Kindly provide information regarding the usage of Spectrofluorimeter and HPLC in the estimation of MDA and AOPP
We have compared measures of TBARS by spectrophotometry and MDA by HPLC. Several HPLC methods for MDA don't work well. The columns foul very quickly.
In serum, we found a close correlation between TBARS and MDA in spite of the spec method being non-specific. When possible, it is always more accurate to go with a selective method to be sure the value generated is due to a specific analyte of interest.
"Homogenate samples were assayed by a modified thiobarbituric acid (TBA)-based method (Girotti et al., 1991). For total MDA, at least 0.15 ml of homogenate clear supernatant were mixed with equal volume of TBA (TBA 0.5% w/v in TCA 20% v/v – 0.33 N HCl) and BHA 0.02% w/v. After incubation at 100 °C for 15 min, samples were brought to room temperature and thereafter, 0.3 ml isobutanol was added, mixed by vigorous vortexing and centrifuged at 15,000 × g for 3 min. Fluorescence of the upper butanol layer was measured (excitation 535 nm, emission 550 nm, Shimadzu RF-1501) against butanol-treated sample and reagent blanks. Emission fluorescence was converted to nmol MDA/mg total protein from a malonaldehyde bis(dimethyl acetal) standard curve (0.2–2 μM)."
The above modified TBARS spectrofluorimetric method is described in the paper:
Konstantinos Grintzalis, Christos D. Georgiou, Stefanos Dailianis,Total thiol redox status as a potent biomarker of PAH-mediated effects on mussels. Marine Environmental Research Volume 81, October 2012, Pages 26–34
Dear collegues!
I would like to note what there are differences between TBARS and MDA. Of course, You could be use manual methods for determination both, but... in ours laboratory for this needs use kit' s produced Cell Biolabs company or (for MDA and 4-HNE) determination kit' s FR 22 (Lubioscience, Switzerland). the best way, I mean, use microplate reader (for ELISA method). Such plate' s need hiting (till +98C), best equipment for it - Thermomixer (Eppendorff). If You have really interest, I can send you Cell Biolabs product catalog for 2015. there are possibility to buy ''training and examination'' kits, and for manual and plate technology. Good luck.
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