Name: evaluation of oxidative stress in biological samples using the thiobarbituric acid reactive substances assay
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Description: Watch this Scientific Journal Video about Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay at JoVE.com

The research reported here was supported in part by the National Cancer Institute of the National Institutes of Health under award no. R33 CA217702 and the Initiative for Maximizing Student Development (IMSD) program. The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health.

Human serum specimens were obtained from consenting volunteers under IRB approval and according to the principles expressed in the Declaration of Helsinki. Specimens were coded and de-identified before transfer to the analytical laboratory.
1. Sample preparation
<ol>
	<li>HepG2 cell lysates
	<ol>
		<li>Seed about 10 x 106 HepG2 cells per flask in 16 T75 flasks with 14 mL of EMEM media supplemented with 10% fetal bovine serum (FBS) and grow cells for 2 days.</li>
	...

<ol>
	<li>Tsikas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+lipid+peroxidation+by+measuring+malondialdehyde+(MDA)+and+relatives+in+biological+samples:+Analytical+and+biological+challenges.">Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples: Analytical and biological challenges.</a> Analytical Biochemistry. 524, 13-30 (2017).</li><li>Ohkawa, H., Ohishi, N., Yagi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction+of+linoleic+acid+hydroperoxide+with+thiobarbituric+acid.">Reaction of linoleic acid hydroperoxide with thiobarbituric acid.</a> Journal of Lipid Research. 19 (8), 1053-1057 (1978).</li><li>Khoubnasabjafari, M., Soleymani, J., Jouyban, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avoid+Using+Spectrophotometric+Determination+of+Malondialdehyde+as+a+Biomarker+of+Oxidative+Stress.">Avoid Using Spectrophotometric Determination of Malondialdehyde as a Biomarker of Oxidative Stress.</a> Biomarkers in Medicine. 12 (6), 551-554 (2018).</li><li>Morales, M., Munn&#233;-Bosch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malondialdehyde:+Facts+and+artifacts.">Malondialdehyde: Facts and artifacts.</a> Plant Physiology. 180 (3), 1246-1250 (2019).</li><li>Devasagayam, T. P. A., Boloor, K. K., Ramasarma, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+estimating+lipid+peroxidation:+An+analysis+of+merits+and+demerits.">Methods for estimating lipid peroxidation: An analysis of merits and demerits.</a> Indian Journal of Biochemistry and Biophysics. 40 (5), 300-308 (2003).</li><li>Dasgupta, A., Klein, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+Measuring+Oxidative+Stress+in+the+Laboratory.">Methods for Measuring Oxidative Stress in the Laboratory.</a> Antioxidants in Food, Vitamins and Supplements. , 19-40 (2014).</li><li>Wade, C. R., van Rij, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+malondialdehyde,+lipid+peroxides,+and+the+thiobarbituric+acid+reaction.">Plasma malondialdehyde, lipid peroxides, and the thiobarbituric acid reaction.</a> Clinical Chemistry. 35 (2), 336-336 (1989).</li><li>Khoubnasabjafari, M., Ansarin, K., Jouyban, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+of+malondialdehyde+as+a+biomarker+of+oxidative+stress+in+psychological+disorders.">Reliability of malondialdehyde as a biomarker of oxidative stress in psychological disorders.</a> BioImpacts. 5 (3), 123-127 (2015).</li><li>Khoubnasabjafari, M., Ansarin, K., Jouyban, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comments+Concerning+"Comparison+of+Airway+and+Systemic+Malondialdehyde+Levels+for+Assessment+of+Oxidative+Stress+in+Cystic+Fibrosis".">Comments Concerning "Comparison of Airway and Systemic Malondialdehyde Levels for Assessment of Oxidative Stress in Cystic Fibrosis".</a> Lung. 193 (5), 867-868 (2015).</li><li>Khoubnasabjafari, M., Ansarin, K., Vaez-Gharamaleki, J., Jouyban, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comments+on+"Salivary+8-hydroxy-2-deoxyguanosine,+malondialdehyde,+vitamin+C,+and+vitamin+E+in+oral+pre-cancer+and+cancer:+diagnostic+value+and+free+radical+mechanism+of+action".">Comments on "Salivary 8-hydroxy-2-deoxyguanosine, malondialdehyde, vitamin C, and vitamin E in oral pre-cancer and cancer: diagnostic value and free radical mechanism of action".</a> Clinical Oral Investigations. 20 (2), 395-396 (2016).</li><li>Khoubnasabjafari, M., Ansarin, K., Jouyban, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comments+on+"An+Investigation+into+the+Serum+Thioredoxin+Superoxide+Dismutase,+Malondialdehyde,+and+Advanced+Oxidation+Protein+Products+in+Patients+with+Breast+Cancer".">Comments on "An Investigation into the Serum Thioredoxin Superoxide Dismutase, Malondialdehyde, and Advanced Oxidation Protein Products in Patients with Breast Cancer".</a> Annals of Surgical Oncology. 24, 573-576 (2017).</li><li>Azizi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+analytical+procedures+on+the+repeatability+of+malondialdehyde+determinations+in+biological+samples.">Effects of analytical procedures on the repeatability of malondialdehyde determinations in biological samples.</a> Pharmaceutical Sciences. 23 (3), 193-197 (2017).</li><li>Azizi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+possible+reason+for+the+low+reproducibility+of+malondialdehyde+determinations+in+biological+samples.">A possible reason for the low reproducibility of malondialdehyde determinations in biological samples.</a> Bioanalysis. 8 (21), 2179-2181 (2016).</li><li>Wasowicz, W., Neve, J., Peretz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+steps+in+fluorometric+determination+of+thiobarbituric+acid-+reactive+substances+in+serum:+Importance+of+extraction+pH+and+influence+of+sample+preservation+and+storage.">Optimized steps in fluorometric determination of thiobarbituric acid- reactive substances in serum: Importance of extraction pH and influence of sample preservation and storage.</a> Clinical Chemistry. 39 (12), 2522-2526 (1993).</li><li>Jentzsch, A. M., Bachmann, H., F&#252;rst, P., Biesalski, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+analysis+of+malondialdehyde+in+human+body+fluids.">Improved analysis of malondialdehyde in human body fluids.</a> Free Radical Biology and Medicine. 20 (2), 251-256 (1996).</li><li>Buege, J. A., Aust, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsomal+lipid+peroxidation.">Microsomal lipid peroxidation.</a> Methods in Enzymology. 52, 302-310 (1978).</li><li>Gutteridge, J. M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free-Radical+Damage+to+Lipids,+Amino-Acids,+Carbohydrates+and+Nucleic-Acids+Determined+by+Thiobarbituric+Acid+Reactivity.">Free-Radical Damage to Lipids, Amino-Acids, Carbohydrates and Nucleic-Acids Determined by Thiobarbituric Acid Reactivity.</a> International Journal of Biochemistry. 14 (7), 649-653 (1982).</li><li>Khoubnasabjafari, M., Ansarin, K., Jouyban, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+malondialdehyde+as+an+oxidative+stress+biomarker+in+oral+and+systemic+diseases.">Salivary malondialdehyde as an oxidative stress biomarker in oral and systemic diseases.</a> J Dent Res Dent Clin Dent Prospects. 10 (2), 71-74 (2016).</li><li>Halliwell, B., Whiteman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+reactive+species+and+oxidative+damage+in+vivo+and+in+cell+culture:+How+should+you+do+it+and+what+do+the+results+mean.">Measuring reactive species and oxidative damage in vivo and in cell culture: How should you do it and what do the results mean.</a> British Journal of Pharmacology. 142 (2), 231-255 (2004).</li><li>Lee, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+oxidative+stress+in+human+cardiovascular+disease:+methodological+aspects+and+considerations.">Evaluating oxidative stress in human cardiovascular disease: methodological aspects and considerations.</a> Current medicinal chemistry. 19 (16), 2504-2520 (2012).</li><li>Morel, D. W., Hessler, J. R., Chisolm, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+density+lipoprotein+cytotoxicity+induced+by+free+radical+peroxidation+of+lipid.">Low density lipoprotein cytotoxicity induced by free radical peroxidation of lipid.</a> Journal of Lipid Research. 24 (8), 1070-1076 (1983).</li><li>Guzm&#225;n-Chozas, M., Vicario-Romero, I. M., Guill&#233;n-Sans, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2-thiobarbituric+acid+test+for+lipid+oxidation+in+food:+Synthesis+and+spectroscopic+study+of+2-thiobarbituric+acid-malonaldehyde+adduct.">2-thiobarbituric acid test for lipid oxidation in food: Synthesis and spectroscopic study of 2-thiobarbituric acid-malonaldehyde adduct.</a> Journal of the American Oil Chemists Society. 75 (12), 1711-1715 (1998).</li><li>Shibata, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+lipid+peroxidation+product+as+a+potential+trigger+of+the+p53+pathway.">Identification of a lipid peroxidation product as a potential trigger of the p53 pathway.</a> Journal of Biological Chemistry. 28 (2), 1196-1204 (2006).</li><li>Skoog, D. A., West, D. M., Holler, F. J., Crouch, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sampling,+standardization,+and+calibration.">Sampling, standardization, and calibration.</a> Fundamentals of Analytical Chemistry. 9th ed. , 153-196 (2014).</li><li>Skoog, D. A., Holler, F. J., Crouch, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction.">Introduction.</a> Principles of Instrumental Analysis. 6th ed. , 1-24 (2007).</li><li>Seibig, S., Van Eldik, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+[FeII(edta)]+Oxidation+by+Molecular+Oxygen+Revisited.+New+Evidence+for+a+Multistep+Mechanism.">Kinetics of [FeII(edta)] Oxidation by Molecular Oxygen Revisited. New Evidence for a Multistep Mechanism.</a> Inorganic Chemistry. 36 (18), 4115-4120 (1997).</li><li>Jeffs, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delta-S-Cys-Albumin:+A+Lab+Test+that+Quantifies+Cumulative+Exposure+of+Archived+Human+Blood+Plasma+and+Serum+Samples+to+Thawed+Conditions.">Delta-S-Cys-Albumin: A Lab Test that Quantifies Cumulative Exposure of Archived Human Blood Plasma and Serum Samples to Thawed Conditions.</a> Molecular &amp; Cellular Proteomics. 18 (10), 2121-2137 (2019).</li><li>Yagi, K., Armstrong, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+Assay+for+the+Level+of+Total+Lipid+Peroxides+in+Serum+or+Plasma.">Simple Assay for the Level of Total Lipid Peroxides in Serum or Plasma.</a> Free Radical and Antioxidant Protocols. Methods in Molecular Biology. , 101-106 (1998).</li><li>Bernheim, F., Bernheim, M. L. C., Wilbur, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reaction+between+thiobarbituric+acid+and+the+oxidation+products+of+certain+lipides.">The reaction between thiobarbituric acid and the oxidation products of certain lipides.</a> Journal of Biological Chemistry. 174 (1), 257-264 (1948).</li><li>Wilbur, K. M., Bernheim, F., Shapiro, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thiobarbituric+acid+reagent+as+a+test+for+the+oxidation+of+unsaturated+fatty+acids+by+various+agents.">The thiobarbituric acid reagent as a test for the oxidation of unsaturated fatty acids by various agents.</a> Archives of Biochemistry. 24 (2), 305-313 (1949).</li><li>Kwon, T. W., Watts, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+malonaldehyde+by+ultraviolet+spectrophotometry.">Determination of malonaldehyde by ultraviolet spectrophotometry.</a> Journal of Food Science. 28 (6), 627-630 (1963).</li><li>Esterbauer, H., Schaur, F. J., Zollner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+and+biochemistry+of+4-hydroxynonenal,+malonaldehyde+and+related+aldehydes.">Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes.</a> Free Radical Biology &amp; Medicine. 11 (1), 81-128 (1991).</li><li>Dalle-Donne, I., Rossi, R., Colombo, R., Giustarini, D., Milzani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+of+oxidative+damage+in+human+disease.">Biomarkers of oxidative damage in human disease.</a> Clinical Chemistry. 52 (4), 601-623 (2006).</li><li>Jentzsch, A. M., Bachmann, H., F&#252;rst, P., Biesalski, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+analysis+of+malondialdehyde+in+human+body+fluids.">Improved analysis of malondialdehyde in human body fluids.</a> Free Radical Biology and Medicine. 20 (2), 251-256 (1996).</li><li>Jo, C., Ahn, D. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorometric+Analysis+of+2-Thiobarbituric+Acid+Reactive+Substances+in+Turkey.">Fluorometric Analysis of 2-Thiobarbituric Acid Reactive Substances in Turkey.</a> Poultry Science. 77 (3), 475-480 (1998).</li><li>Tsikas, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development,+validation+and+biomedical+applications+of+stable-isotope+dilution+GC-MS+and+GC-MS/MS+techniques+for+circulating+malondialdehyde+(MDA)+after+pentafluorobenzyl+bromide+derivatization:+MDA+as+a+biomarker+of+oxidative+stress+and+its+relation+to+15(S)-8-iso-prostaglandin+F2&#945;+and+nitric+oxide+(NO).">Development, validation and biomedical applications of stable-isotope dilution GC-MS and GC-MS/MS techniques for circulating malondialdehyde (MDA) after pentafluorobenzyl bromide derivatization: MDA as a biomarker of oxidative stress and its relation to 15(S)-8-iso-prostaglandin F2&#945; and nitric oxide (NO).</a> Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences. 1019, 95-111 (2016).</li><li>Barden, A. E., Mas, E., Croft, K. D., Phillips, M., Mori, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimizing+artifactual+elevation+of+lipid+peroxidation+products+(F+2-isoprostanes)+in+plasma+during+collection+and+storage.">Minimizing artifactual elevation of lipid peroxidation products (F 2-isoprostanes) in plasma during collection and storage.</a> Analytical Biochemistry. 449 (1), 129-131 (2014).</li><li>Jeffs, J. W., Ferdosi, S., Yassine, H. N., Borges, C. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malondialdehyde+in+Stored+Plasma.">Malondialdehyde in Stored Plasma.</a> Biochemical and Biophysical Research Communications. 95 (4), 1663-1672 (1980).</li><li>Tsikas, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+GC-MS/MS+measurement+of+malondialdehyde+and+4-hydroxy-2-nonenal+in+human+plasma:+Effects+of+long-term+L-arginine+administration.">Simultaneous GC-MS/MS measurement of malondialdehyde and 4-hydroxy-2-nonenal in human plasma: Effects of long-term L-arginine administration.</a> Analytical Biochemistry. 524, 31-44 (2017).</li></ol>

Despite its limitations1,3,4,7,8,9,10,12,13,14,15,19 and a lack of suitability for comparison between laboratories, t...

Lipid peroxidation is a process in which free radicals, such as reactive oxygen species and reactive nitrogen species, attack carbon-carbon double bonds in lipids, a process that involves the abstraction of a hydrogen from a carbon and insertion of an oxygen molecule. This process leads to a mixture of complex products including, lipid peroxyl radicals, and hydroperoxides as the primary products, as well as malondialdehyde (MDA) and 4-hydroxynonenal as predominant secondary products1.
MDA has been widely used in biomedical research as a marker of lipid peroxidation due to its facile reaction with thiobarbituric acid (TBA). The reaction leads to the formation of MDA-TBA2, a conjugate that absorbs in the visible spectrum at 532 nm and produces a red-pink color2. Other molecules derived from lipid peroxidation besides MDA can also react with TBA and absorb light at 532 nm, contributing to the overall absorption signal that is measured. Similarly, MDA can react with most other major classes of biomolecules, potentially limiting its accessibility for reaction with TBA3,4. As such, this traditional assay is simply considered to measure &#8220;thiobarbituric acid reactive substances&#8221; or TBARS5.
When correctly applied and interpreted, the TBARS assay is generally considered a good indicator of the overall levels of oxidative stress in a biological sample6. Unfortunately, as documented by Khoubnasabjafari and others, the TBARS assay is often conducted and interpreted in ways that facilitate dubious conclusions3,4,7,8,9,10,11. The causes for this are rooted primarily in sample-related pre-analytical variables and a lack of assay ruggedness that prohibits seemingly minor variations in assay protocol without substantial changes in assay results1,7,12,13.
Preanalytical variables related to biospecimen handling and storage (e.g., blood plasma kept temporarily at -20 &#176;C)14,15 can have a major impact on TBARS assay results16,17; so much so, that TBARS assay results should not be compared across different laboratories unless warranted by explicit interlaboratory analytical validation data. This recommendation is akin to how western blots are commonly used and interpreted. Comparisons of band densities are valid for within-blot and perhaps within-laboratory studies, but comparing band densities between laboratories is generally considered an invalid practice.
Some researchers have suggested that MDA as measured by the TBARS assay simply does not meet the analytical or clinical criteria required of an acceptable biomarker3,9,10,18,19. Indeed, if the assay had not been developed over 50 years ago, it probably would not have gained the widespread use and tacit acceptability that it has today. Although there are other assays with greater analytical sensitivity, specificity, and ruggedness used for determining oxidative stress, TBARS assay based on absorbance at 532 nm remains by far one of the most commonly used assays for the determination of lipid peroxidation20, and thereby assessment of oxidative stress.
The TBARS assay can only be found as an expensive kit (over 400 U.S. dollars), in which the instructions do not provide detailed information on most concentrations of the reagents used. Additionally, the reagents provided can only be used for one experiment, because only one colorimetric standard curve can be made per kit. This can be problematic for researchers who intend to determine levels of oxidation within a few samples at different timepoints, because the same standard curve cannot be used at multiple times. Hence, multiple kits need to be purchased for multiple experiments. Currently, unless an expensive kit is purchased, there is not a detailed protocol available for how to perform a TBARS assay. Some researchers in the past have vaguely described how to perform a TBARS assay21,22, but neither a fully detailed protocol or comprehensive video on how to conduct the TBARS assay without an expensive kit is available in the literature.
Here we report a detailed, analytically validated for-purpose methodology on how to perform a TBARS assay in a simple, reproducible, and inexpensive way. Changes in the lipid peroxidation of human serum, HepG2 lysates, and low density lipoproteins upon treatment with Cu(II) ions are demonstrated as illustrative applications for the TBARS assay. Results demonstrate that this TBARS assay is consistent and reproducible on a day-to-day basis.

<table>
	<tbody>
		<tr>
			<td>1x Sterile PBS pH 7.4 1 L</td>
			<td>VWR, PA</td>
			<td>101642--262</td>
			<td>cell lysis reagent</td>
		</tr>
		<tr>
			<td>50 mL self-standing centrifuge tube</td>
			<td>Corning, NY</td>
			<td>CLS430897</td>
			<td>General material</td>
		</tr>
		<tr>
			<td>96 well plate, Non-Treated, clear, with lid, Non-sterile</td>
			<td>Thermo Fisher Scientific, MA</td>
			<td>280895</td>
			<td>To measure absorbance</td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 100 kD centrifugal spin filter device</td>
			<td>Fisher Scientific, NH</td>
			<td>UFC510024</td>
			<td>LDL purification</td>
		</tr>
		<tr>
			<td>Caps for glass tubes</td>
			<td>Thermo Fisher Scientific, MA</td>
			<td>14-930-15D</td>
			<td>for TBARS assay</td>
		</tr>
		<tr>
			<td>Copper II Chloride</td>
			<td>SIGMA, MO</td>
			<td>222011-250G</td>
			<td>to induce oxidation</td>
		</tr>
		<tr>
			<td>Culture tubes, Disposable, with Screw-Cap Finish, Borosilicate Glass (13 x 100 mm)</td>
			<td>VWR, PA</td>
			<td>53283-800</td>
			<td>for TBARS assay</td>
		</tr>
		<tr>
			<td>Eagle&#39;s Minimum Essential Medium (EMEM)</td>
			<td>ATCC, VA</td>
			<td>HB-8065</td>
			<td>HepG2 cell media</td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes, 1.5 mL</td>
			<td>eppendorf, NY</td>
			<td>22363204</td>
			<td>General material</td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes, 2.0 mL</td>
			<td>Genesee Sceitific, CA</td>
			<td>22363352</td>
			<td>General material</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum US Source</td>
			<td>Omega Scientific, CA</td>
			<td>FB-11</td>
			<td>for cell culture</td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>SIGMA, MO</td>
			<td>27225-1L-R</td>
			<td>TBARS Reagent</td>
		</tr>
		<tr>
			<td>Halt Protease Inhibitor Cocktail (100x)</td>
			<td>Thermo Scientific, MA</td>
			<td>87786</td>
			<td>cell lysis reagent</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>SIGMA, MO</td>
			<td>H3375-250G</td>
			<td>LDL solvent</td>
		</tr>
		<tr>
			<td>HepG2 Cells</td>
			<td>ATCC, VA</td>
			<td>HB-8065</td>
			<td>Biological matrix prototype</td>
		</tr>
		<tr>
			<td>Hydrocloric acid (HCl)</td>
			<td>Fisher Scientific, NH</td>
			<td>A144-212</td>
			<td>cell lysis reagent</td>
		</tr>
		<tr>
			<td>Legend Micro 17 Centrifuge</td>
			<td>Thermo Scientific, MA</td>
			<td>75002431</td>
			<td>General material</td>
		</tr>
		<tr>
			<td>Low Density Lipoprotein, Human Plasma</td>
			<td>Athens Research &#38; Technology, GA</td>
			<td>12-16-120412</td>
			<td>Biological matrix prototype</td>
		</tr>
		<tr>
			<td>Magnetic Stir Bars, Octagon 6-Assortment</td>
			<td>VWR, PA</td>
			<td>58948-025</td>
			<td>General material</td>
		</tr>
		<tr>
			<td>Malondialdehyde bis (dimethyl acetal)</td>
			<td>SIGMA, MO</td>
			<td>8207560250</td>
			<td>TBARS Standard</td>
		</tr>
		<tr>
			<td>Multiskan Go Microplate Spectrophotometer</td>
			<td>Fisher Scientific, NH</td>
			<td>51119200</td>
			<td>To measure absorbance</td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>EMD Millipore Corp, MA</td>
			<td>492016-100ML</td>
			<td>cell lysis reagent</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>SIGMA, MO</td>
			<td>S7653-1KG</td>
			<td>cell lysis reagent</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>SIGMA, MO</td>
			<td>436143-100G</td>
			<td>TBARS Reagent</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>SIGMA, MO</td>
			<td>367176-2.5KG</td>
			<td>TBARS Reagent</td>
		</tr>
		<tr>
			<td>SpeedVac Concentrator</td>
			<td>Thermo Scientific, MA</td>
			<td>SC250EXP</td>
			<td>For concentrating cell lysates</td>
		</tr>
		<tr>
			<td>T-75 Flask, Tissue Culture Treated, 250 mL, w/filter cap</td>
			<td>USA Scientific, FL</td>
			<td>658175</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>Thiobarbituric Acid</td>
			<td>SIGMA, MO</td>
			<td>T5500-100G</td>
			<td>TBARS Reagent</td>
		</tr>
		<tr>
			<td>TRIS base</td>
			<td>Fluka, GA</td>
			<td>93362</td>
			<td>cell lysis reagent</td>
		</tr>
		<tr>
			<td>Trypsin (1x)</td>
			<td>VWR, PA</td>
			<td>16777-166</td>
			<td>To detach HepG2 cells</td>
		</tr>
	</tbody>
</table>

evaluation of oxidative stress in biological samples using the thiobarbituric acid reactive substances assay

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic metric of lipid peroxidation in biological fluids. It is often considered a good indicator of the levels of oxidative stress within a biological sample, provided that the sample has been properly handled and stored. The assay involves the reaction of lipid peroxidation products, primarily malondialdehyde (MDA), with thiobarbituric acid (TBA), which leads to the formation of MDA-TBA2 adducts called TBARS. TBARS yields a red-pink color that can be measured spectrophotometrically at 532 nm. The TBARS assay is performed under acidic conditions (pH = 4) and at 95 &#176;C. Pure MDA is unstable, but these conditions allow the release of MDA from MDA bis(dimethyl acetal), which is used as the analytical standard in this method. The TBARS assay is a straightforward method that can be completed in about 2 h. Preparation of assay reagents are described in detail here. Budget-conscious researchers can use these reagents for multiple experiments at a low cost rather than buying an expensive TBARS assay kit that only permits construction of a single standard curve (and thus can only be used for one experiment). The applicability of this TBARS assay is shown in human serum, low density lipoproteins, and cell lysates. The assay is consistent and reproducible, and limits of detection of 1.1 &#956;M can be reached. Recommendations for the use and interpretation of the spectrophotometric TBARS assay are provided.

Under acidic conditions (pH = 4) and at 95 &#176;C, malondialdehyde (MDA) bis(dimethyl acetal) yields MDA23. MDA and closely related chemical congeners react with two molecules of thiobarbituric acid (TBA) to produce compounds called thiobarbituric acid reactive substances (TBARS), which give a red-pink color and have an absorbance &#955;max at 532 nm (Figure 1, Figure 2). Using MDA bis (dimethyl acetal) as the standard, standa...

Watch this Scientific Journal Video about Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay at JoVE.com

Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay

The goal of the thiobarbituric acid reactive substances assay is to assess oxidative stress in biological samples by measuring the production of lipid peroxidation products, primarily malondialdehyde, using visible wavelength spectrophotometry at 532 nm. The method described here can be applied to human serum, cell lysates, and low density lipoproteins.

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic ...

So the TBARS assay is best used for intra-laboratory general assessment of oxidative stress in biological samples, in which relative TBARS levels are directly compared between samples that were stored and processed together. These assay has an extremely low cost of about 3 for testing 96 samples compared to commercially available kits, which costs about 400, and permit only a single batch samples to be analyzed. The method described here can be further adapted for specific applications including biospecimens stability studies, and those for which the addition of antioxidants or other types of sample preservatives prior to analysis is appropriate or mandated.To avoid highly variable results, prepare fresh reagents, making sure that the pH is not greater than four for the color reagents and remove any bubbles in the nidese swell plate before measuring the absorbance. Begin by preparing a 0.8%aqueous solution of thiobarbituric acid. Prepare a five molar sodium hydroxide solution by dissolving four grams of sodium hydroxide beads in 20 milliliters of water.Store the solution in a plastic container. It should be freshly prepared for each batch. Add four grams of thiobarbituric acid to 450 milliliters of deionized water and use a magnetic stir bar to gently dissolve it.Slowly add approximately four milliliters of the sodium hydroxide solution in 100 microliter increments, checking the pH as the thiobarbituric acid particles dissolve. Continue adding the sodium hydroxide until all particles dissolve. Verify that the pH of the solution is no higher than four with a pH strip, then bring the final volume to 500 milliliters with deionized water and store the aqueous thiobarbituric barbaric acid solution at room temperature.Prepare MDA bis dimethyl acetal standard curve solutions in two milliliter snap cap polypropylene tubes. By diluting the 200 micromolar stock solution in water as described in the text manuscript. Vortex the samples.Label the glass tubes for the samples to be analyzed, then add 100 microliters of the prepared sample to each tube. Add 200 microliters of 8.1%SDS to each sample and gently swirl the glass tube in a circular motion to mix. Add 1.5 milliliters of 3.5 molar sodium acetate buffer to each sample, then add 1.5 milliliters of the aqueous 0.8%thiobarbituric acid solution.Bring the final in each tube to four milliliters by adding 700 microliters of deionized water. Tightly cap the glass tubes and incubate them in a heating block set to 95 degrees Celsius for one hour. Cover the tubes with aluminum foil to prevent condensation, then remove the tubes from the block and incubate them on ice for 30 minutes.After the incubation, centrifuge the samples and standards at 1, 500 times G for 10 minutes at four degrees Celsius. Transfer 150 microliters of supernatant from each tube to a well in a 96 well plate, removing any air bubbles with the pipette tip. Measure the absorbance of the samples at 532 nanometers, then subtract the average absorbent reading of the blank samples from all other absorbents readings.Create a standard curve and use it to calculate unknown sample concentrations. MDA bis dimethyl acetol standard curves were generated to determine the limits of detection and sensitivity of the assay and levels of oxidation in three different biological samples. A total of 9 TBARS assays were performed on the three samples on different days.To determine the limits of detection, absorbance of blank samples was measured on three different days. The minimum concentration of TBARS substance needed to give a detectable non noise absorbent signal is 1.1 micromolar. And the sensitivity of the assay is about 0.0016 absorbents units per micromolar increase in concentration.The TBARS assay can be used to detect changes in lipid peroxidation of various biological matrices. To demonstrate this copper two chloride was used to induce the invitro oxidation of human serum HEPG2 cell lysates and low density lipoproteins it. A TBARS assay was performed on LDL samples containing EDTA and the levels of TBARS did not change between the copper two treated and untreated LDL samples.However, after EDTA was removed by spin filtration, LDL underwent copper two mediated oxidation. To illustrate the dynamic range of the TBARS assay in human serum, a concentration of two millimolar copper two ions was added to the samples, which resulted in a six to seven fold increase in TBARS. When attempting this protocol, do not leave the samples on ice for longer than 10 minutes, and leave them at room temperature after centrifugation.For batches or subsets of biospecimens that were not originally collected, processed, and stored together, one should statistically evaluate the data for batch effects to ensure that artifactual oxidation did not contribute to the results. Intentional quantifiable invitro oxidation off low density lipoprotein makes it possible to understand how oxidation affects it's functioning biology.

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