Jump to ContentJump to Main Navigation
Show Summary Details
More options …

Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Ed. by Gillery, Philippe / Greaves, Ronda / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter

IMPACT FACTOR 2018: 3.638

CiteScore 2018: 2.44

SCImago Journal Rank (SJR) 2018: 1.191
Source Normalized Impact per Paper (SNIP) 2018: 1.205

See all formats and pricing
More options …
Volume 42, Issue 8


Preventing in vitro lipoperoxidation in the malondialdehyde-thiobarbituric assay

Ricardo Gonzalo
  • Centre d’Investigacions en Bioquímica i Biologia Molecular, Hospital Vall d’Hebron, Barcelona, Spain
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Cristofol Vives-Bauza
  • Centre d’Investigacions en Bioquímica i Biologia Molecular, Hospital Vall d’Hebron, Barcelona, Spain
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Antonio L. Andreu
  • Centre d’Investigacions en Bioquímica i Biologia Molecular, Hospital Vall d’Hebron, Barcelona, Spain
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Elena García-Arumí
  • Centre d’Investigacions en Bioquímica i Biologia Molecular, Hospital Vall d’Hebron, Barcelona, Spain
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
Published Online: 2005-06-01 | DOI: https://doi.org/10.1515/CCLM.2004.146


The malondialdehyde-thiobarbituric acid assay is widely used to study lipid peroxidation. Among the various methods used to perform the assay, the most widely accepted is the quantification of malondialdehyde using the thiobarbituric acid reaction, followed by reversed-phase chromatography. However, unacceptable results may be obtained as malondialdehyde can be produced in vitro. To study the conditions that inhibit in vitro lipid peroxidation, malondialdehyde levels were measured in cultured cells using different concentrations of butylated hydroxytoluene, EDTA or a combination of both. Butylated hydroxytoluene alone inhibits in vitro lipid peroxidation effectively. EDTA reduces artificially produced malondialdehyde, but not totally. Finally, the combination of EDTA and butylated hydroxytoluene does not improve the results obtained using butylated hydroxytoluene alone. The conclusion is that in the malondialdehyde-thiobarbituric acid assay it is necessary to add an inhibitor of the in vitro lipid peroxidation and assay the necessary concentration depending on the specimen used.

Keywords: butylated hydroxytoluene (BHT); high pressure liquid chromatography (HPLC); lipid peroxidation; malondialdehyde; oxidative stress


  • 1

    Pryor WA, Stanley JP, Blair E. Autoxidation of polyunsaturated fatty acids: II. A suggested mechanism for the formation of TBA-reactive materials from prostaglandin-like endoperoxides. Lipids 1976; 11:370–9.CrossrefGoogle Scholar

  • 2

    Halliwell B. Free radicals, reactive oxygen species and human disease: a critical evaluation with special reference to atherosclerosis. Br J Exp Pathol 1989; 70:737–57.Google Scholar

  • 3

    Yagi K. A simple fluorometric assay for lipoperoxide in blood plasma. Biochem Med 1976; 15:212–6.CrossrefGoogle Scholar

  • 4

    Yu LW, Latriano L, Duncan S, Hartwick RA, Witz G. High-performance liquid chromatography analysis of the thiobarbituric acid adducts of malonaldehyde and trans,trans-muconaldehyde. Anal Biochem 1986; 156:326–33.Google Scholar

  • 5

    Sinhuber RO, Yu TC. Characterization of the red pigment formed in the 2-thiobarbituric acid determination of oxidative rancidity. Food Res 1958; 23:626–34.Google Scholar

  • 6

    Slater TF, Sawyer BC. The stimulatory effects of carbon tetrachloride on peroxidative reactions in rat liver fractions in vitro. Inhibitory effects of free-radical scavengers and other agents. Biochem J 1971; 123:823–8.Google Scholar

  • 7

    Bird RP, Draper HH. Comparative studies on different methods of malonaldehyde determination. Methods Enzymol 1984; 105:299–305.Google Scholar

  • 8

    Gilbert HS, Stump DD, Roth EF Jr. A method to correct for errors caused by generation of interfering compounds during erythrocyte lipid peroxidation. Anal Biochem 1984; 137:282–6.Google Scholar

  • 9

    Bird RP, Hung SS, Hadley M, Draper HH. Determination of malonaldehyde in biological materials by high-pressure liquid chromatography. Anal Biochem 1983; 128:240–4.Google Scholar

  • 10

    Jordan RA, Schenkman JB. Relationship between malondialdehyde production and arachidonate consumption during NADPH-supported microsomal lipid peroxidation. Biochem Pharmacol 1982; 31:393–400.CrossrefGoogle Scholar

  • 11

    Yagi K. Assay for serum lipid peroxide level and its clinical significance. In Yagi K, editor. Lipid peroxides in biology and medicine. New York: Academic Press, 1982:223–42.Google Scholar

  • 12

    Young LS, Trimble ER. Measurement of malondialdehyde in plasma by high performance liquid chromatography with fluorimetric detection. Ann Clin Biochem 1991; 28:504–8.CrossrefGoogle Scholar

  • 13

    Fraga CG, Leibovitz BE, Tappel AL. Lipid peroxidation measured as thiobarbituric acid-reactive substances in tissue slices: characterization and comparison with homogenates and microsomes. Free Radic Biol Med 1988; 4:155–61.Google Scholar

  • 14

    Rice-Evans CA, Diplock AT, Symons MC. Techniques in free radical research. In: Burdon RH, van Knippenberg PH, editors. Laboratorytechniques in biochemistry and molecular biology. Amsterdam: Elsevier, 1991:125–84.Google Scholar

  • 15

    Knight JA, Voorhees RP, Martin L. The effect of metal chelators on lipid peroxidation in stored erythrocytes. Ann Clin Lab Sci 1992; 22:207–13.Google Scholar

  • 16

    Petrusca JM, Wong SH, Sunderman Jr FW, Mossman BT. Detection of lipid peroxidation in lung and in bronchoalveolar lavage cells and fluid. Free Radic Biol Med 1990; 9:51–8.Google Scholar

  • 17

    Lepage G, Munoz G, Champagne J, Roy CC. Preparative steps necessary for the accurate measurement of malondialdehyde by high-performance liquid chromatography. Anal Biochem 1991; 197:277–83.Google Scholar

  • 18

    Nielsen F, Borg Mikkelsen B, Bo Nielsen J, Andersen HR, Grandjean P. Plasma malondialdehyde as biomarker for oxidative stress: reference interval and effects of lifestyle factors. Clin Chem 1997; 43:1209–17.Google Scholar

  • 19

    Li XY, Chow CK. An improved method for the measurement of malondialdehyde in biological samples. Lipids 1994; 29:73–5.CrossrefGoogle Scholar

  • 20

    Pang CY, Lee HC, Wei YH. Enhanced oxidative damage in human cells harboring A3243G mutation of mitochondrial DNA: implication of oxidative stress in the pathogenesis of mitochondrial diabetes. Diabetes Res Clin Pract 2001; 54:S45–6.CrossrefGoogle Scholar

  • 21

    Volpi N, Tarugi P. Improvement in the high-performance liquid chromatography malondialdehyde level determination in normal human plasma. J Chromatogr B Biomed Sci Appl 1998; 713:433–7.Google Scholar

  • 22

    Suttnar J, Masova L, Dyr JE. Influence of citrate and EDTA anticoagulants on plasma malondialdehyde concentrations estimated by high-performance liquid chromatography. J Chromatogr B Biomed Sci 2001; 751:193–7.Google Scholar

  • 23

    Richard MJ, Guiraud P, Meo J, Favier A. High-performance liquid chromatographic separation of malondialdehyde-thiobarbituric acid adduct in biological materials (plasma and human cells) using a commercially available reagent. J Chromatogr Biomed Appl 1992; 577:9–18.Google Scholar

  • 24

    Agarwal R, Chase SD. Rapid, fluorimetric-liquid chromatographic determination of malondialdehyde in biological samples. J Chromatogr B Biomed Sci 2002; 775:121–6.Google Scholar

  • 25

    Fukunaga K, Suzuki T, Takama K. Highly sensitive high-performance liquid chromatography for the measurement of malondialdehyde in biological samples. J Chromatogr 1993; 621:77–81.Google Scholar

  • 26

    Jentzsch AM, Bachmann H, Fürst P, Biesalski HK. Improved analysis of malondialdehyde in human body fluids. Free Radic Biol Med 1996; 20:251–6.Google Scholar

  • 27

    Lambert CR, Black HS, Truscott TG. Reactivity of butylated hydroxytoluene. Free Radic Biol Med 1996; 21:395–400.Google Scholar

About the article

Corresponding author: Elena García-Arumí, PhD, Centre d’Investigacions en Bioquímica i Biologia Molecular, Hospital Vall d’Hebron, Passeig Vall d’Hebron 119-129, 08035 Barcelona, Spain. Phone: (+34)934894054, Fax: (+34)934894040, E-mail:

Received: 2004-02-02

Accepted: 2004-07-16

Published Online: 2005-06-01

Published in Print: 2004-08-01

Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), Volume 42, Issue 8, Pages 903–906, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/CCLM.2004.146.

Export Citation

© Walter de Gruyter.Get Permission

Citing Articles

Here you can find all Crossref-listed publications in which this article is cited. If you would like to receive automatic email messages as soon as this article is cited in other publications, simply activate the “Citation Alert” on the top of this page.

Ricardo Gonzalo, Elena Garcia-Arumi, David Llige, Ramon Marti, Abelardo Solano, Julio Montoya, Joaquín Arenas, and Antonio L. Andreu
FEBS Letters, 2005, Volume 579, Number 30, Page 6909
Cristofol Vives-Bauza, Ricardo Gonzalo, Giovanni Manfredi, Elena Garcia-Arumi, and Antonio L. Andreu
Neuroscience Letters, 2006, Volume 391, Number 3, Page 136
Carole B. Rudra, Chunfang Qiu, Robert M. David, J. Alexander Bralley, Scott W. Walsh, and Michelle A. Williams
Clinical Biochemistry, 2006, Volume 39, Number 7, Page 722

Comments (0)

Please log in or register to comment.
Log in