent equipment), including a gradient pump system, degasser, and autosampler
  • Triple quadrupole or triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems MDS Sciex 4000 QTrap, equipped with a TurboV electrospray source and Analyst 1.4.1 data acquisition software, or equivalent equipment)

Support Protocol 5: Synthesis of LPO‐MA and LPO‐MAd3 Conjugates

  Materials
  • MA (Sigma‐Aldrich) and MAd 3 ( protocol 5)
  • Sodium phosphate buffer, 0.1 M, pH 8 (see recipe)
  • LPO products HNE and ONE (Cayman Chemical), ONA ( protocol 2), HNA ( protocol 3), and ONO ( protocol 4)
  • Ethanol
  • 5 M sodium borohydride in 1 N NaOH
  • 1 N HCl
  • Ethyl acetate
  • 2:8 mobile phase A/mobile phase B
  • 1.5‐ml microcentrifuge tubes
  • Stir plate with warming
  • Zymark TurboVap LV nitrogen evaporation system (Caliper Life Sciences)

Support Protocol 6: Constructing a Calibration Curve

  Materials
  • HNE‐MA, ONO‐MA, and DHN‐MA (as prepared in protocol 6)
  • HNE‐MAd 3, ONO‐MAd 3, and DHN‐MAd 3 (as prepared in protocol 6)
  • 2:8 Mobile phase A/Mobile phase B
  • Small‐volume HPLC autosampler vials with 300‐µl glass inserts and caps

Support Protocol 7: Quantitation of Urinary Creatinine

  Materials
  • Urine sample (100 µl aliquot of the sample used for the protocol 1)
  • Creatinine assay kit (Cayman Chemical or equivalent)
  • Spectrophotometer reading at 500 nm (SpectraMax 190 or equivalent)
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Figures

Videos

Literature Cited

   Alary, J., Bravais, F., Cravedi, J.P., Debrauwer, L., Rao, D., and Bories, G. 1995. Mercapturic acid conjugates as urinary end metabolites of the lipid peroxidation product 4‐hydroxy‐2‐nonenal in the rat. Chem. Res. Toxicol. 8:34‐39.
   Alary, J., Debrauwer, L., Fernandez, Y., Cravedi, J.P., Rao, D., and Bories, G. 1998. 1,4‐Dihydroxynonene mercapturic acid, the major end metabolite of exogenous 4‐hydroxy‐2‐nonenal, is a physiological component of rat and human urine. Chem. Res. Toxicol. 11:130‐135.
   Amunom, I., Stephens, L.J., Tamasi, V., Cai, J., Pierce, W.M.J., Conklin, D.J., Bhatnagar, A., Srivastava, S., Martin, M.V., Guengerich, F.P., and Prough, R.A. 2007. Cytochromes P450 catalyze oxidation of α,β‐unsaturated aldehydes. Arch. Biochem. Biophys. 464:187‐196.
   Annangudi, S.P., Sun, M., and Salomon, R.G. 2005. An efficient synthesis of 4‐oxoalkenoic acids from 2‐alkylfurans. Synlett. 9:1468‐1470.
   Barbin, A. 1998. Formation of DNA etheno adducts in rodents and humans and their role in carcinogenesis. Acta Biochim. Pol. 45:145‐161.
   Benedetti, A., Comporti, M., and Esterbauer, H. 1980. Identification of 4‐hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal lipids. Biochim. Biophys. Acta 620:281‐296.
   Blair, I.A. 2010. Analysis of endogenous glutathione‐adducts and their metabolites. Biomed. Chromatogr. 24:29‐38.
   Boon, P.J., Marinho, H.S., Oosting, R., and Mulder, G.J. 1999. Glutathione conjugation of 4‐hydroxy‐trans‐2,3‐nonenal in the rat in vivo, the isolated perfused liver and erythrocytes. Toxicol. Appl. Pharmacol. 159:214‐223.
   Butterfield, D.A. and Sultana, R. 2007. Redox proteomics identification of oxidatively modified brain proteins in Alzheimer's disease and mild cognitive impairment: Insights into the progression of this dementing disorder. J. Alzheimers Dis. 12:61‐72.
   Cohn, J.A., Tsai, L., Friguet, B., and Szweda, L.I. 1996. Chemical characterization of a protein‐4‐hydroxy‐2‐nonenal cross‐link: Immunochemical detection in mitochondria exposed to oxidative stress. Arch. Biochem. Biophys. 328:158‐164.
   Doorn, J.A. and Petersen, D.R. 2002. Covalent modification of amino acid nucleophiles by the lipid peroxidation products 4‐hydroxy‐2‐nonenal and 4‐oxo‐2‐nonenal. Chem. Res. Toxicol. 15:1445‐1450.
   Doorn, J.A., Srivastava, S.K., and Petersen, D.R. 2003. Aldose reductase catalyzes reduction of the lipid peroxidation product 4‐oxonon‐2‐enal. Chem. Res. Toxicol. 16:1418‐1423.
   Doorn, J.A., Maser, E., Blum, A., Claffey, D.J., and Petersen, D.R. 2004. Human carbonyl reductase catalyzes reduction of 4‐oxonon‐2‐enal. Biochemistry 43:13106‐13114.
   Doorn, J.A., Hurley, T.D., and Petersen, D.R. 2006. Inhibition of human mitochondrial aldehyde dehydrogenase by 4‐hydroxynon‐2‐enal and 4‐oxonon‐2‐enal. Chem. Res. Toxicol. 19:102‐110.
   Enoiu, M., Herber, R., Wennig, R., Marson, C., Bodaud, H., Leroy, P., Mitrea, N., Siest, G., and Wellman, M. 2002. γ‐Glutamyltranspeptidase‐dependent metabolism of 4‐hydroxynonenal‐glutathione conjugate. Arch. Biochem. Biophys. 397:18‐27.
   Esterbauer, H. and Zollner, H. 1989. Methods for determination of aldehydic lipid peroxidation products. Free Radic. Biol. Med. 7:197‐203.
   Esterbauer, H., Schaur, R.J., and Zollner, H. 1991. Chemistry and biochemistry of 4‐hydroxynonenal, malonaldehyde and related aldehydes. Free Radic. Biol. Med. 11:81‐128.
   Facchinetti, F., Amadei, F., Geppetti, P., Tarantini, F., Di Serio, C., Dragotto, A., Gigli, P.M., Catinella, S., Civelli, M., and Patacchini, R. 2007. α,β‐unsaturated aldehydes in cigarette smoke release inflammatory mediators from human macrophages. Am. J. Respir. Cell. Mol. Biol. 37:617‐623.
   Guéraud, F., Peiro, G., Bernard, H., Alary, J., Créminon, C., Debrauwer, L., Rathahao, E., Drumare, M.F., Canlet, C., Wal, J.M., and Bories, G. 2006. Enzyme immunoassay for a urinary metabolite of 4‐hydroxynonenal as a marker of lipid peroxidation. Free Radic. Biol. Med. 40:54‐62.
   Guichardant, M. and Lagarde, M. 2009. Hydroxy‐alkenals from the peroxidation of n‐3 and n‐6 fatty acids and urinary metabolites. Eur. J. Lipid Sci. Technol. 111:75‐82.
   Honzatko, A., Brichac, J., and Picklo, M.J. 2007. Quantification of trans‐4‐hydroxy‐2‐nonenal enantiomers and metabolites by LC‐ESI‐MS/MS. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 857:115‐122.
   Iles, K.E. and Liu, R.M. 2005. Mechanisms of glutamate cysteine ligase (GCL) induction by 4‐hydroxynonenal. Free Radic. Biol. Med. 38:547‐556.
   Jensson, H., Guthenberg, C., Alin, P., and Mannervik, B. 1986. Rat glutathione transferase 8‐8, an enzyme efficiently detoxifying 4‐hydroxyalk‐2‐enals. FEBS Lett. 203:207‐209.
   Jian, W., Arora, J.S., Oe, T., Shuvaev, V.V., and Blair, I.A. 2005. Induction of endothelial cell apoptosis by lipid hydroperoxide‐derived bifunctional electrophiles. Free Radic. Biol. Med. 39:1162‐1176.
   Knight, J.A., Pieper, R.K., and McClellan, L. 1988. Specificity of the thiobarbituric acid reaction: Its use in studies of lipid peroxidation. Clin. Chem. 34:2433‐2438.
   Kuiper, H.C., Miranda, C.L., Sowell, J.D., and Stevens, J.F. 2008. Mercapturic acid conjugates of 4‐hydroxy‐2‐nonenal and 4‐oxo‐2‐nonenal metabolites are in vivo markers of oxidative stress. J. Biol. Chem. 283:17131‐17138.
   Kuiper, H.C., Langsdorf, B.L., Miranda, C.L., Joss, J., Jubert, C., Mata, J.E., and Stevens, J.F. 2010. Quantitation of mercapturic acid conjugates of 4‐hydroxy‐2‐nonenal and 4‐oxo‐2‐nonenal metabolites in a smoking cessation study. Free Radic. Biol. Med. 48:65‐72.
   Lang, J., Celotto, C., and Esterbauer, H. 1985. Quantitative determination of the lipid peroxidation product 4‐hydroxynonenal by high‐performance liquid chromatography. Anal. Biochem. 150:369‐378.
   Lee, S.H., Rindgen, D., Bible, R.H. Jr., Hajdu, E., and Blair, I.A. 2000. Characterization of 4‐oxo‐2‐nonenal as a novel product of lipid peroxidation. Chem. Res. Toxicol. 13:565‐574.
   LoPachin, R.M., Gavin, T., Petersen, D.R., and Barber, D.S. 2009. Molecular mechanisms of 4‐hydroxy‐2‐nonenal and acrolein toxicity: Nucleophilic targets and adduct formation. Chem. Res. Toxicol. 22:1499‐1508.
   Lovell, M.A. and Markesbery, W.R. 2007. Oxidative DNA damage in mild cognitive impairment and late‐stage Alzheimer's disease. Nucleic Acids Res. 35:7497‐7504.
   Mally, A., Amberg, A., Hard, G.C., and Dekant, W. 2007. Are 4‐hydroxy‐2(E)‐nonenal derived mercapturic acids and (1)H NMR metabonomics potential biomarkers of chemically induced oxidative stress in the kidney? Toxicology 230:244‐255.
   Mitchell, D.Y. and Petersen, D.R. 1987. The oxidation of alpha‐beta unsaturated aldehydic products of lipid peroxidation by rat liver aldehyde dehydrogenases. Toxicol. Appl. Pharmacol. 87:403‐410.
   Picklo, M.J.S. and Montine, T.J. 2007. Mitochondrial effects of lipid‐derived neurotoxins. J. Alzheimers Dis. 12:185‐193.
   Poli, G., Schaur, R.J., Siems, W.G., and Leonarduzzi, G. 2008. 4‐Hydroxynonenal: A membrane lipid oxidation product of medicinal interest. Med. Res. Rev. 28:569‐631.
   Pollack, M., Oe, T., Lee, S.H., Silva Elipe, M.V., Arison, B.H., and Blair, I.A. 2003. Characterization of 2′‐deoxycytidine adducts derived from 4‐oxo‐2‐nonenal, a novel lipid peroxidation product. Chem. Res. Toxicol. 16:893‐900.
   Rahman, I., van Schadewijk, A.A., Crowther, A.J., Hiemstra, P.S., Stolk, J., MacNee, W., and De Boer, W.I. 2002. 4‐Hydroxy‐2‐nonenal, a specific lipid peroxidation product, is elevated in lungs of patients with chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 166:490‐495.
   Rathahao, E., Peiro, G., Martins, N., Alary, J., Guéraud, F., and Debrauwer, L. 2005. Liquid chromatography‐multistage tandem mass spectrometry for the quantification of dihydroxynonene mercapturic acid (DHN‐MA), a urinary end‐metabolite of 4‐hydroxynonenal. Anal. Bioanal. Chem. 381:1532‐1539.
   Rindgen, D., Nakajima, M., Wehrli, S., Xu, K., and Blair, I.A. 1999. Covalent modifications to 2′‐deoxyguanosine by 4‐oxo‐2‐nonenal, a novel product of lipid peroxidation. Chem. Res. Toxicol. 12:1195‐1204.
   Rindgen, D., Lee, S.H., Nakajima, M., and Blair, I.A. 2000. Formation of a substituted 1,N(6)‐etheno‐2′‐deoxyadenosine adduct by lipid hydroperoxide‐mediated generation of 4‐oxo‐2‐nonenal. Chem. Res. Toxicol. 13:846‐852.
   Sayre, L.M., Arora, P.K., Iyer, R.S., and Salomon, R.G. 1993. Pyrrole formation from 4‐hydroxynonenal and primary amines. Chem. Res. Toxicol. 6:19‐22.
   Slatter, J.G., Rashed, M.S., Pearson, P.G., Han, D.H., and Baillie, T.A. 1991. Biotransformation of methyl isocyanate in the rat. Evidence for glutathione conjugation as a major pathway of metabolism and implications for isocyanate‐mediated toxicities. Chem. Res. Toxicol. 4:157‐161.
   Spiteller, G. 2007. The important role of lipid peroxidation processes in aging and age dependent diseases. Mol. Biotechnol. 37:5‐12.
   Srivastava, S., Dixit, B.L., Cai, J., Sharma, S., Hurst, H.E., Bhatnagar, A., and Srivastava, S.K. 2000. Metabolism of lipid peroxidation product, 4‐hydroxynonenal (HNE) in rat erythrocytes: Role of aldose reductase. Free Radic. Biol. Med. 29:642‐651.
   Van Kuijk, F.J., Thomas, D.W., Stephens, R.J., and Dratz, E.A. 1986. Occurrence of 4‐hydroxyalkenals in rat tissues determined as pentafluorobenzyl oxime derivatives by gas chromatography‐mass spectrometry. Biochem. Biophys. Res. Commun. 139:144‐149.
   Warnke, M.M., Wanigasekara, E., Singhal, S.S., Singhal, J., Awasthi, S., and Armstrong, D.W. 2008. The determination of glutathione‐4‐hydroxynonenal (GSHNE), E‐4‐hydroxynonenal (HNE), and E‐1‐hydroxynon‐2‐en‐4‐one (HNO) in mouse liver tissue by LC‐ESI‐MS. Anal. Bioanal. Chem. 392:1325‐1333.
   Winter, C.K., Segall, H.J., and Haddon, W.F. 1986. Formation of cyclic adducts of deoxyguanosine with the aldehydes trans‐4‐hydroxy‐2‐hexenal and trans‐4‐hydroxy‐2‐nonenal in vitro. Cancer Res. 46:5682‐5686.
Key References
   Annangudi et al., 2005. See above.
  Key reference for the synthesis of ONA. A modification of this method is described in .
   Kuiper et al., 2008. See above.
  Key reference for the distinction between isomeric LPO‐MA conjugates HNE‐MA and ONO‐MA. One of the methods on which the and Support Protocols 1 to 5 and 7 are based.
   Kuiper et al., 2010. See above.
  Key reference for the quantitation of LPO‐MA conjugates using isotope‐dilution mass spectrometry. One of the methods on which the and Support Protocols 1 to 7 are based.
   Slatter et al., 1991. See above.
  Key reference for the synthesis of MAd3. A modification of this method is described in .
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