10.
8) Estimation of oxidant-antioxidant status in diabetic rats
After the treatment period of 24 hrs, liver was isolated from the rats, washed in tris buffer (pH
7.4) and weighed. It was then placed in tris buffer and homogenized at a speed of 2500 RPM
(Remi motors, Mumbai). The homogenate was centrifuged (Remi- motors Ltd., Mumbai) and
the supernatant was taken. It was used for the study of following biochemical parameters by
spectrophotometric method using Spectrophotometer (Uv-250 Shimadzu, Japan).
10.8.1. Estimation of lipid peroxidation (LPO) [173]
Lipid peroxidation was estimated by the method of Slater et al., (1971).
Principle: The method estimates Malondialdehyde (MDA), a product of lipid peroxidation. One
molecule of MDA reacts with two molecules of thiobarbituric acid (TBA) under mildly acidic
conditions to form a pink colored chromogen, whose intensity is measured colorimetrically at
535 nm.
Reagents:
1. 2 ml of tissue supernatant was taken for the study.
2. Thiobarbituric acid (0.067% in 1M Tris hydrochloride, pH 7): 0.0067 g of TBA was
dissolved in 100 ml of 1M Tris hydrochloride, pH 7.
3. Trichloroacetic acid (20%): 20 g of trichloroacetic acid (TCA) was dissolved in 100 ml of
distilled water.
Procedure: Supernatant was mixed with TCA and centrifuged. The supernatant was mixed with
TBA and absorbance was taken at 535 nm.
Blank Test
1.5 ml of distilled water 2 ml of supernatant
1.5 ml of TCA (20%) 2 ml of TCA (20%)
Cool for 15 minute, centrifuge and take the supernatant
3 ml of supernatant 2 ml of supernatant
3 ml of TBA (0.067% in Tris) 2 ml TBA
Keep in boiling water bath for 10 minutes, cool. Read the absorbance of test against blank at 505
nm using Spectrophotometer.
Calculation:
Equation obtained from standard curve of lipid peroxidation:
Y= 0.0113X – 1.0061
Y= absorbance of test sample
X= Concentration of malondialdehyde in test sample
X= Y + 1.0041/0.00113
Unit: nmol/L
10.8.2. Estimation of reduced glutathione (GSH) [174]
Reduced glutathione was used by the method of Moran et al., (1979).
Principle: Glutathione present in RBC consists of some sulfhydryl groups. 5, 5 dithio bis 2-
nitrobenzoic acid (DTNB), a disulphide compound gets easily attacked by these sulphydryl
groups and formed a yellow colored anion which is measured colorimetrically at 412 nm.
Reagents:
• Trichloro acetic acid 10% (TCA): 10 g TCA was dissolved in 100 ml of distilled water.
• DTNB Reagents: 60 mg of DTNB was mixed in 100 ml of 1 % sodium citrate solution.
• Phosphate buffer (0.2M, pH 8)
a) Dissolve 1.36 g KH2PO4 in 100 ml of distilled water.
b) 0.8 g NaOH was dissolved in 100 ml of distilled water.
25 ml of KH2PO4 and 22.5 ml of NaOH were mixed and the volume was made upto 100 ml
with distilled water
• Reduced glutathione standard solution: 10 mg of reduced glutathione solution was dissolved
in 100 ml of distilled water (100 mcg/ml).
Procedure: Tissue supernatant was mixed with TCA and centrifuged. The supernatant was
mixed with DTNB and phosphate buffer and estimated at 412 nm.
Blank Test
1 ml distilled water 1 ml supernatant
1ml TCA (10%0) 1 ml TCA (10%)
Cool For 10 minutes and centrifuge at 2000 rpm.
0.5ml supernatant 0.5ml supernatant
4 ml DTNB 4 ml DTNB
1.5 ml phosphate buffer 1.5 ml phosphate buffer
Mix well and keep at room temperature for 5 minutes. Read the absorbance against blank using
Spectrophotometer.
Calculations: Equation obtained from standard curve of reduced glutathione:
Y = 0.0002X + 0.0046
Y = Absorbance of test sample
X = Concentration of reduced glutathione in test sample.
X = Y - 0.0046 / 0.0002
Units: μg of GSH / mg of tissue.
10.8.3. Estimation of superoxide dismutase (SOD) [175]
Superoxide dismutase was estimated by the method Misra et al., (1972).
Principle: Superoxide dismutase has the ability to inhibit autooxidation of epinephrine to
adenochrome at pH 10.2. This inhibition can be measured colorimetrically at 480 nm.
Reagents:
• Carbonate buffer (0.05M, pH 10.2): 16.8 g of sodium bicarbonate (NaHCO 3) and 22 g of
sodium carbonate (Na2CO3) were dissolved in 500 ml of distilled water and final volume was
made up the final 1000 ml with distilled water.
• Ethylene Diamine Tetra Acetic Acid (EDTA), 0.49 M: 1.82 g of EDTA was dissolved in
1000 ml of distilled water
3. Epinephrine (Freshly prepared): 0.99 g of epinephrine bitartarate was dissolved in 1000 ml
of 0.1N HCl.
rocedure:
All reagents should be in cold condition. The supernatant was mixed with ethanol, chloroform
and centrifuged. The supernatant was mixed with carbonate buffer, EDTA and epinephrine and
adsorbance was observed at 480 nm.
Blank Test
1.5 ml distilled water 0.5 ml supernatant
0.5 ml DW
0.38 ml ethanol 0.25 ml ethanol
0.15 ml chloroform 0.15 ml chloroform.
Shake and centrifuge at 2000 rpm for 10 minutes and separate the supernatant.
1.2 ml ST 0.5 ml ST
3.6 ml carbonate buffer 1.5 ml carbonate buffer
1.2 ml EDTA 0.5 ml EDTA
0.4 ml epinephrine 0.4 ml epinephrine
Add epinephrine just before taking OD. Note the initial absorbance at zero minutes and take for
3 minutes with 30 seconds intervals at 480 nm using Spectrophotometer.
Calculations:
SOD = 0.25 – X x 50 x 100
X = Final absorbance – initial absorbance.
Units: EU/dl
10.8.4. Estimation of catalase [176]
Catalse was estimated by the method of Aebi H (1984).
Priciple: Catalase exerts a dual function, because it catalyses the following reaction.
A) 2H2O2 2H2O + 02
Catalase
b) ROOH + AH2 H2O + ROH + A
In the ultra violet range H2O2 shows a continued increase in absorption with decreasing
wavelength. The decomposition of H2O2 can be followed directly by the decrease in absorbance
at 240 nm. The difference in the absorbance per unit time is a measure of the catalase activity.
Reagents:
• Phosphate buffer ( 50 mmol /L, pH 7.0)
• 6.81 g KH2PO4 was dissolved in distilled water and the volume was made up to 1000
ml with distilled water.
• 8.9 g Na2HPO4 was dissolved in distilled water and the volume was made upto 1000
ml with distilled water.
Both the solutions were stored in the refrigerator. a and b were mixed in the proportion of 1: 1.5
at the time of estimation.
• Hydrogen peroxide ( 30 nmol/ L): 0.34 ml of 30 % H 2O2 was diluted with phosphate
buffer to 100 ml (freshly prepared).
Procedure:
1 ml of the homogenate was taken and diluted 20 times with phosphate buffer (0.4 ml to 8 ml)
immediately before assay. The diluted homogenate was used as a sample for catalase estimation.
Blank Test
4 ml diluted homogenate 2 ml diluted homogenate
2 ml phosphate buffer 1 ml phosphate buffer
1 ml H2O2 (8.5 ml in 2.5 ml of DW)
Add H2O2 just before taking OD at 240 nm. Take the initial absorbance at zero minutes and then
take for 3 minutes with 15 seconds intervals.
Calulations:
Catalase = log A1/ log A2 x 2297.3 (factor) x dilution factor.
A1 = Initial absorbance
A2 = Final absorbance
Units: µmol of H2O2 / min/mg
