effect of infection of the filarial parasite brugia malayi - Pondicherry ...

4.1.1.10 Lipid peroxidation
Lipid peroxidation in the tissue was estimated by the method of Stoch and
Dormandy (197 1 ).
Malondialdehyde (MDA), produced during peroxidation of lipids, served as an index
of lipid pemxidation. In this method MDA reacts with thiobarbihuic acid to generate a
coloured product, which is read at 532 nm.
Reagents
I. Isotonic phosphate buffered saline (PBS), pH 7.4.
2. TBA : 1% in 0.05 mMit sodium hydroxide.
3. TCA : 10°/o solution in 0.1 moVlit dmn arsenite.
4. Standard MDA : stock solution of MDA (400 nmol/ml) was prepared using 1,1,3,3
tetraethoxy propane. This was stored at 4OC and diluted with distilled water to make
working standard of 50 dm1 concentration.
5. EDTA : 0.1 momit.
One rnl of the homogenate was suspended in 0.1 ml of PBS and 0.5 ml of TCA,
followed by 2.0 ml TBA and 0.075 ml EDTA. Tubes were mixed and kept in a boiling water
bath for I5 minutes. Tubes were cooled at R.T. and centrifuged. Absorbance was read at 532
nm. Each test sample had its own blank tube, which was not boiled. Subtraction of
absorbance of unboiled sample from boiled sample eliminated absorbance increase due to
any non-TBA reactive material in the sample. EDTA was added to chelate any ironlother
metal in the ahact which, otherwise can initiate lipid peroxidation during boiling and may
result in falsely elevated TBA reactivity. Results were expressed as nmoles of MDA
fonndmg protein! 20 minutes.
4.1.1.11 Protein
Protein was estimated by the method of Lowry et al. (I%])