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 (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 TBA^3,4. As such, this traditional assay is simply considered to measure “thiobarbituric acid reactive substances” 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 conclusions^{3,4,7,8,9,10,11}. 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 results^1,7,12,13.

Preanalytical variables related to biospecimen handling and storage (e.g., blood plasma kept temporarily at -20 °C)^14,15 can have a major impact on TBARS assay results^16,17; 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 biomarker^3,9,10,18,19. 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 assay^21,22, 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.

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 volume to 50 mL with DI water.
   3. Prepare lysis buffer: aliquot 20 mL of RIPA buffer into a 50 mL tube and add 200 μL of a 100x protease inhibitor solution to inhibit protein and lipid degradation. Store at 4 °C.
      NOTE: Lysis buffer is compatible with TBARS reagents and does not interfere with absorbance at 532 nm. If planning to use a different lysis buffer or add additional ingredients to the lysis buffer, preliminary validation studies need to be done to verify that lysis buffer components are compatible with the TBARS assay.
   4. Remove media containing 10% FBS and wash cells 2x with 5 mL of cold, sterile 1x PBS.
   5. Add 1 mL of lysis buffer to the T75 flasks containing the cells and incubate them for 10 min at room temperature (RT) with constant swirling to ensure the buffer is well-distributed.
   6. Collect lysates into appropriately labeled 2 mL snap-cap polypropylene tubes and incubate on ice for 10 min.
   7. Spin the lysates at 5,000 x g for 10 min at RT to collect cell debris, and aspirate supernatants into a single 15 mL tube.
   8. Concentrate cell lysate supernatant four-fold using a Speed Vac at 50 °C and 3 mbar and make aliquots of 94 μL each into 2 mL snap-cap polypropylene tubes. Store samples at -80 °C until they are used for in vitro oxidation and/or TBARS assay.
      NOTE: To avoid concentrating the cell lysate supernatant, cells can also be detached using 3 mL of 1x trypsin, neutralized with 6 mL of media, and washed 2x with 5 mL of cold PBS. Cell pellets can then be reconstituted in 250 μL of lysis buffer, and steps 1.1.6 and 1.1.7 can then be performed.
   9. Prepare a 35 mM CuCl₂ stock solution in acetic acid (pH = 4).
      1. Prepare acetic acid solution (pH = 4): Dilute 1 μL of glacial acetic acid in 100 mL of DI water (pH should be approximately 4 but confirm this with a pH meter). Add more water or acetic acid to adjust the pH to 4.
      2. Weight out about 0.1936 g of copper II chloride and dissolve in 10 mL of the acetic acid solution (pH = 4) to make a 144 mM CuCl₂ stock. Aliquot 490 μL from this solution and add to 1,510 μL of acetic acid (pH = 4) to make a 35 mM CuCl₂ solution.
   10. Aliquot 6 μL from the 35 mM CuCl₂ stock solution and add it to six samples containing 94 μL of cell lysate to make a final CuCl₂ concentration of about 2 mM. Add 6 μL of an acetic acid solution (pH = 4) that does not have CuCl₂ to six samples containing 94 μL of cell lysates to use as controls. The final volume of cell lysate should be 100 μL, which is what will be used for the TBARS assay.
       NOTE: Making the 35 mM CuCl₂ stock solution in acetic acid (pH = 4) is necessary to prevent precipitation of copper hydroxide.
   11. Incubate samples in an oven at 37 °C for 24 h and perform a TBARS assay on each sample containing a final volume of 100 μL.
   12. Repeat steps 1.1.9 and 1.1.11 2x on separate days to check the reproducibility of the TBARS assay for HepG2 cell lysates.
2. Low density lipoproteins
   NOTE: Typically, pre-purified low density lipoprotein (LDL) contains some amount of EDTA. LDL samples used here contain 0.01% EDTA. EDTA can inhibit the in vitro Cu(II)-mediated oxidation of LDL. Hence, it may be necessary to remove EDTA from LDL samples prior to experiments or analysis. Steps 1.2.1–1.2.5 describe this process.
   CAUTION: Sodium hydroxide is corrosive and causes irritation in skin and eyes. Use proper personal protective equipment.
   1. Aliquot 24 μL from a 5.51 mg/mL LDL stock (protein concentration determined by modified Lowry method using BSA as a standard) into appropriately labeled 1 mL snap-cap polypropylene tubes. Make as many aliquots as needed and store at 4 °C until use in oxidation and/or TBARS assay.
   2. Prepare a 10 mM HEPES buffer in 0.15 M NaCl adjusted to pH = 7 with NaOH beads: dissolve 4.39 g of NaCl in 0.49 L of water, then add 1.19 g of HEPES. Dissolve well with a stir bar. Add sodium hydroxide beads until pH is 7. Dilute to 0.5 L with water. Store buffer at 4 °C and use within 3 months.
   3. Add 476 μL of the 10 mM HEPES buffer in 0.15 M NaCl (pH = 7) to the aliquoted LDL samples to bring final volume to 500 μL. Add diluted LDL sample to a 0.5 mL centrifugal spin filter device with a 100K molecular weight cutoff.
   4. Spin samples at 14,000 x g for 10 min at RT, leaving a final retentate volume of about 30 μL. Reconstitute samples in 480 μL of the 10 mM HEPES buffer in 0.15 M NaCl (pH = 7) and spin again at 14,000 x g for 10 min at RT. Perform this step 2x for a total of four spin-throughs.
   5. Place filter device upside down into a new 2 mL snap-cap polypropylene tube, and centrifuge at 1000 x g for 2 min to collect LDL sample (final volume = about 30 μL).
   6. Aliquot sample into appropriately labeled 1 mL tube and add 20 μL of water to each sample to achieve a final volume of 50 μL.
   7. Preparation of 200 μM CuCl₂ stock solution in acetic acid (pH = 4)
      1. Prepare acetic acid solution (pH = 4): see step 1.1.9.1.
      2. Prepare a 144 mM CuCl₂ stock solution (see step 1.1.9.2), then aliquot 5.5 μL from the 144 mM CuCl₂ stock and dissolve in a final volume of 4 mL of acetic acid (pH = 4) to make the 200 μM solution.
   8. Aliquot 2.7 μL from the 200 μM CuCl₂ stock solution and add it to six samples containing 50 μL of LDL to achieve a final CuCl₂ concentration of ~10 μM. Add 2.7 μL from an acetic acid solution (pH = 4) that does not contain CuCl₂ to six samples containing 50 μL of LDL to be used for the controls.
   9. Incubate LDL samples for 2 h in an oven at 37 °C. After 2 h, bring the final volume to 100 μL for each sample with 10 mM HEPES buffer in 0.15 M NaCl (pH = 7). Immediately perform a TBARS assay.
   10. Repeat steps 1.2.3–1.2.9 2x on two different days to test reproductivity of the TBARS assay.
3. Human serum
   1. From a human serum sample, make aliquots of 94 μL each into 2 mL snap-cap polypropylene tubes and store samples at -80 °C.
   2. Prepare a 35 mM CuCl₂ stock solution in acetic acid (pH = 4): see step 1.1.9.
   3. Aliquot 6 μL from the CuCl₂ stock solution and add it to six samples containing 94 μL of human serum to make a final CuCl₂ concentration of about 2 mM. Add 6 μL of an acetic acid solution (pH = 4) that does not have CuCl₂ to six samples containing 94 μL of human serum to use as controls.
   4. Incubate human serum samples for 24 h in an oven at 37 °C and determine levels of oxidation with TBARS assay (section 4).
   5. Repeat steps 1.3.2–1.3.4 2x on two separate days to determine reproducibility of the TBARS assay.

2. Reagent preparation

CAUTION: Thiobarbituric acid causes skin and eye irritation and maybe harmful by inhalation or skin absorption. Acetic acid can damage internal organs if inhaled. Prepare all acid solutions in a fume hood.

1. Preparation of 8.1% (w/v) sodium dodecylsulfate (SDS) solution
   1. Weight out 32.4 g of SDS and dissolve in 350 mL of DI water in a beaker. Use a magnetic stir bar to gently dissolve SDS and avoid making bubbles. Bring final volume to 400 mL with DI water and store SDS solution at RT.
      NOTE: Here, excess 8.1% SDS solution is prepared; however, for 96 samples, only about 20 mL of the 8.1% SDS solution are needed. Prepare this solution according to the number of samples being analyzed.
2. Preparation of 3.5 M sodium acetate buffer (pH = 4)
   1. Dilute 100 mL of glacial acetic acid in 350 mL of DI water in a beaker. Use a magnetic stir bar to gently dissolve it.
   2. Prepare a 6.5 M NaOH solution using sodium hydroxide beads in water. Dissolve 13 g of NaOH beads in 40 mL of DI water and bring to a final volume of 50 mL with DI water.
   3. Slowly add about 46 mL of the 6.5 M NaOH solution to the acetic acid solution while mixing with the stir bar (this should raise the pH to 4, but confirm by slowly adding the NaOH solution while measuring using a pH meter).
   4. Bring final volume to 500 mL with DI water and store sodium acetate buffer at RT.
3. Preparation of 0.8% aqueous solution of thiobarbituric acid (adjusted to pH = 4)
   NOTE: In this step, preparation of thiobarbituric acid is optimized for large volumes, since a large number of samples is going to be analyzed (108 samples, not including the standards). Prepare this solution depending on the number of samples planned for analysis.
   1. Prepare a 5 M sodium hydroxide solution using sodium hydroxide beads and water: dissolve 4 g of sodium hydroxide beads in a final volume of 20 mL of water. Store in a plastic container. This solution should be freshly prepared for each batch.
   2. Weight 4 g of thiobarbituric acid and add 450 mL of DI water. Use a magnetic stir bar to gently dissolve it.
      NOTE: This solution will eventually be brought to a 500 mL total volume.
   3. While dissolving thiobarbituric acid with a stir bar, add (slowly and in a dropwise manner) about 3 mL of the 5 M NaOH solution in 100 μL increments. After adding the NaOH solution, the thiobarbituric acid particles will start to dissolve.
   4. If the thiobarbituric acid particles still have not fully dissolved, add more of the 5 M NaOH solution in 100 μL increments until all thiobarbituric acid particles are fully dissolved. For this particular volume of solution, a total of 4 mL of the 5 M NaOH solution is added to fully dissolve the thiobarbituric acid particles.
      NOTE: At this concentration, thiobarbituric acid will not fully dissolve unless the pH is nearly 4.
   5. Stop adding NaOH after all the thiobarbituric acid has fully dissolved. Avoid exceeding a pH of 4. The final pH can be verified by taking 1 μL from the mixing thiobarbituric acid solution and placing it onto pH paper.
   6. Bring final volume to 500 mL with DI water and store aqueous 0.8% thiobarbituric acid solution at RT.

3. Malondialdehyde bis(dimethyl acetal) standard sample preparation

NOTE: Malondialdehyde (MDA) is unstable and not commercially available. However, there are different chemical forms of MDA that are commercially available, such as MDA tetrabutylammonium salt, MDA bis(dimethyl acetal), and MDA bis(diethyl acetal). Of these three chemical forms, MDA bis(dimethyl acetal) is used here, because a majority of studies use this same standard^21,22. If choosing to use the other two chemical forms of MDA, prior validation of their suitability should be carried out.

1. Prepare a 550 μM MDA bis(dimethyl acetal) stock solution by diluting 92 μL of pure MDA bis(dimethyl acetal) in 1 L of DI water. Use a magnetic stir bar to mix the solution thoroughly for 10 min. Store solution at 4 °C and use within 1 month.
2. Prepare a 200 μM MDA bis(dimethyl acetal) by diluting 726 μL from the 550 μM MDA bis(dimethyl acetal) stock in 1274 μL of DI water. This 200 μM MDA bis(dimethyl acetal) solution should be prepared fresh every time a TBARS assay is performed.
3. Standard curve preparation: take eight 2 mL snap-cap polypropylene tubes and label them with letters A through H. Add MDA bis(dimethyl acetal) from the 200 μM stock and dilute in water as described in Table 1.
4. Take eight glass tubes (13 mm x 100 mm) and label them A–H, then add 100 μL of standard to the corresponding tubes. Perform six replicates for the blank standard (sample A) to calculate the limits of detection of the TBARS assay.
   NOTE: The protocol can be paused here for no more than 1 h.

4. TBARS assay

NOTE: Once the TBARS assay is started, it should be finished without stopping.

1. Take as many glass tubes as needed for the number of samples to be analyzed and label them with the names of the samples. Then, add 100 μL of each prepared sample (as described above) to each glass tube.
2. Add 200 μL of 8.1% SDS to each sample and standard and gently swirl the glass tube in a circular motion to mix the sample.
3. Add 1.5 mL of the 3.5 M sodium acetate buffer (pH = 4) to each sample and standard.
4. Add 1.5 mL of the aqueous 0.8% thiobarbituric acid solution (pH = 4) to each sample and standard.
5. Bring the final volume to 4 mL for each sample and standard by adding 700 μL of DI water.
6. Tightly cap each glass tube and incubate in a heating block set to 95 °C for 1 h. Cover the glass tubes with aluminum foil to prevent condensation at the tops of the tubes.
7. Remove the glass tubes from the heating block and incubate on ice for 30 min.
8. Centrifuge samples and standards at 1500 x g for 10 min at 4 °C. After centrifugation, keep the glass tubes containing the samples and standards at RT.
   NOTE: Keeping the samples on ice or at 4 °C will cause the entire sample or standard to precipitate.
9. Immediately after centrifugation, aliquot 150 μL of supernatant from each tube and place into a separate well of a 96 well plate.
10. Remove any bubbles from each well using a pipette tip.
    NOTE: The presence of bubbles will yield inconsistent absorbance readings, leading to high assay imprecision.
11. Read absorbances at 532 nm. Subtract the average absorbance reading of the blank samples from all other absorbance readings.
12. Create a standard curve by plotting the blank-subtracted absorbance readings at 532 nm vs. the known concentration of each standard. Fit the data points using linear regression. Calculate unknown sample concentrations by using the equation of the linear regression line obtained from the standard curve.

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