Please have a look at enclosed documents...hope you like them...

1. Determination of glutathione (GSH) content in blood

Reduced glutathione in blood was determined according to the method of Moron et al. (1979) after preparing a haemolysate in water. Principle Reduced glutathione forms a yellow colored complex with dithionitrobenzene (DTNB). The absorbance of the colored complex was measured at 412 nm. Procedure A 20 % haemolysate of heparinised blood was prepared in distilled water and proceed for the glutathione determination as described in the section 3.2.6. The GSH level was expressed as micromoles/ml of blood.

2. Determination of serum lipid peroxidation

Serum lipid peroxidation was determined by Ohkawa et al. (1979) after precipitating the protein according to the method of Satoh (1987). Principle Lipids were isolated by precipitating them with serum protein using 0.02 % trichloroacetic acid. The level of lipid peroxidation was measured as malondialdehyde by reacting with TBA in acetic acid solution. The reaction product was assayed by measuring absorption at 532 nm. Procedure To 0.5 ml serum, 2.5 ml of 0.02 % TCA was added and the tube is left to stand for 10 min at room temperature. After centrifugation at 3500 rpm for 10 min, the precipitate was washed once with 0.05 M H2SO4. The precipitate was suspended in distilled water and estimated the TBARS by procedure given under tissue lipid peroxidation determination (section 3.2.9). The result was expressed as n mol/ ml of serum.

3. Determination of tissue lipid peroxidation

The level of lipid peroxidation in the tissue was measured as malondialdehyde (MDA) according to the method of Ohkawa et al. (1979). Principle The tissue malondialdehyde was allowed to react with TBA. The MDA-TBA adduct formed during the reaction in acidic medium was extracted to the organic layer and the absorbance was measured at 532 nm. Procedure 4 ml of reaction mixture containing 0.4 ml of the tissue homogenate, 1.5 ml of 0.8 % TBA, 1.5 ml of acetic acid (20 %, pH 3.5) and distilled water was kept for 1 h in a boiling water bath at 95oC. After 1 hr, the reaction mixture was removed from the water bath, cooled and added 1 ml of distilled water. 5 ml of butanol: pyridine mixture (15:1) was added to the reaction tube, mixed well 52 and centrifuged at 3000 rpm for 10 min. Absorbance of the clear supernatant was measured at 532 nm against butanol: pyridine mixture. The MDA was calculated with the help of a standard graph prepared by using different concentrations (1-10 nmol) of 1'1'3'3'-tetramethoxypropane in 1 ml distilled water and is expressed as nmol of MDA/mg protein.

I would perform a pilot experiment first. But, it seems that it will work fine. The reason is that the homogenisation protocols for glutathione measurement in tissues often use acid protein-precipitating solutions (e.g. trichloroacetic acid, sulfosalycilic acid and perchloric acid).

Here in the lab we used to homogenise tissue samples in 10% (w/v) trichloroacetic acid and use the same homogenate for TBARS (whole crude homogenate; using Buege and Aust 1978 method) and glutathione assays (homogenate supernatant; by the recycling method Tietze 1969; Rahman et al. 2006).

Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. Methods Enzymol. 52, 302–310. doi:10.1016/S0076-6879(78)52032-6

Tietze, F., 1969. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem. 27, 502–522.

Rahman, I., Kode, A., Biswas, S.K., 2006. Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat. Protoc. 1, 3159–3165. doi:10.1038/nprot.2006.378

For glutathione determination it is essential to use deproteination samples to prevent interference of thiol containing protein with GSH determination while for MDA determination you can used whole crud extract.

for methods you can see our work in RG good luck

Se the attached PDFs. Hopefully this will solve your problem.

Dear Praneeti

I never worked in brain so I do not know if there is any additional difficulty of this tissue to prepare the homogenates. In my experience you can use the same homogenate to make all the determinations: superoxide dismutase, catalase, MDA, proteins and glutathione. The buffer used to homogenize can be phosphate buffer as mentioned by Anoop or also PBS without the addition of antibiotics or any protein compound. Another buffer that I have used in homogenates of uterus and mouse ovaries, that has the advantage that it allows freezing and thawing, but the disadvantage of containing 2-mercaptoethanol can therefore interfere in the determination of GSH, it is:

Tris-Base 20 mM buffer, pH = 7.6:

EDTA 1mM

Tris-Base 20 mM

150 mM KCl

Sucrose 500 mM

b-mercaptoethanol 1 mM

Homogenize with teflon pestle and centrifuge cold at 14,000 X g for 10 min. Save the supernatants can be stored at -20 ° C or -80 ° C.

I hope this information helps you.

Greetings and luck!

   Carolina