I might be a little bit biased here, since we measure on a daily base MDA in our lab.

The always referred TBA-assay is not quantitative by any means - it can maybe give you a rough idea. This is due to the nature of this assay, since many molecules other than MDA will bind TBA and they are often indistinguishably even by HPLC-UV. If you want to quantify MDA I highly recommend the use of an internal standard (heavy labelled) and MS.

Especially for plants I would recommend you the following publications:

http://doi.wiley.com/10.1111/j.1365-313X.2003.02013.x --> describes an appropriate method

http://www.ncbi.nlm.nih.gov/pubmed/17928299 --> a way to visualize MDA pools in plants

http://www.ncbi.nlm.nih.gov/pubmed/22298768 --> latest news about quantification of MDA in plants

Again, research showed that the TBA-assay overestimates MDA levels by up to 10-fold and might even generate artificial MDA.

http://pubs.acs.org/doi/abs/10.1021/jf00021a018

This is an alternative solution buffer caompared to TCA, I do not know if a similar approach could be useful in your case.

TBA & TCA are best practised methods....its better to depend on TBA only.....

MDA content can be estimated by Thiobarbituric assay method (Sharma and Krishna Murti, 1968).

Procedure:

1.0 ml of homogenate prepared in KCl solution was incubated at 37C for 30 minutes. Proteins were precipitated by adding 1 ml of 10% TCA and then centrifuged at 2,000 rpm for 15 minute. 1 ml supernatant was taken as an aliquot in a separate tube to which 1 ml of TBA solution was added. The tubes were kept in boiling water bath for 10 minutes. After cooling the tubes, the optical density was read at λ 535 nm.

Calculation : OD X1000

OD of Standard X mg protein

https://www.researchgate.net/publication/235902815_Lipid_peroxidation_as_a_biochemical_marker_for_oxidative_stress_during_drought._An_effective_tool_for_plant_breeding

Article Lipid peroxidation as a biochemical marker for oxidative str...

I used Hodges et al (1997) method in my previous research. He was develope the procedue in extraction and calculation for an accurate result. I got a sucess lab with his procedue. You should try to test his procedue .

Hodges et al. 1997. Crop Science vol. 37 no.3 pages 857-863

I might be a little bit biased here, since we measure on a daily base MDA in our lab.

The always referred TBA-assay is not quantitative by any means - it can maybe give you a rough idea. This is due to the nature of this assay, since many molecules other than MDA will bind TBA and they are often indistinguishably even by HPLC-UV. If you want to quantify MDA I highly recommend the use of an internal standard (heavy labelled) and MS.

Especially for plants I would recommend you the following publications:

http://doi.wiley.com/10.1111/j.1365-313X.2003.02013.x --> describes an appropriate method

http://www.ncbi.nlm.nih.gov/pubmed/17928299 --> a way to visualize MDA pools in plants

http://www.ncbi.nlm.nih.gov/pubmed/22298768 --> latest news about quantification of MDA in plants

Again, research showed that the TBA-assay overestimates MDA levels by up to 10-fold and might even generate artificial MDA.

To tie in with the previous claimed potential HPLC-UV artifacts, other lipid oxidation adducts such as 2-alkenals or hexanal were shown to produce similar absorption spectra:

http://www.ncbi.nlm.nih.gov/pubmed/22061167

http://www.springerlink.com/index/V11N145472070843.pdf

http://www.clinchem.org/content/34/12/2433.short

Susanne, I absolutely agree on the HPLC-MS as reliable system - I never doubted that.

I only wanted to draw the attention to potential artifacts, if no unambiguous detection system such as MS can be employed.

I guess we can agree on the problem, that the classical TBARS-assay itself should rather be described as an assay to detect lipid oxidation products than specifically MDA

The TBARS reaction to determine MDA, I think, necessarily needs a very acidic medium, e.g. with TCA. This medium is necessary to minimize interfering reactions with TBA. Then if you need to measure TBA-MDA adducts, because there could be many others interfering substances during detection (spectrometric or fluorometric), I recommend you a chromatographic technique, e.g. HPLC.

Another way to determine MDA is measuring BSA-MDA (BSA=Bovine serum albumin) adducts. This technique is base on immunoassays, e.g. immunoblot, ELISA, and also require calibration curve (as same as TBARS). Also you could find many commercial kits for these techniques.

Remember that MDA is not a very stable compound, even in frozen samples.

Ses causevic et al Planta 2006

Prepare an extract using trichloroacetic acid (1%), 50 mg of tissue in 1 mL. Centrifugate at 10000 rpm during 10 min, use 250 uL of each extract and mix with 750 uL of TCA 20% with thiobarbituric acid (TBA) 5%, maintain 30 mins at 100ºC in water bath, and measure the absorbance at 532 and 600 nm. Use the difference of 532-600 nm and the 155 mM-1 cm-1 extinction coefficient. Greetings

I am agreed with Dr Mirza Hasanuzzaman. You have to test all the possible protocols in your model plant to test which one gives you best biochemical signal to measure TBARS. You may use butylated hydroxy toluene during incubation to restrict auto-peroxidation of the sample.

nice

Dear All

What I read your discussion regarding quantification of MDA it is too many approaches which is confusing I am afraid,  that should be very simple and reliable. Too  

I used 1.5mg leaf samples and grinded in 3 mL of 0.1% TCA .The Homogenate was centrifuged at 10000 rpm and enzyme extract was recovered which was then mixed with 0.5 %TBA and 20% TCA in a water bath at 95 degrees for 50 minutes .After that it was again centrifuged and absorbance was read at 532 nm and 600 nm

how much is TCA and TBA ?