Biochemical assessment
Biochemical tests were conducted immediately after last behavioral test. The animals
were sacrificed by decapitation. Brains were removed and rinsed with ice-cold
isotonic saline. Brains were then homogenized with ice-cold 0.1mmol/L phosphate
buffer (pH 7.4). The homogenates (10%w/v) were then centrifuged at 10,000 g for 15
min and supernatants so formed were used for the biochemical estimations.
Measurement of lipid peroxidation
The extent of lipid peroxidation in the brain was determined quantitatively by
performing the method as described by Wills [12]. The amount of malondialdehyde
(MDA) was measured by reaction with thiobarbituric acid at 532 nm using Perkin
Elmer Lambda 20 UV VIS Spectrophotometer (Norwalk, CT, USA). The values were
calculated using the molar extinction co-efficient of chromophore (1.56 x 105 (mol/l)1
cm-1).
Estimation of nitrite
The accumulation of nitrite in the supernatant, an indicator of the production of nitric
oxide was determined by a colorimetric assay with Greiss reagent (0.1 % N-(1napththyl) ethylene diamine dihydrochloride, 1% sulphanilamide and 5% phosphoric
acid.) [4]. Equal volumes of the supernatant and Greiss reagent were mixed and
incubated for 10 min at room temperature in the dark. The absorbance was measured
at 540 nm using Perkin Elmer Lambda 20 UV VIS Spectrophotometer (Norwalk, CT,
USA). The concentration of nitrite in the supernatant was determined from sodium
nitrite standard curve.
Estimation of reduced glutathione
Reduced glutathione was estimated according to the method described by Ellman et al
[1]. A 1 ml supernatant was precipitated with 1 ml of 4% sulphosalicylic acid and
cold digested for 1 hour at 4°C. The samples were then centrifuged at 1,200 g for 15
min at 4°C. To 1 ml of the supernatant obtained, 2.7 ml of phosphate buffer
(0.1mmol/l, pH 8) and 0.2 ml of 5, 5’ dithio-bis (2-nitrobenzoic acid) (DTNB) was
added. The yellow color developed was measured at 412 nm using Perkin Elmer
Lambda 20 UV VIS Spectrophotometer (Norwalk, CT, USA). Results were calculated
using molar extinction co-efficient of the chromophore (1.36× 104 (mol/l)-1cm-1).
Superoxide dismutase activity
Superoxide dismutase (SOD) activity was assayed by the method of Kono [8]. The
assay system consists of EDTA 0.1 mM, sodium carbonate 50 mM and 96 mM of
nitro blue tetrazolium (NBT). In the cuvette, 2ml of the above mixture, 0.05 ml of
hydroxylamine and 0.05 ml of the supernatant was added and auto-oxidation of
hydroxylamine was measured for 2 min at 30s interval by measuring absorbance at
560 nm using Perkin Elmer Lambda 20 UV VIS Spectrophotometer (Norwalk, CT,
USA).
Catalase activity
Catalase activity was assessed by the method of Luck [10], wherein the breakdown of
H2O2 is measured. Briefly, assay mixture consists of 3 ml of H2O2 phosphate buffer
and 0.05 ml of the supernatant of the tissue homogenate. The change in absorbance
was recorded for 2 min at 30 s interval at 240 nm using Perkin Elmer Lambda 20
spectrophotometer. The results are expressed as micromoles of hydrogen peroxide
decomposed per min per mg of protein.
Glutathione-S-transferase activity
The activity of glutathione–s-transferase was assayed by the method of Habig and
Jakoby [5]. Briefly, the assay mixture consisted of 2.7 ml of phosphate buffer, 0.1 ml
of reduced glutathione, 0.1 ml of 1-chloro-2, 4-dinitrobenzene (CDNB) as substrate
and 0.1 ml of supernatant. The increase in the absorbance was recorded at 340 nm for
5 min at 1 min interval using Perkin Elmer Lambda 20 UV VIS Spectrophotometer
(Norwalk, CT, USA). The results are expressed as nmoles of CDNB
conjugated/min/mg of protein.
Estimation of Acetyl cholinesterase (AChE) activity
AchE is a marker of loss of cholinergic neurons in the forebrain. The AChE activity
was assessed by Ellman method [2]. The assay mixture contained 0.05 ml of
supernatant, 3 ml of sodium phosphate buffer (pH 8), 0.1 ml of acetylthiocholine
iodide and 0.1 ml of DTNB (Ellman reagent). The change in absorbance was
measured for 2 min at 30s interval at 412 nm using Perkin Elmer Lambda 20 UV VIS
Spectrophotometer (Norwalk, CT, USA). Results are expressed as micromoles of
acetylthiocholine iodide hydrolyzed per min per mg of protein.
Protein estimation
The protein content was estimated by Biuret method [3] using bovine serum albumin
as a standard
Aluminum estimation
The aluminium was analyzed by wet acid digestion method of Zumkley [13] in
hippocampus and cortex of the brain. A mixture of 2.5 ml of per choleric acid/nitric
acid (1:4 in volume) was added to brain part which is then placed in sand bath for 44
h until the point of a white ash or residue obtained. Residues were dissolved in 2.5 ml
of 10mM nitric acid. Then this sample (in liquid form) was placed in the sample
holder of atomic absorption spectrophotometer (Perkin Elmer, India). The total
concentration of aluminium was calculated in μg/gm of tissue.
Mitochondrial Complex Estimation
Isolation of rat brain mitochondria
The whole brain (excluding cerebellum) was used for mitochondrial isolation. Rat
brain mitochondria were isolated by differential centrifugation. The brain is
homogenized in 10 ml of homogenizing buffer containing 225 mM mannitol,75 mM
sucrose, 5 mM HEPES, 1mM EGTA, 1 mg/ml BSA, pH 7.4. The homogenate is
brought to 30 ml with the same buffer and centrifuged at 2000 g for 3 min at 40C. The
pellet is discarded and the supernatant is divided into 2 tubes and centrifuged at
12000g for 10 min. The pellet containing the mixture of synaptosomes and
mitochondria is suspended in 10 ml of homogenization buffer containing 0.02%
digitonin to lyse the synaptosomes followed by centrifugation at 12000g for 10 min to
pellet down both extra synaptosomal and intra synaptosomal mitochondria. The
mitochondrial pellet is washed twice in the same buffer without EGTA, BSA and
digitonin.
Complex-I (NADH Dehydrogenase activity)
Complex-I was measured spectrophotometrically by the method of King and Howard
[7]. The method involves catalytic oxidation of NADH to NAD+ with subsequent
reduction of cytochrome C. The reaction mixture contained 0.2 M glycyl glycine
buffer pH 8.5, 6 mM NADH in 2 mM glycyl Glycine buffer and 10.5 mM cytochrome
C. The reaction was initiated by addition of requisite amount of solubilized
mitochondrial sample and followed absorbance change at 550 nm for 2 min.
Complex-II (Succinate Dehydrogenase (SDH) activity)
SDH was measured spectrophotometrically according to King [6]. The method
involves oxidation of succinate by an artificial electron acceptor, potassium
ferricyanide. The reaction mixture contained 0.2 M phosphate buffer pH 7.8, 1%
BSA, 0.6 M succinic acid, and 0.03 M potassium ferricyanide. The reaction was
initiated by the addition of mitochondrial sample and absorbance change was
followed at 420 nm for 2 min.
Complex-III (MTT ability)
The MTT assay is based on the reduction of (3-(4, 5- dimethylthiazol-2-yl)-2, 5diphenyl-H-tetrazolium bromide (MTT) by hydrogenase activity in functionally intact
mitochondria. The MTT reduction rate was used to assess the activity of the
mitochondrial respiratory chain in isolated mitochondria by the method of Liu et al.
[9]. Briefly, 100 μl mitochondrial samples were incubated with 10 μl MTT for 3 hour
at 37 °C. The blue formazan crystals were solubilized with dimethylsulfoxide and
measured by an ELISA reader at 580 nm filter
Complex-IV (Cytochrome oxidase assay)
Cytochrome oxidase activity was assayed in brain mitochondria according to the
method of Sotocassa [11]. The assay mixture contained 0.3 mM reduced cytochrome
C in 75 mM phosphate buffer. The reaction was started by the addition of solubilized
mitochondrial sample and absorbance change was recorded at 550 nm for 2 min.
Reference
1. Ellman GL (1959) Tissue Sulfhydryl Groups. Archives of Biochemistry and
Biophysics 82:70-77.
2. Ellman GL, Courtney KD, Andres V, Featherstone RM (1961) A New and
Rapid
Colorimetric
Determination
of
Acetylcholinesterase
Activity.
Biochemical Pharmacology 7:88-&.
3. Gornall AG, Bardawill CJ, David MM (1949) Determination of Serum
Proteins by Means of the Biuret Reaction. Journal of Biological Chemistry
177:751-766.
4. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum
SR (1982) Analysis of Nitrate, Nitrite, and [N-15]-Labeled Nitrate in
Biological-Fluids. Analytical Biochemistry 126:131-138.
5. Habig WH, Jakoby WB (1981) Assays for differentiation of glutathione Stransferases. Methods Enzymol 77:398-405.
6. King TE (1967) [58] Preparation of succinate dehydrogenase and
reconstitution of succinate oxidase. In: Methods in Enzymology, vol. Volume
10 (Ronald W. Estabrook, M. E. P., ed), pp 322-331: Academic Press.
7. King TE, Howard RL (1967) [52] Preparations and properties of soluble
NADH dehydrogenases from cardiac muscle. In: Methods in Enzymology,
vol. Volume 10 (Ronald W. Estabrook, M. E. P., ed), pp 275-294: Academic
Press.
8. Kono Y (1978) Generation of Superoxide Radical during Autoxidation of
Hydroxylamine and an Assay for Superoxide-Dismutase. Archives of
Biochemistry and Biophysics 186:189-195.
9. Liu Y, Peterson DA, Kimura H, Schubert D (1997) Mechanism of cellular 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.
J Neurochem 69:581-593.
10. Luck H (1971) Catalase. Methods of Enzymatic analysis Bergmeyer HU
(Eds). Academic Press, New York. 885-893.
11. Sottocasa GL, Kuylenstierna B, Ernster L, Bergstrand A (1967) An electrontransport system associated with the outer membrane of liver mitochondria. A
biochemical and morphological study. The Journal of cell biology 32:415-438.
12. Wills ED (1966) Mechanisms of lipid peroxide formation in animal tissues.
Biochem J 99:667-676.
13. Zumkley H, Bertram HP, Lison A, Knoll O, Losse H (1979) Aluminum, zinc
and copper concentrations in plasma in chronic renal insufficiency. Clin
Nephrol 12:18-21.