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

Summary

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.

Abstract

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 °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 μM can be reached. Recommendations for the use and interpretation of the spectrophotometric TBARS assay are provided.

Introduction

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 ....

Protocol

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

1. HepG2 cell lysates
   1. 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.
   2. Prepare RIPA buffer: in a 50 mL tube, add 1.5 mL of 5 M NaCl, 2.5 mL of 1 M Tris-HCl (pH = 7), 500 μL of NP-40 reagent, then bring the final....

Results

Under acidic conditions (pH = 4) and at 95 °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 λ_max at 532 nm (Figure 1, Figure 2). Using MDA bis (dimethyl acetal) as the standard, standard curves .......

Discussion

Despite its limitations^{1,3,4,7,8,9,10,12,13,14,15,19} and a lack of suitability for comparison between laboratories, t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1x Sterile PBS pH 7.4 1 L	VWR, PA	101642--262	cell lysis reagent
50 mL self-standing centrifuge tube	Corning, NY	CLS430897	General material
96 well plate, Non-Treated, clear, with lid, Non-sterile	Thermo Fisher Scientific, MA	280895	To measure absorbance
Amicon Ultra-0.5 100 kD centrifugal spin filter device	Fisher Scientific, NH	UFC510024	LDL purification
Caps for glass tubes	Thermo Fisher Scientific, MA	14-930-15D	for TBARS assay
Copper II Chloride	SIGMA, MO	222011-250G	to induce oxidation
Culture tubes, Disposable, with Screw-Cap Finish, Borosilicate Glass (13 x 100 mm)	VWR, PA	53283-800	for TBARS assay
Eagle's Minimum Essential Medium (EMEM)	ATCC, VA	HB-8065	HepG2 cell media
Eppendorf Safe-Lock Tubes, 1.5 mL	eppendorf, NY	22363204	General material
Eppendorf Safe-Lock Tubes, 2.0 mL	Genesee Sceitific, CA	22363352	General material
Fetal Bovine Serum US Source	Omega Scientific, CA	FB-11	for cell culture
Glacial Acetic Acid	SIGMA, MO	27225-1L-R	TBARS Reagent
Halt Protease Inhibitor Cocktail (100x)	Thermo Scientific, MA	87786	cell lysis reagent
HEPES	SIGMA, MO	H3375-250G	LDL solvent
HepG2 Cells	ATCC, VA	HB-8065	Biological matrix prototype
Hydrocloric acid (HCl)	Fisher Scientific, NH	A144-212	cell lysis reagent
Legend Micro 17 Centrifuge	Thermo Scientific, MA	75002431	General material
Low Density Lipoprotein, Human Plasma	Athens Research & Technology, GA	12-16-120412	Biological matrix prototype
Magnetic Stir Bars, Octagon 6-Assortment	VWR, PA	58948-025	General material
Malondialdehyde bis (dimethyl acetal)	SIGMA, MO	8207560250	TBARS Standard
Multiskan Go Microplate Spectrophotometer	Fisher Scientific, NH	51119200	To measure absorbance
NP-40	EMD Millipore Corp, MA	492016-100ML	cell lysis reagent
Sodium Chloride	SIGMA, MO	S7653-1KG	cell lysis reagent
Sodium dodecyl sulfate (SDS)	SIGMA, MO	436143-100G	TBARS Reagent
Sodium hydroxide	SIGMA, MO	367176-2.5KG	TBARS Reagent
SpeedVac Concentrator	Thermo Scientific, MA	SC250EXP	For concentrating cell lysates
T-75 Flask, Tissue Culture Treated, 250 mL, w/filter cap	USA Scientific, FL	658175	cell culture
Thiobarbituric Acid	SIGMA, MO	T5500-100G	TBARS Reagent
TRIS base	Fluka, GA	93362	cell lysis reagent
Trypsin (1x)	VWR, PA	16777-166	To detach HepG2 cells