I do TBARS assay by following the protocol of Ohkawa et al, 1979 (Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction). I need a clarification on the working protocol this method.
The following is the protocol as per original Ohkawa Paper, i.e.
1. 0.2ml of sample is added with 0.2ml of 8.1% SDS, 1.5ml 20% acetic acid at pH3.5, 1.5ml of 0.8% of TBA
2. This mixture is made up to 4ml and boiled for 1 hour at 95-degree celsius.
3. After cooling, 1 of distilled water and 5 ml of n-butanol & pyridine solution have to be added and shaken vigorously.
4. After centrifugation (4000rpm) for 10 minutes and a supernatant was read at 532nm.
I used to do this protocol by eliminating the adding of distilled water and n-butanol & pyridine solution (The step 3). However, I can get the standard curve with R2 value 0.99. I also performed with 3rd step, but I didn't get the proper standard curve. Please let me clarify that, Is following the original protocol mandatory (with 3rd step) or following this protocol without 3rd step is sufficient.
Thanks in advance for your valuable suggestions.
All photometric TBARS assays are non specific for MDA because they measure other aldehydes as well, which absorb at near same wavelength as MDA does. To measure specifically MDA you will have to go with fluorometric assays. Such is the following:
Grintzalis, K., Zisimopoulos, D., Grune, T., Weber, D., Georgiou, C. D. (2013). Method for the simultaneous determination of free/protein malondialdehyde and lipid/protein hydroperoxides. Free Radical Biology Medicine 59: 27-35.
If interested, you could get a free copy from my Researchgate site
The TBARS assay is a low sensitivity non-specific assay for MDA, and its value is very limited when used for the evaluation of lipid peroxidation. Its problems are addressed in the study (which also offers specific MDA assays): Grintzalis, K., Zisimopoulos, D., Grune, T., Weber, D., Georgiou, C. D. (2013). Method for the simultaneous determination of free/protein malondialdehyde and lipid/protein hydroperoxides. Free Radical Biology Medicine 59: 27-35.
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