Encyclopedia of Experiments: Biology
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Transcript
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.
