This article discusses the generation of free radicals during metabolic processes and their role in oxidative stress. It details the lipid peroxidation process and the detection of oxidative stress in human serum using the TBARS assay.
Cells generate low levels of free radicals during metabolic processes.
In pathological conditions, free radical overproduction occurs, causing oxidative stress. These free radicals attack polyunsaturated fatty acids in cellular membrane phospholipids in a process called lipid peroxidation. This forms toxic oxidation products, including lipid hydroperoxides and malondialdehyde (MDA), which cause membrane damage and cell death.
To detect oxidative stress in human serum in vitro using the thiobarbituric acid reactive substance (TBARS) assay, begin with a tube containing human serum treated with an oxidation-inducing compound. The treated serum majorly comprises lipid peroxidation products — MDA and lipid hydroperoxides — along with phospholipids, lipids, and proteins.
Next, add sodium dodecyl sulfate (SDS) and mix to solubilize the lipids and release the MDA. SDS denatures any contaminating proteins. Add a suitable acidic buffer, followed by acidic thiobarbituric acid (TBA). Incubate at a high temperature.
Under acidic conditions and high temperatures, TBA molecules react with MDA, forming a stable red-pink MDA-TBA complex, called TBARS. Incubate on ice to stop the reaction and stabilize the existing complexes.
Centrifuge. Collect and transfer the MDA-TBA-containing supernatant into a multi-well plate. Using a microplate reader, measure the absorbance of the complex.
A higher absorbance suggests a high TBARS concentration, which indicates higher levels of lipid peroxidation products and oxidative stress in serum.
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 aqueous 0.8% thiobarbituric acid solution. Bring the final volume in each tube to 4 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 1 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 x g for 10 minutes at 4 degrees Celsius. Transfer 150 microliters of supernatant from each tube to a well in a 96-well plate, removing any air bubbles with a pipette tip.
Measure the absorbance of the samples at 532 nanometers. Then, subtract the average absorbance reading of the blank samples from all other absorbance readings.
Create a standard curve and use it to calculate unknown sample concentrations.
繁體中文
