Chemistry
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Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay
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Summary May 12th, 2020
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.
Transcript
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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