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 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.
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 “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 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 °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.
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
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.
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 standard21,22. If choosing to use the other two chemical forms of MDA, prior validation of their suitability should be carried out.
4. TBARS assay
NOTE: Once the TBARS assay is started, it should be finished without stopping.
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 λmax at 532 nm (Figure 1, Figure 2). Using MDA bis (dimethyl acetal) as the standard, standard curves were generated (Figure 3, Table 1) to determine the limits of detection and sensitivity of the assay and levels of oxidation in three different biological samples. A total of nine TBARS assays were performed to determine the levels of oxidation in the three different samples on different days. Hence, a total of nine standard curves were generated, as shown in Figure 3. The least squares procedure24 was used to determine the standard deviations of the slope and the y-intercept, which were 8.67 x 10-6 and 5.66 x 10-4, respectively.
The limits of detection of the TBARS assay were determined according to standard analytical procedures25 by measuring absorbances of the blank samples (six experimental replicates with two technical replicates per experimental replicate) on three different days. The minimum distinguishable analytical signal (Sm) was determined by summing the mean of the blank signal (S̄bl) plus a multiple k of the standard deviation of the blank (ksbl), where k = 3. That is, Sm = S̄bl + ksbl. Using Sm and the slope of the standard curve (m), the detection limit (cm) was calculated as cm = (Sm – S̄bl)/m. The resulting data of the blank samples on three different days shows that the minimum concentration of TBARS substance needed to give a detectable non-noise absorbance signal is 1.1 μM (Table 2). The sensitivity of the TBARS assay is about 0.00160 absorbance units/μM, which is the ability of the assay to distinguish differences in analyte concentration (Table 2).
To illustrate the applicability of the TBARS assay in detecting changes in lipid peroxidation in various biological matrices, CuCl2 was used to induce the in vitro oxidation of human serum, HepG2 cell lysates, and low density lipoproteins. These biological samples used here are prototypes of biological matrices. For example, based on the results presented here for HepG2 cell lysates, it is reasonable to expect that this assay will work with other types of cell lysate; however, it would need to be analytically validated for this purpose. Also, of the three biological matrices used here, it is common for certain types of samples to exhibit low endogenous concentrations of TBARS. For example, TBARS for HepG2 cell lysates that were not treated with CuCl2 were just above the limit of detection of the assay (about 2 μM; Figure 4). As would be expected in the presence of low signal-to-noise ratios, the standard deviation and coefficient of variation for this particular sample is relatively high (Table 3). However, as the signal increases as a result of Cu(II) mediated oxidation, the coefficient of variation becomes lower. In general, as the absorbance increases beyond the detection limit, assay reproducibility improves (Table 3).
For the purposes of this protocol, there was no desire to use antioxidants to mask the in vitro Cu(II)-mediated oxidation of biological samples. Commercially prepared low density lipoprotein (LDL) may contain 0.01% EDTA. EDTA will prevent Cu(II)-mediated oxidation of LDL but not necessarily other metal-mediated oxidation reactions26,27. A TBARS assay was performed on LDL samples containing EDTA, and the levels of TBARS did not change between the Cu(II)-treated and untreated LDL samples (Figure 5A). However, after EDTA was removed by spin filtration (see step 1.2.3–1.2.5), LDL underwent Cu(II)-mediated oxidation, as detected by the TBARS assay (Figure 5B).
The normal range of lipid peroxidation products in the human serum from healthy donors expressed in terms of MDA is between 1.80–3.94 μM28. To illustrate the dynamic range of the TBARS assay in human serum, a concentration of 2 mM Cu(II) ions was added to the samples, followed by incubation for 24 h at 37 °C. This resulted in a 6x–7x increase in TBARS (Figure 6).
Figure 1: Thiobarbituric acid reactive substances assay schematic.
One hundred microliters of sample or standard are added to a 13 mm x 100 mm glass tube, followed by addition of thiobarbituric acid reactive substances (TBARS) reagents. After incubation at 95 °C for 1 h, samples and standards are incubated in ice for 30 min, then centrifuged at 1,500 x g for 10 min at 4 °C. One hundred fifty microliters of sample or standard supernatant are loaded onto a 96 well plate, and absorbance is measured at 532 nm. Unknown sample concentration is calculated using the equation of the standard curve. Please click here to view a larger version of this figure.
Figure 2: Archetype thiobarbituric acid reactive substances reaction.
Malondialdehyde bis(dimethyl acetal) yields malondialdehyde under acid-catalyzed hydrolysis1. Released Malondialdehyde (MDA) then reacts with two molecules of 2-thiobarbituric acid (TBA) (pH = 4 and 95 °C) to form MDA-TBA2 adducts that give a red-pink color and can be measured spectrophotometrically at 532 nm. Because other molecules besides MDA that are derived from oxidized lipids can also react with TBA, the absorbance measurement at 532 nm is simply referred to as a measurement of thiobarbituric acid reactive substances, or TBARS. Please click here to view a larger version of this figure.
Figure 3: Malondialdehyde bis(dimethyl acetal) colorimetric standard curves.
Figure shows nine standard curves as created on different days. Some points overlap and cannot be distinguished from one another. Malondialdehyde bis(dimethyl acetal) was fortified into calibrator samples at 0, 2.5, 5, 10, 20, 40, 80, and 160 μM (as shown in Table 1; n = 1 per concentration point per day). Absorbance was measured at 532 nm, with the average absorbance of the blank samples subtracted from all measurements in that batch, including unknowns. Each day, the equation generated by least squares linear regression was used to determine TBARS in biological samples. For all nine standard curves combined, the standard deviation of the slope was 8.67 x 10-6, and the standard deviation of the y-intercept was 5.66 x 10-4. Standard deviations of the slope and y-intercept were calculated using the least squares procedure24. Please click here to view a larger version of this figure.
Figure 4: Oxidation in HepG2 lysates detected by TBARS.
Six HepG2 cell lysate samples were incubated with 2 mM CuCl2 [HepG2 cell lysate + 2 mM Cu(II)] and six samples were incubated in a solution without CuCl2 (HepG2 cell lysate) for 24 h at 37 °C. After incubation, the TBARS assay was performed on the 12 samples. This procedure was repeated 2x for a total of three different days. Error bars represent SD. Asterisk indicates statistically significant differences between control and Cu(II)-treated lysates (p < 0.001). Statistical significance was determined using a Mann Whitney U test in GraphPad. Please click here to view a larger version of this figure.
Figure 5: Oxidation in low density lipoprotein detected by TBARS.
(A) TBARS assay conducted in LDL samples containing 0.01% EDTA. Six LDL samples were incubated with 10 μM CuCl2 [LDL + 10 μM Cu(II)], and six samples were incubated with a control solution with no CuCl2 added (Native LDL) for 2 h at 37 °C. Then, a TBARS assay was performed on the 12 samples. “ns” represents no statistical significance. (B) LDL was spin filtered using a centrifugal spin filter device to remove EDTA. Then, incubation with and without added Cu(II) was performed again as described for (A). The TBARS assay was performed immediately afterward. This same procedure was repeated 2x for a total of 3 days. Error bars represent SD. Asterisk indicates statistically significant differences between control and Cu(II)-treated LDL samples (p < 0.001). Statistical significance was determined using the Mann Whitney U test in GraphPad. Please click here to view a larger version of this figure.
Figure 6: Lipid peroxidation in human serum samples detected by TBARS.
Six human serum samples were incubated with 2 mM CuCl2 [serum + 2 mM Cu(II)], and six samples were incubated with a solution that did not have any added CuCl2 (normal serum) for 24 h at 37 °C. After incubation, the TBARS assay was performed on the 12 samples. This procedure was repeated on two additional days. Error bars represent SD. Asterisk indicates statistically significant differences between control and Cu(II)-treated serum samples (p < 0.001). Statistical significance was determined using the Mann Whitney U test in GraphPad. Please click here to view a larger version of this figure.
Glass Tube | 200 μM MDA bis (dimethyl acetal) (μL) | Water (μL) | MDA bis (dimethyl acetal) Final Concentration (μM) |
Aa | 0 | 1000 | 0 |
B | 12.5 | 987.5 | 2.5 |
C | 25 | 975 | 5 |
D | 50 | 950 | 10 |
E | 100 | 900 | 20 |
F | 200 | 800 | 40 |
G | 400 | 600 | 80 |
H | 800 | 200 | 160 |
Table 1: Malondialdehyde bis(dimethyl acetal) standard sample preparation. From the freshly prepared 200 μM malondialdehyde bis(dimethyl acetal), aliquot the suggested volumes to reach the final concentration for the standard curve. It is recommended to perform at least six replicates of the blank sample (A) per day to determine the limits of detection of the method.
Day | Absorbancea | Sblb | Smc | Sensitivity (absorbance units/μM)d | cm (μM)e |
1 (n = 6) | 0.0412 | 0.000612 | 0.0430 | 0.00160 | 1.14 |
2 (n = 6) | 0.0415 | 0.000632 | 0.0433 | 0.00160 | 1.18 |
3 (n = 6) | 0.0413 | 0.000605 | 0.0431 | 0.00160 | 1.13 |
All three days (n = 18) | 0.0413 | 0.000589 | 0.0431 | 0.00160 | 1.10 |
aAbsorbance of the blank samples on three different days with 6 replicates per day. | |||||
bSbl = Standard deviation of the absorbance of the blank samples. | |||||
cSm = Minimum distinguishable analytical signal, which was determined by summing the mean of the blank signal (S̄bl) plus a multiple k of the standard deviation of the blank (ksbl), where k = 3. That is; Sm = S̄bl + ksbl. | |||||
dSensitivity of the TBARS assay, which is the slope of the standard curve. | |||||
ecm = Limits of detection, which was calculated as cm = (Sm – S̄bl)⁄m, where m = the slope of the standard curve. |
Table 2: Detection limits of the TBARS assay.
Low density lipoprotein | Human Serum | HepG2 Cell Lysate | |||
Day | % CV | Day | % CV | Day | % CV |
1 (n = 6) | 5.6 | 1 (n = 6) | 7.9 | 1 (n = 6) | 12.6 |
2 (n = 6) | 5.4 | 2 (n = 6) | 7.2 | 2 (n = 6) | 15.8 |
3 (n = 6) | 3.9 | 3 (n = 6) | 7.0 | 3 (n = 6) | 17.7 |
All three days (n = 18)a | 7.4 | All three days (n = 18) | 9.8 | All three days (n = 18) | 15.5b |
With 10 μM CuCl2 | With 2 mM CuCl2 | With 2 mM CuCl2 | |||
1 (n = 6) | 4.5 | 1 (n = 6) | 6.0 | 1 (n = 6) | 5.8 |
2 (n = 6) | 6.5 | 2 (n = 6) | 4.3 | 2 (n = 6) | 6.0 |
3 (n = 6) | 6.7 | 3 (n = 6) | 6.2 | 3 (n = 6) | 8.0 |
All three days (n = 18) | 6.1 | All three days (n = 18) | 5.6 | All three days (n = 18) | 7.3 |
aInterday precision was calculated by pooling data from all three days. | |||||
bPrecision was limited due to results being near the assay LOD. |
Table 3: Analytical reproducibility of TBARS in three different biological samples.
Despite its limitations1,3,4,7,8,9,10,12,13,14,15,19 and a lack of suitability for comparison between laboratories, the TBARS assay is one of the oldest29,30 but most widely used assays to measure oxidative stress in biological samples. The TBARS assay is a straightforward method that only takes about 2 h to perform, once all the required reagents have been prepared. Here, we have described in detail how this assay, including standard curve, can be performed many times in an economical way (about $3.50 USD for 96 samples), without having to buy an expensive kit for every batch of samples.
All steps of the assay are critical, but there are some steps that require extra attention. For instance, the pH of the thiobarbituric acid should not be higher than 4. Precautions should be taken when adding the sodium hydroxide solution to the thiobarbituric acid and avoid obtaining a pH of greater than 4. An acidic environment is required for the reaction between MDA and TBA to occur, and the MDA standard is released from MDA bis (dimethyl acetal) by acid-catalyzed hydrolysis. Hence, a high pH may lead to unpredictable and highly variable results31.
Also, while this may be obvious to some readers, it is also critical to remove any bubbles in the 96 well plate before measuring the absorbance. The presence of bubbles will yield high absorbance values and differences between replicates, leading to high percentage of CVs. Additionally, after the 1 h incubation at 95 °C, samples should not be incubated longer than 30 min on ice, since this will precipitate the entire sample, and collecting a precipitate-free supernatant will be difficult to accomplish. Notably, there are no good stopping points once the TBARS assay has been started. It should be completed once initiated. Finally, there are many possible methodological variations that can be applied to this assay. The general protocol described here can be further adapted (and validated) for specific applications, including those in which the addition of radical scavengers or other types of antioxidants prior to analysis is required.
While the TBARS assay is popular, it is important to realize that it is not a molecularly specific assay. Numerous chemically reactive carbonyl-containing organic molecules, including those derived from oxidized biomolecules other than lipids, can react with TBA and are thus counted as TBARS1,32,33,34. In addition, the limits of detection of the absorbance-based TBARS assay do not get much better than about 1.1 μM, as determined by this method. However, the limits of detection can be improved by using other detection methods. For instance, spectrofluorometry with excitation at 520 nm and emission at 550 nm offer higher sensitivity and better limits of detection, as previously suggested by Jo and Ahn35. Mass spectrometry-based methods can dramatically improve both specificity and limits of detection. For example, a GC-MS/MS with electron-capture negative-ion chemical ionization (ECNICI) method has been used to detect the pentafluorobenzyl derivative of MDA in human serum and urine samples, with limits of detection of 2 x 10-18 mol MDA on column36. Here, the chromatographic separation, in combination with tandem mass spectrometry, dramatically improves the molecular specificity of the assay, as well.
Nevertheless, as with other measurements of oxidative processes within biological samples37,38, preanalytical sample handling is critical to the outcome of TBARS measurements. For example, blood plasma storage at -20 °C results in slow but dramatic increases in MDA concentrations39,40. Thus, exposure of biological samples to thawed or even partially thawed conditions for anything but a minimal amount of time should be assumed to cause artifactual elevation of TBARS levels. This means that even modest variability in the preanalytical handling and storage of biospecimens that are to be compared using the TBARS assay must be avoided.
Given these caveats related to preanalytical variability as well as limited sensitivity and specificity, it is recommended that the absorbance-based TBARS assay only be used for intra-laboratory general assessment or range-finding experiments. These applications include studies in which relative TBARS levels are directly compared between one or more groups of biologically similar samples that are processed or stored together and separated by only a single variable that is fully controlled by researchers.
The authors have nothing to disclose.
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.
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 |