Despite its limited analytical specificity and robustness, the Thiobarbituric Acid Reactive Substances Assay (TBARS) has been widely used as a generic metric of lipid peroxidation in biological fluids. It is often considered a good indicator of oxidative stress levels within a biological sample, provided the sample has been properly handled and stored. The assay involves the reaction of lipid peroxidation products, primarily malondialdehyde (MDA), with thiobarbituric acid (TBA), leading to the formation of MDA-TBA2 adducts called TBARS. TBARS produces 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 bis MDA (dimethyl acetal), which is used as an analytical standard in this method. The TBARS assay is a simple method that can be completed in approximately 2 hours. The preparation of the assay reagents is described in detail here. Budget-conscious researchers can use these reagents for multiple experiments at low cost instead of purchasing an expensive TBARS assay kit that only allows the construction of a single standard curve (and therefore 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 detection limits of 1.1 μM can be achieved. Recommendations are provided for the use and interpretation of the spectrophotometric TBARS assay.
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 the insertion of an oxygen molecule. This process leads to a mixture of complex products that include lipid peroxyl radicals and hydroperoxides as 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 for lipid peroxidation due to its easy 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 in addition to 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 of the 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 general levels of oxidative stress in a biological sample6. Unfortunately, as Khoubnasabjafari et al. Documented, the TBARS trial is often conducted and interpreted in ways that lead to dubious conclusions3,4,7,8,9,10,11. The causes of this are mainly based on the preanalytical variables related to the sample and the lack of rigidity of the assay that prohibits seemingly minor variations in the assay protocol without substantial changes in the assay results1,7,12,13.
Preanalytical variables related to the handling and storage of biological samples (eg, blood plasma temporarily kept at -20 ° C) 14,15 can have a significant impact on the results of the TBARS assay16,17; So much so, that TBARS assay results should not be compared between different laboratories unless warranted by explicit interlaboratory analytical validation data. This recommendation is similar to the way Western blots are commonly used and interpreted. Band density comparisons are valid for intra-blot studies and perhaps within the laboratory, but comparing band densities between laboratories is generally considered invalid practice.
Some investigators have suggested that the MDA measured by the TBARS trial simply does not meet the required clinical or analytical criteria of an acceptable biomarker3,9,10,18,19. In fact, if the trial had not been developed more than 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 robustness that are used to determine oxidative stress, the TBARS assay based on absorbance at 532 nm remains by far one of the most widely used assays for the determination of lipid peroxidation20 and, therefore, the evaluation of oxidation. stress.
The TBARS assay can only be found as an expensive kit (over US $ 400), in which the instructions do not provide detailed information on most of the concentrations of the reagents used. Also, the reagents provided can only be used for one run, because only one colorimetric standard curve can be made per kit. This can be problematic for researchers seeking to determine oxidation levels in a few samples at different time points, because the same standard curve cannot be used at multiple time points. Therefore, it is necessary to buy multiple kits for multiple experiments. Currently, unless an expensive kit is purchased, there is no detailed protocol available on how to conduct a TBARS assay. In the past, some investigators have vaguely described how to conduct a TBARS trial21,22, but a fully detailed protocol and complete video on how to conduct the TBARS trial without an expensive kit is not available in the literature.
Here we present a detailed, analytically validated methodology for a purpose on how to perform a TBARS assay in a simple, reproducible, and inexpensive manner. Changes in peroxidation of human serum lipids, HepG2 lysates, and low-density lipoproteins after treatment with Cu (II) ions are demonstrated as illustrative applications for the TBARS assay. The results demonstrate that this TBARS assay is consistent and reproducible from day to day.