Fluorescence Microplate Reader Measurement of Tissue Susceptibility to Lipid Peroxidation

Jin‐Gang Zhang1, Marc W. Fariss2

1 BD Biosciences, Woburn, Massachusetts, 2 University of Colorado Health Sciences Center, Denver, Colorado
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 17.3
DOI:  10.1002/0471140856.tx1703s18
Online Posting Date:  February, 2004
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This unit describes a method for the measurement of cellular membrane antioxidant capacity or susceptibility of tissue samples to lipid peroxidation using a fluorescence microplate reader. The assay is simple and has the advantage of monitoring susceptibility to lipid peroxidation in a large number of samples in real time.

Keywords: antioxidant; susceptibility to lipid peroxidation; antioxidant capacity; fluorescence plate reader; vitamin E; vitamin E succinate

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • Tissue, cells, or subcellular organelles
  • 50 mM potassium phosphate buffer, pH 7.4 (see recipe)
  • Glycine/ascorbate solution (see recipe)
  • Initiator solution (see recipe), fresh
  • Sonicator
  • Flat‐bottomed 96‐well microplates (Rainin Instrument)
  • Cytofluor 4000 fluorescence multiwell microplate reader (PerSeptive Biosystems) with temperature control
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Literature Cited

Literature Cited
   Benzie, I.F.F. and Strain, J.J. 1996. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Anal. Biochem. 239:70‐76.
   Cervato, G., Viani, P., Cazzola, R., and Cestaro, B. 1999. A fluorescence method for the determination of plasma susceptibility to lipid peroxidation. Clin. Biochem. 32:171‐177.
   Chevion, S., Roberts, M.A., and Chevion, M. 2000. The use of cyclic voltammetry for the evaluation of antioxidant capacity. Free Radic. Biol. Med. 28:860‐870.
   Fariss, M.W., Merson, M.H., and O' Hara, T.M. 1989. Alpha tocopheryl succinate protects hepatocytes from chemical‐induced toxicity under physiological calcium conditions. Toxicol. Lett. 47:61‐75.
   Fariss, M.W., Nicholls‐Grzemski, F.A., Tirmenstein, M.A., and Zhang, J‐G. 2001. Enhanced antioxidant and cytoprotective abilities of vitamin E succinate is associated with a rapid uptake advantage in rat hepatocytes and mitochondria. Free Rad. Biol. Med. 31:530‐541.
   Ghiselli, A., Serafini, M., Natella, F., and Scaccini, C. 2000. Total antioxidant capacity as a tool to assess redox status: Critical view and experimental data. Free Rad. Biol. Med. 29:1106‐1114.
   Hovius, R., Lambrechts, H., Nicolay, K., and De Kruijff, B. 1990. Improved methods to isolate and subfractionate rat liver mitochondria. Lipid composition of the inner and outer membrane. Biochim. Biophys. Acta. 1021:217‐226.
   Lesnefsky, E.J., Slabe, T.J., Stoll, M.S.K., Minkler, P.E., and Hoppel, C.L., 2001. Myocardial ischemia selectively depletes cardiolipin in rabbit heart subsarcolemmal mitochondria. Am. J. Physiol. Heart Circ. Physiol. 280:H2770‐H2778.
   Niki, E. and Noguchi, N., 2000. Evaluation of antioxidant capacity. What capacity is being measured by which method? IUBMB Life 50:323‐329.
   Re, R., Pellegrini, N., Proteggente, A, Pannala, A., Yang, M., and Rice‐Evans, C. 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Rad. Biol. Med. 26:1231‐1237.
   Tirmenstein, M.A., Pierce, C.A., Leraas, T.L., and Fariss, M.W. 1998. A fluorescence plate reader assay for monitoring the susceptibility of biological samples to lipid peroxidation. Anal. Biochem. 265:246‐252.
   Wayner, D.D.M., Burton, G.W., Ingold, K.U., and Locke, S. 1985. Quantitative measurement of the total, peroxyl radical‐trapping antioxidant capacity of human blood plasma by controlled peroxidation. FEBS Lett. 187:33‐37.
   Wiswedel, I., Ulbricht, O., and Augustin, W. 1989. Studies of lipid peroxidation in isolated rat heart mitochondria. Biomed. Biochim. Acta 48:S73‐S76.
   Woodford, F.P. and Whitehead, T.P. 1998. Is measuring serum antioxidant capacity clinically useful? Ann. Clin. Biochem. 35:48‐56.
   Yu, T‐W. and Ong, C.N. 1999. Lag‐time measurement of antioxidant capacity using myoglobin and 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid): Rationale, application, and limitation. Anal. Biochem. 275:217‐223.
   Zhang, J.G. and Fariss, M.W. 2002. Thenoyltrifluoroacetone, a potent inhibitor of carboxylesterase activity. Biochem. Pharmacol. 63:753‐757.
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