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CHAPTER - 4

Material and Methods


Institute of Pharmaceutical Technology, SPMVV, Tirupati 65

Materials

i) Drugs

Carbamazepine (Intas Pharmaceuticals, Ahmedabad, India), Phenytoin (Anglo French

Drugs and Industries Ltd., Bangalore, India), Vitamin C (Qualigens Fine Chemicals,

Mumbai, India), Vitamin E (E. Merck, Mumbai, India), Alpha Lipoic Acid (Sami

Labs, Bangalore, India), N Acetyl Cysteine (Fourts India laboratories, Pvt. Ltd, Tamil

Nadu, India).

ii) Chemicals

Tris buffer, ethylenediamine tetra acetate (EDTA), Pyrogallol, hydrogen peroxide,

trichloro acetic acid (TCA), Ellman’s reagent (Hi- Media Laboratories, Mumbai,

India), 5,5-dithiobis-2-nitrobenzoic acid (DTNB), sodium citrate, Dinitro phenyl

hydrazine (DNPH), thiourea, H2SO4, methanol, diphenyl picryl hydrazine (DPPH),

thio barbituric acid (TBA) (Himedia Laboratories, Mumbai, India), 1, 1, 3,

3-tetraethoxypropane, paraformaldehyde, acetyl thiocholine (Sigma Aldrich), HPLC

grade methanol (E. Merck, Mumbai, India), HPLC grade ethyl acetate (E. Merck,

Mumbai, India), HPLC grade glacial acetic acid (S.D. Fine Chem Ltd., Mumbai,

India).

iii) Assay Kits

SGOT, SGPT, bilirubin, ALP, albumin, total protein, TC, HDL, TG, blood glucose

estimation kits (AGAPPE, India).



iv) Instrumentation

Rota-Rod (INCO, Ambala, India), Hole board, Actophotometer (INCO, Ambala,

India), Elevated plus maze, Autoanalyser (Chemistry Analyser (CA 2005), B4B

Diagnostic Division, China), Automated Haematology Analyzer XT-1800i (Sysmex),

UV-visible, Shimadzu 1601 Spectrophotometer, Digital balance, Tissue homogenizer

RQ 127A (REMI Motors Ltd., Mumbai), Centrifuge (REMI Motors Ltd., Mumbai),

micropipettes (20 µl, 100 µl, 200 µl and 1000 µl).

HPLC system consisted of LC-8A solvent delivery module (Shimadzu, Kyoto, Japan),

SPD-10AVP UV-Visible Spectrophotometric detector, Chromotech N 2000, China

(soft ware), Rheodyne Injection Port (Rheodyne, Cotati, CA USA) with a 20 µl

sample loop and reverse phase C18 column (Grace-alltima, USA Inc., 250 x 4.6 mm,

5µm). Hamilton syringe 50 µl (Switzerland), Cyclomixer (Remi Equipments,

Mumbai, India), Biofuge Fresco centrifuge (Heracus, Germany), cooling centrifuge

(Remi Instruments, Mumbai, India), Ultra sonicator and Electrically heated water bath

were used.

Animals

Pathogen free adult male albino rats weighing 150-200 g were used. The rats were

housed in polypropylene cages at room temperature (25 ± 3oC) with 12/12 hours light

and dark cycle and were fed with a balanced diet and tap water ad libitum. The study

protocol was approved by the Institutional Animal Ethical Committee of M.S.

Ramaiah College of Pharmacy, Bangalore, Karnataka (Ref. No. MSRCP/P-09/2009,

Reg.No. 220/abc/CPCSEA).



Study Protocol

The rats were divided into fourteen groups; each group consisted of twelve animals.

Six out of twelve animals were used for the assessment of behavioural,

haematological and biochemical parameters. After the collection of blood samples on

45th day, the animals were sacrificed, liver and brain were harvested for further

histopathological analysis. The other six animals were subjected to investigation of

pharmacokinetic and pharmacodynamic interactions i.e the animals were subjected to

maximal electro shock after which blood samples were collected for estimation of

serum CBZ concentration.

Control group: Received 0.2% carboxy methyl cellulose (CMC) orally for 45 days.

CBZ group: Received CBZ (50 mg/Kg p.o) daily for 45 days.

Low dose Vit C group: Received co-administration of 50 mg/Kg of Vit C p.o, daily

for 45 days along with CBZ (50 mg/Kg).

Medium dose Vit C group: Received supplementation of 100 mg/Kg of Vit C p.o,

daily for 45 days along with CBZ (50 mg/Kg).

High dose Vit C group: Received 200 mg/Kg of Vit C p.o, daily for 45 days along

with CBZ (50 mg/Kg).

Low dose Vit E group: Received 50 mg/Kg of Vit E in 0.2% CMC orally for 45 days

daily along with CBZ (50 mg/Kg).

Medium dose Vit E group: Received 100 mg/Kg of Vit E in 0.2% CMC orally for

45 days along with CBZ (50 mg/Kg).

High dose Vit E group: Received 200 mg/Kg of Vit E in 0.2% CMC orally for 45

days with CBZ (50 mg/Kg).



Low dose ALA group: Received 50 mg/Kg of ALA in 0.2% CMC orally for 45 days

along with CBZ (50 mg/Kg).

Medium dose ALA group: Received 100 mg/Kg of ALA in 0.2% CMC orally for 45

days along with CBZ (50 mg/Kg).

High dose ALA group: Received 200 mg/Kg of ALA in 0.2% CMC orally for 45

days along with CBZ (50 mg/Kg).

Low dose NAC group: Received 50 mg/Kg of NAC p.o, daily for 45 days along with

CBZ (50 mg/Kg).

Medium dose NAC group: Received 100 mg/Kg of NAC p.o, daily for 45 days

along with CBZ (50 mg/Kg).

High dose NAC group: Received 200 mg/Kg of NAC p.o, daily for 45 days along

with CBZ (50 mg/Kg).

The dose of CBZ and antioxidants were chosen from previous investigations. CBZ

(Thaakur and Puspha, 2008), Vit C (Alsaif, 2009; Shahidi, et al., 2008; Afkhami-

Ardekani and Shojaoddiny-Ardekani, 2007), Vit E (Cadenas, et al., 1995; Naghibi, et

al., 2006), ALA (Pari and Murugavel, 2004; Thaakur and Himabindhu, 2009;

Arivazhagan, et al., 2006), NAC (Prakash and Kumar, 2009; Kamalakkannan, et al.,

2005; Raza, et al., 2003).

The animals (six out of twelve) were subjected to the following behavioural tests on

0, 15th, 30

th and 45

th day. Only one behavioural parameter was assessed at a given

time. The behavioural parameters were analyzed between 9.00 to 11.00 A.M.

Memory was assessed using Elevated Plus Maze, motor co-ordination was studied

using Rota Rod, locomotor activity was assessed with Actophotometer and alertness

was evaluated using Hole Board apparatus. On 45th day, after the investigation of

behavioural parameters the blood samples were collected from retro orbital plexus



under light ether anaesthesia for the estimation of enzymatic and non enzymatic

antioxidants, MDA content, total antioxidant status, liver enzymes, bilirubin, total

protein, albumin, LDL, HDL, VLDL, TG, blood glucose along with haematological

profiles which included the estimation of total RBC, total WBC, platelet count,

haemoglobin content and packed cell volume.

The animals were then sacrificed and the brain and liver were isolated, rinsed with

cold phosphate buffer (100 mM, pH 7.4), weighed, sliced for histopathological studies

and stored at -40ºC. The stored tissues were homogenized and the homogenate was

centrifuged at 10,000 rpm for 10 minutes at 4°C and was used for the estimation of

lipid peroxidation.

The other six animals were treated with CBZ and antioxidants according to the above

protocol for 45 days. On 45th

day, three hours after the last dose of CBZ and

antioxidant supplementation the animals were subjected to maximal electro shock

(MES) induced convulsions to compare the degree of protection offered by CBZ in

CBZ alone treated group and groups subjected to combination of CBZ and

antioxidants. Immediately after MES the blood samples were collected from retro

orbital plexus under light ether anaesthesia for the estimation of serum CBZ level.



Work Plan

Selection of antioxidants

Investigation of influence of selected antioxidants on the adverse

effects of CBZ

Vitamin C Vitamin E

Alpha Lipoic Acid N Acetyl Cysteine

1. Parameters to

assess

haematotoxicity

2. Parameters to

assess behavioural

abnormalities

3. Parameters

to assess

hepatotoxicity

4. Parameters to

assess metabolic

disorder

5. Investigation of

pharmacokinetic

& pharmacodynamic

interaction

Control

Study Protocol

CBZ

(50 mg/Kg)

Vitamin C Vitamin E

Alpha Lipoic Acid N Acetyl Cysteine

50 mg/Kg

100 mg/Kg

200 mg/Kg

50 mg/Kg

100 mg/Kg

200 mg/Kg

50 mg/Kg

100 mg/Kg

200 mg/Kg

50 mg/Kg

100 mg/Kg

200 mg/Kg

Estimation

1.2 Antioxidants

1.2.1 Enzymatic antioxidants i) Superoxide dismutase

ii) Catalase

1.2.2 Non-enzymatic antioxidants i)Reduced glutathione

ii) Vitamin C

1.2.3 Total antioxidant status

1.2.4 Lipid peroxidation

1. PARAMETERS TO ASSESS HAEMATOTOXICITY

1.1 Estimation of

Haematological

Profile

i) Haemoglobin

ii) Total erythrocyte

iii) Total leukocyte

iv) Platelet count

v) Packed cell volume



2. PARAMETERS TO ASSESS BEHAVIOURAL ABNORMALITIES

2.1 Assessment of

behavioural

parameters

i) Motor co-ordination test

ii) Test for alertness

iii) Test for memory

impairment

iv) Test for locomotor activity

2.2 Assessment of

oxidative stress in

brain tissues

Regional brain lipid

peroxidation

2.3 Brain

Histopathology

3. PARAMETERS TO ASSESS HEPATOTOXICITY

3.1 Assessment of liver

function test

i) SGPT

ii) SGOT

iii) ALP

v) Bilirubin

vi) Total protein v) Albumin

3.2 Assessment of

oxidative stress in liver i) Liver lipid peroxidation

3.3 Liver

histopathology

4. PARAMETERS TO ASSESS METABOLIC DISORDER

4.1 Blood glucose

estimation

4.2 Assessment of lipid

profile

i) Total cholesterol

ii) Triglycerides

iii) HDL

vi) LDL

v) VLDL

5. INVESTIGATION OF PHARMACOKINETIC

& PHARMACODYNAMIC INTERACTION

5.1 Maximal electroshock

induced seizures

5.2 Estimation of plasma

CBZ concentration by

HPLC method



1. PARAMETERS TO ASSESS HAEMATOTOXICITY

1.1 Assessment of Haematological profile

Haematological parameters include haemoglobin estimation, total erythrocyte, total

leukocyte and platelet count along with packed cell volume using Automated

Haematology Analyzer XT-1800i (Sysmex, USA).

1.2 Assessment of antioxidant status

1.2.1 Estimation of enzymatic antioxidants

i) Superoxide dismutase

Principle: Assay of superoxide dismutase (SOD) was based on the ability of the

enzyme to inhibit the autooxidation of pyrogallol.

The assay was performed by using 1.5 ml tris buffer (0.05 M) and 0.5ml EDTA

(1 mM) as blank. 1.5 ml tris buffer, 0.5 ml EDTA (1 mM) and 1 ml pyrogallol (0.2

mM) as control and the test sample consisted of 1.5 ml tris buffer (0.05 M), 0.5 ml

EDTA (1 mM), 0.05 ml serum and 1 ml pyrogallol (0.2 mM). Change in optical

absorbance of sample per minute with reference to blank was recorded at a

wavelength of 420 nm using SICO Spectrophotometer. The enzyme inhibition caused

by the serum was calculated and the enzyme activity was expressed as superoxide

anion reduced/mg protein/min (Marklund and Marklund, 1974).

ii) Catalase

Principle: Catalase (hydrogen peroxide oxidoreductase) catalyzes the following

reactions.

Decomposition of H2O2 to form H2O and O2

2 H2O2 2 H2O + O2 Catalase



Catalase activity is measured either by decomposition of H2O2 or by liberation of O2.

The decrease in the absorbance by H2O2 as a function of time is used to follow the

catalase-peroxide reaction. The spectral region for hydrogen peroxide is 210-240 nm.

The difference in absorbance per minute is a measure of catalase activity.

2.5 ml of phosphate buffer was added to 0.1 ml of serum and incubated at 25°C for 30

min. After transferring into a cuvette the absorbance was measured at 240 nm, 650 µl

of hydrogen peroxide solution was added to initiate the reaction, the change in

absorbance was measured for 3 min (Beer and Sizer, 1952).

1.2.2 Estimation of non-enzymatic antioxidants

i) Reduced glutathione

Principle: The most widely used method for the determination of GSH in biological

samples is by Ellman reagent (DTNB), which reacts with sulfydryl compounds to

give a relatively stable yellow color. This compound is water soluble and the color

formed is proportional to the amount of GSH.

To 0.5 ml of citrated blood, 0.5 ml of 5% trichloro acetic acid (TCA) solution was

added to precipitate the proteins and centrifuged at 3000 rpm for 20 min. To 0.1 ml of

supernatant, 1 ml of sodium phosphate buffer and 0.5 ml of DTNB reagent was

added. The absorbance of the yellow color developed was measured at 412 nm

(Ellman, 1959).

ii) Vitamin C

To 0.5 ml of plasma, 1.5 ml of 6% TCA was added and centrifuged at 3500 rpm for

20 min. To 0.5 ml of the supernatant, 0.5 ml of DNPH reagent (2 % DNPH and 4 %

thiourea in 9 N H2SO4) was added and developed colour was read at 530 nm after 30

min (Omaye, et al., 1979).



1.2.3 Total antioxidant status

0.1 ml of serum was deproteinated by the addition of 1 ml of methanol, vortexed for

30 s. It was then centrifuged at 3000 rpm for 30 min to separate the proteins. To the

clear supernatant 1.5 ml of methanol and 0.5 ml of DPPH solutions were added,

mixed thoroughly and absorbance was read at 517 nm against blank. Blank was

prepared in an identical way but without the addition of serum (Blois, 1958).

1.2.4 Estimation of extent of oxidative stress in blood

i) Determination of MDA content (Lipid peroxidation)

0.1 ml of plasma was treated with 2 ml of TBA 0.37%, 0.25 N HCl and 15% TCA

(1:1:1) and heated in water bath for 15 min, cooled and centrifuged and then clear

supernatant was measured at 535 nm against reference blank (Niehaus and

Samuelson, 1968).

2. PARAMETERS TO ASSESS BEHAVIOURAL ABNORMALITIES

2.1 Assessment of behavioural parameters

i) Motor co-ordination test

Motor co-ordination test was conducted in rats using a Rota-Rod apparatus (Inco-

Ambala, India). The animals were screened for motor co-ordination and the animals

which stayed on the rotating rod without falling for 120 sec were chosen for the study.

Each animal was placed on the Rota rod and the time taken by the animal to fall down

was noted (Kulkarni, 1999).



ii) Test for alertness (Exploratory Behaviour)

This test was done using Hole Board, which consisted of a 0.5 m3 wooden board with

16 holes (3 cm in diameter). Each rat was placed individually on the board for a

period of 6 minutes. In first 2 minutes the animal was allowed for acclimatization and

then the number of head dipping performed in the next 4 minutes was noted for each

animal (Takeda, et al., 1998).

iii) Test for memory impairment

Elevated plus maze test was used for the assessment of memory. The elevated plus

maze consists of two closed arms and two open arms forming a cross, with a

quadrangular centre and has a height of 50 cm. The rats were placed individually at

the end of one open arm facing away from central platform and the time it took to

move from the open arm to either of the enclosed arms (transfer latency) was recorded

on the day of acquisition trial. Transfer latency is the time taken by the rats to move

from one end of the open arm to enclosed arm. The rat was allowed to move freely in

the plus maze regardless of open and closed arms for 10 sec after the measurement of

transfer latency. The rat was then gently taken out of the plus maze and was returned

to its home cage. On the test day, the transfer latency test was performed in the same

manner as in the acquisition trial (Sharma and Kulkarni, 1992).

iv) Test for locomotor activity

Spontaneous motor activity was monitored using Actophotometer. Each animal was

subjected to an adaptation period of 2-5 minutes, because the first measure of

animal’s activity is the rate of habituation to a novel environment. Thus, during

prolonged exposure to a new environment, animals typically spend less time in

movement and exploration, so the second measure was considered as the rate of

spontaneous activity of the rats. The counting was started following 5 minutes of



adaptation period. Increase in count was regarded as central nervous system stimulant

activity. Decrease in count was considered as central nervous system depressant

activity (Kulkarni, 1999).

2.2 Assessment of oxidative stress in brain tissues

The brains were quickly removed, cleaned with chilled saline, dissected into cortex,

midbrain, medulla, pons and cerebellum according to the method of Glownski and

Iversen (1966). The separated brain regions were stored at −40°C, homogenized and

regional brain lipid peroxidation was estimated.

i) Estimation of lipid peroxidation in brain regions

The extent of lipid peroxidation in tissues was assessed by measuring the level of

MDA according to the method of Ohkawa, et al., (1979). Briefly, 1 ml (10%) tissue

homogenate was added to the reaction mixture containing 1 ml of TCA (15%) and 2

ml of TBA (0.38%). The reaction mixture was heated for 60 min at 90o

C, cooled and

centrifuged at 6900 rpm for 15 min. The absorbance of supernatant was measured at

532 nm against blank, which contained all reagents except homogenate. MDA was

quantified and expressed as μmol of MDA per mg of wet tissue (Ohkawa, et al.,

1979).

2.3 Histopathological investigation on brain tissues

Brain tissues were dissected out carefully and were kept in 10% formalin solution

prepared with normal saline. Histopathological studies were conducted by Dr. Kishore

Alwa, Isha Diagnostics, Malleswaram, Bangalore. Tissues were stained using

Hematoxylin and Eosin stain (H and E).



i) Processing of isolated brain: The animals were sacrificed and the brain of each

animal was isolated. The isolated brain was cut into small pieces and fixed in 10%

formalin for two days. The brain pieces were washed in running water for about 12

hours. This was followed by dehydration with isopropyl alcohol of increasing strength

(70%, 80% and 90%) for 12 h each. Then the final dehydration was carried out using

absolute alcohol with about three changes for 12 h each. The clearing was done by

using chloroform with two changes for 15 to 20 min each. After clearing, the brain

pieces were subjected to paraffin infiltration in automatic tissue processing unit. The

brain pieces were washed with running water to remove formalin completely. To

remove the water, alcohol of increasing strengths was used. Further alcohol was

removed by using chloroform and chloroform was removed by paraffin infiltration.

ii) Embedding in paraffin vacuum: Hard paraffin was melted and the hot paraffin

was poured into L-shaped blocks. The brain pieces were then dropped into the molten

paraffin quickly and allowed to cool.

iii) Sectioning: The blocks were cut using microtone to get sections of 5 m

thickness. The sections were taken on a micro slide on which egg albumin (sticking

substance) was applied. The sections were allowed to remain in an oven at 60o

C for 1

hour. Paraffin melts and egg albumin denatures, thereby fixes tissues to slide.

iv) Staining: Eosin is an acid stain. Hence it stains all the cell constituents pink

which are basic in nature, like Cytoplasm. Haematoxyllin basic stain, stains acidic cell

components blue (e.g) DNA.

v) Procedure: The sections were deparaffinized, by washing with chloroform, for

about 15 min. They were then hydrated by washing in isopropyl alcohol of decreasing

strength (100%, 90%, 80%, 70%), followed by washing with water. The sections were



then stained with hemotoxylin for 15 min. After 15 min they were rinsed in tap water.

The sections were then differentiated in 1% acid alcohol by 10 quick dips. The

differentiation was examined under a microscope. Again the sections were washed in

tap water and dipped in lithium carbonate until they become bright blue (3-5 dips).

This was followed by washing in running tap water for 10 to 20 min, if washing is

inadequate eosin does not stain evenly. Eosin staining was done for 15 seconds to 2

min depending on the age of the eosin and the depth of the counter stain desired. For

even staining results, the slides were dipped several times before allowing them to set

in the eosin for the desired time. They were dehydrated in 95% isopropyl and absolute

isopropyl alcohol, until excess eosin was removed and mounted in Desterene dibutyl

phthalate xylene (Li, et al., 1998; Luna, 1986).

3. PARAMETERS TO ASSESS HEPATOTOXICITY

3.1 Assessment of liver function test

Serum was separated by centrifuging blood at 2500 rpm for 10 minutes and the levels

of SGOT, SGPT, bilirubin, ALP, albumin and total protein were analyzed by using a

commercially available enzymatic kit (AGAPPE, India) and an autoanalyser

(Chemistry Analyser (CA 2005), B4B Diagnostic Division, China).

i) Estimation of Serum Glutamate Pyruvate Transaminases (SGPT/ ALT)

Principle: Alanine aminotransferase catalyses the transfer of amino group from

alanine to 2-oxoglutarate, resulting in the formation of pyruvate and glutamate. The

catalytic concentration is determined from the rate of decrease of NADH, measured at

340 nm, by means of lactate dehydrogenase coupled reaction (Gella, et al., 1985)

The enzymatic reaction employed in the assay of SGPT is as follows:



L- Alanine + 2-oxoglutarate Pyruvate + L- Glutamate

Pyruvate + NADH+ H+ D- Lactate + NAD

+

Reagent Preparation

Reagent A: Tris-150 mmol/l, L-Alanine-750 mmol/l, Lactate dehydrogenase >1350

U/l, pH-7.3.

Reagent B: NADH-1.3 mmol/l, 2-oxoglutarate-75 mmol/l, Sodium hydroxide-148

mmol/l, Sodium azide-9.5 g/l.

Auxillary Reagent – Reagent C: Pyridoxal phosphate-10 mmol/l.

Working Reagent: Reagent A (4 parts) is mixed with 1 part of Reagent B. The

combined reagent was stable for 2 months at 2-8° C. The mixed reagent was stored in

a dark place and protected from light.

Working Reagent with Pyridoxal phosphate: 10ml of working reagent was mixed with

0.1 ml of reagent C. The solution was stable for 6 days at 2-8° C.

Procedure: Animal serum was used as the sample. 50 µl of serum was mixed with

1000 µl of mixed reagent and estimated in kinetic mode using a Biochemical

Analyzer.

Calculations

SGPT/ ALT concentration (U/l ) = delta A/min x Vt x 106

Ε x L x Vs

Molar absorbance (E) of NADH at 340 nm is 6300

L - Light path 1cm

ALT

LDH



Vt - Total reaction volume is 1.05 at 37° C

Vs - Sample volume is 0.05 at 37° C

ii) Estimation of Serum Glutamate Oxaloacetate Transaminases (SGOT/

AST)

Principle: Aspartate aminotransferase catalyzes the transfer of the amino group from

aspartate to 2-oxoglutarate, forming oxaloacetate and glutamate. The catalytic

concentration is determined from the rate of decrease of NADH, measured at 340 nm,

by means of malate dehydrogenase (MDH) coupled reaction (Gella, et al., 1985).

The enzymatic reaction employed in the assay of SGOT is as follows.

2-oxoglutarate Glutamate + Oxaloacetate

Oxaloacetate + NADH+ H+ D-Malate + NAD

+

Reagent Preparation

Reagent A: Tris 121 mmol/l, L- aspartate 362 mmol/l, malate dehydrogenase >460

U/l, lactate dehydrogenase > 660 U/l, sodium hydroxide 255 mmol/l, pH 7.8.

Reagent B: NADH 1.3 mmol/l, 2-oxoglutarate 75 mmol/l, sodium hydroxide

148 mmol/l, sodium azide 9.5 g/l.

Auxillary Reagent – Reagent C: Pyridoxal phosphate 10 mmol/l.

Working Reagent: Reagent A (4 parts) is mixed with 1 part of Reagent B. The

combined reagent is stable for 2 months at 2-8° C. The mixed reagent was stored

protected from light.

MDH

AST



Working Reagent with Pyridoxal phosphate: 10ml of working reagent was mixed with

0.1 ml of reagent C. Stable for 6 days at 2-8° C. Animal serum was used as the

sample.

Procedure: 50 µl of serum was mixed with 1000 µl of mixed reagent and estimated

in kinetic mode using a Biochemical Analyzer.

Calculations

SGOT/ AST concentration (U/l ) = delta A/min x Vt x 106

Ε x L x Vs


L - Light path 1cm



iii) Estimation of Alkaline Phosphatase

Principle

Alkaline Phosphatase catalyses in alkaline medium the transfer of phosphate group

from 4-nitrophenyl phosphate to 2-amino-2-methyl-1-propanol, liberating

4-nitrophenol. The catalytic concentration is determined from the rate of

4-nitrophenol formation, measured at 405 nm (Rosalki, et al., 1993).

The enzymatic reaction employed in the assay of Alkaline Phosphatase is as follows:

4- Nitrophenyl phosphate + H2O Phosphate + 4- Nitrophenol

Reagents

Reagent A:

2- Amino-2- methyl- 1- propanol 0.4 mol/l

ALP



Zinc sulphate 1.2 mmol/l

N hydroxy ethylene diamine tri aceticacid 2.5 mmol/l

Magnesium acetate 2.5 mmol/l, pH 10.4.

Reagent B

4- Nitrophenyl phosphate 60 mmol/l.

Working Reagent: 4 parts of Reagent A is mixed with 1 part of Reagent B. The

combined reagent is stable for 2 months at 2-8° C.

Animal serum was used as the sample.

20 µl of serum was mixed with 1000 µl of mixed reagent and estimated in kinetic

mode using a Biochemical Analyzer.

Calculations

ALP concentration (U/l) = delta A/min x Vt x 106

Ε x L x Vs


L- Light path 1cm



iv) Total bilirubin

Principle: Direct bilirubin in the sample reacts with diazotised sulfanilic acid forming

a coloured complex that can be measured by spectrophotometry. Both direct and

indirect bilirubin couple diazo in the presence of cetrimide (Pearlman and Lee, 1974).

The terms direct and total refer to the reaction characteristics of serum bilirubin in the



absence or presence of solubilizing reagents. The direct and indirect bilirubin is

approximately equivalent to the conjugated and unconjugated fractions.

Composition

Bilirubin (Total)

Reagent A

Sulfanilic acid 29 mmol/l

Hydrochloric acid 0.2 mol/l

Cetrimide 50 mmol/l

Reagent B

Sodium nitrite- 11.6 mmol/l. Stored at 15-30° C. Reagents were stable until the expiry

date shown on the label when stored tightly closed and if contaminations was avoided

during use. Presence of particulate matter, turbidity, absorbance over 0.05 at 540 nm,

indicate deterioration.

Working Reagent preparation: Mixture of 1 ml of Reagent B and 4 ml of Reagent A.

This was Stable for 20 days at 2-8° C.

Particulars Reagent Blank Sample Blank Sample Standard

Distilled water

Sample

Standard

Reagent A

Working Reagent

100 µl

--

--

--

1000 µl

--

100 µl

--

1000 µl

--

--

100 µl

--

--

1000 µl

--

--

100 µl

--

1000 µl



Mixed thoroughly and was allowed to stand for 2 min at room temperature.

Absorbance of sample blank was read at 540 nm against distilled water and

absorbance of sample was read at 540 nm against reagent blank.

Calculations

Bilirubin content in the sample =A (sample) – A (sample blank) x C (standard)

A (standard)

Mass concentration (mg/dl) x 17.1 = Substance concentration in µmol/l.

v) Total protein estimation

Principle: The enzymatic reaction sequence employed in the assay of total protein

was as follows:

Protein + Cu2+

Cu – Protein complex

(Biuret)

Total proteins were estimated using Total protein reagent from Agappe Diagnostics,

Kerala, India (Gornall, et al., 1949; Lowry, et al., 1951).

Composition of Total protein reagent

Potassium iodide- 6 mmol/l

Potassium sodium tartrate- 21 mmol/l

Copper sulphate- 6 mmol/l

Sodium hydroxide- 58 mmol/l

Total Protein Standard- 6 g/dl.

The reagent was stable for 18 months when stored at 2-8° C.



To 20 µl of serum, 1ml of total protein reagent was added and mixed. The mixture

was incubated at 37oC for 15 minutes and the absorbance was measured at 546 nm

using a Biochemical Analyzer. The protein content was calculated by using the

following formula and expressed as total protein in g/dl.

Total protein in g/dl = Absorbance of sample x C

Absorbance of standard

where C refers to the protein concentration in standard protein solution in g/dl.

vi) Albumin estimation

Principle: The reaction between albumin in serum or plasma and the dye bromocresol

green produces a change in colour, which is proportional to albumin concentration.

(Dumas, et al., 1997).

Reagent Composition

Albumin reagent

Succinate buffer (pH- 4.2) - 75 mmol/l

Bromocresol green - 0.14 g/l

Albumin Standard

Albumin Standard concentration - g/dl

The reagent is stable for 18 months when stored at 2-8o C.

Animal serum was used as sample. 10 µL of serum was mixed with 1000 µL of

reagent, mixed and incubated for 1 minute. The absorbance was measured against

blank at 630 nm.



Calculation

Albumin (g/dl) = Absorbance of sample x C


where C refers to the albumin concentration in standard albumin solution in g/dl.

3.2 Assessment of oxidative stress in liver

i) Preparation of liver homogenates

Livers were dissected, stored at −40°C, homogenized and the homogenates were

subjected to estimation of lipid peroxidation. Liver homogenate was prepared in 0.15

M potassium chloride buffer and centrifuged at 8000 rpm for 10 minutes. The

supernatant was used for estimation of MDA.

ii) Estimation of extent of lipid peroxidation

Principle: The extent of liver lipid peroxidation was assessed by measuring the

amount of MDA. In free radical induced hepatic damage, free radicals attack PUFAs,

which results in the formation of lipid radical. The lipid radicals readily react with

molecular oxygen to produce peroxyl radicals which initiate lipid peroxidation. This

is considered as the major factor influencing the breakdown and turnover of

biomembranes. Inhibition of lipid peroxidation implies hepato-protection. The chief

secondary product of lipid peroxidation reaction is MDA. This reacts with

thiobarbituric acid to form a chromogenic adduct with two molecules of TBA, which

is a pink colored complex and can be measured at 532 nm (Chatterjee and Sil, 2006;

Maté, et al., 2000).

To 500 µl 10% w/v liver homogenate in potassium chloride buffer, 1 ml of

TBA:TCA:HCl reagent (TBA 0.38%, 15 % trichloroacetic acid and 0.25 N



hydrochloric acid in ratio 1:1:1) was added, boiled for 15 min and cooled. The

mixture was then centrifuged at 10,000 rpm for 5 min, absorbance of the supernatant

solution was measured at 532 nm against reagent blank. The MDA content was

calculated as TBARS and expressed in terms of nmol/g of tissue, using the molar

extinction co-efficient, 1.56 105 moles/cm.

Concentration of TBARS (nmol/g) = A / t

Where A = Absorbance of sample, t = Path length

= Molar extinction coefficient of MDA (1.56 105 /Moles/cm)

3.3 Histopathological investigation on liver tissues

Liver tissues were dissected out carefully and were kept in 10% formalin solution

prepared with normal saline. Histopathological studies were conducted by Dr. Kishore

Alwa, Isha Diagnostics, Malleswaram, Bangalore. Tissues were stained using

Hematoxylin and Eosin stain (H and E).

4. PARAMETERS TO ASSESS METABOLIC DISORDERS

4.1 Estimation of glucose

Principle: The substrate β-D-glucose is oxidized by glucose oxidase to from gluconic

acid and hydrogen peroxide. The hydrogen peroxide so generated oxidizes the

chromogen system consisting of 4-aminoantipyrine and phenolic compound to a red

quinoeimine dye. The intensity of the colour produced is proportional to the glucose

concentration and is measured at 505 nm (490-530 nm) or with green filter (Trinder,

1969).

Glucose + O2 Gluconic acid + H2O2

H2O2+Phenolic compound+4-Aminoantipyrine Red Quinoemine+H2O

Glucose

Oxidase Peroxidase



Kit contents

Reagent 1: Glucose reagent

Reagent 2: Glucose Standard (For calibration)

Procedure for estimation of glucose

Pipetted into micro-

centrifuge tubes Blank (µl) Standard (µl) Test (µl)

Glucose Reagent 500 500 500

Calibrator (Standard) -- 5 --

Sample (Serum) -- -- 5

Distilled water 5 -- --

Mixed and incubated at 37oC for 10 minutes. Absorbance of the Test (AT), Standard

(AS) and Reagent Blank (AB) at 505 nm was read against distilled water using

Biochemical Analyzer.

Calculations

Glucose (mg/dl) = (AT-AB/AS-AB) x100

Where 100 = Standard concentration of Glucose (mg/dl)

4.2 Estimation of lipid profile

i) Total cholesterol

Principle: Enzymatic determination of total cholesterol was performed according to

the following equation (Allain, et al., 1974).

Cholesterol ester+H2O Cholesterol + Fatty acids

Cholesterol + O2 4- Cholesten- 3- one + H2O2

2 H2O2 + Phenol + 4- aminoantipyrene Red quinine + 4H2O

Cholesterol

esterase

Cholesterol

Oxidase

Peroxidase



Cholesterol reagent

Pipes buffer, pH 6.7- 50 mmol/l

Phenol- 24 mmol/l

Sodium cholate- 0.5 mmol/l

4- aminoantipyrene- 0.5 mmol/l

Cholesterol esterase > 180 U/l

Cholesterol oxidase > 200 U/l

Peroxidase > 1000 U/l

Cholesterol standard solution 200 mg/dl

The reagent is stable for 18 months when stored at 2-8° C. Animal serum was used as

the sample.

10 µl of serum was mixed with 1000 µl of reagent, incubated for 5 min at 37 °C and

estimated at 630 nm using a Biochemical Analyzer.

Calculation

Cholesterol (mg/dl) = Absorbance of sample x 200


ii) Triglycerides

Principle: Enzymatic determination of triglycerides was performed according to the

following equation (Schettler and Nussel, 1975).

Triglyceride + H2O Glycerol + Fatty acids

Glycerol + ATP Glycerol-3- phosphate + ADP

Glycerol-3-PO4 + O2 Dihydroxy acetone phosphate+ H2O2

Lipoprotein lipase

Glycerol kinase

Glycerol-3-

phosphate oxidase



H2O2 + 4- aminoantipyrine + p- chlorophenol Red quinoneimine

Reagent Composition

Pipes buffer, pH 7.0- 50 mmol/l

p-chlorophenol- 5.3 mmol/l

Potassium ferrocyanate- 10 mmol/l

Magnesium salt- 17 mmol/l

4- aminoantipyrine- 0.9 mmol/l

ATP- 3.15 mmol/l

Lipoprotein lipase > 1800 U/l

Glycerol kinase > 450 U/l

Glycerol-3- phosphate oxidase > 3500 U/l

Peroxidase > 450 U/l

Triglyceride standard solution- 200 mg/dl

The reagent was stable for 18 months when stored at 2-8° C.

Animal serum was used as the sample. 10 µl of serum was mixed with 1000 µl of

reagent, incubated for 5 min at 37 ° C and estimated at 630 nm using a Biochemical

Analyzer.

Calculation:

Triglyceride (mg/dl) = Absorbance of sample x 200


iii) HDL cholesterol

Principle: The chylomicrons, VLDL and LDL of serum were precipitated by

phosphotungstic acid and magnesium ions. After centrifugation, HDL in the

supernatant solution was measured by enzymatic method (Gordon, et al., 1977).

Peroxidase



HDL cholesterol reagent

Phosphotungstate- 14 mmol/l

Magnesium chloride- 1 mmol/l

Preservative

HDL cholesterol standard- 50 mg/dl.

Animal serum was used as the sample. 300 µl of serum was mixed with 300 µl of

HDL reagent, allowed to stand for 10 min at room temperature, mixed again and

centrifuged for 10 min at 4000 rpm. After centrifugation the clear supernatant was

separated from the precipitate within 1hr and HDL was determined using cholesterol

reagent. 50 µl of supernatant was mixed with 1000 µl of cholesterol reagent,

incubated for 5 min at 37 ° C and estimated at 630 nm using a Biochemical Analyzer.

Calculation

HDL cholesterol conc (mg/dl) = Absorbance of sample x N x 2


N= Standard concentration (50 mg/dl)

iv) Estimation of LDL

Formula

LDL = TC/1.19 + TG/1.9 – HDL/1.1 – 38 (mg/dl) (Ahmadi, et al., 2008).

v) Estimation of VLDL

Formula

VLDL = Triglycerides (mg/dl) (Ahmadi, et al., 2008).

5



5. INVESIGATION OF PHARMACOKINETIC AND PHARMACO-

DYNAMIC INTERACTIONS

5.1 Maximal electroshock induced seizures (MES)

Electroconvulsions were induced by ear electrodes (current intensity-150 mA,

duration - 0.2 sec). The animals were observed for tonic hind limb extension i.e., the

hind limbs of animals outstretched 180° to the plane of the body axis (Kulkarni,

1999).

5.2 Estimation of plasma carbamazepine concentration by HPLC method

i) Chromatographic conditions

Mobile phase consisting of acetonitrile: water (30:70 v/v) was prepared and mixed

thoroughly, degassed and was used for the HPLC analysis. 1.0 ml per minute flow

rate was maintained throughout the analysis. The eluent was monitored using a UV-

VIS detector set at 270 nm and sensitivity was set at 0.001 a.u.f.s (Demrikaya and

Kadioglu, 2005).

ii) Preparation of standard graph

a) Standard solutions

Stock solution of 100 µg/ml of CBZ was prepared in methanol and diluted with

methanol to the required concentration. The solutions were stored at –4ºC. For the

preparation of standard graph 2, 4, 6, 8, 10, 12, 14, 16, 18 20 µg/ml of pure CBZ was

used.



b) Plasma extraction procedure

To each 100 µl of plasma sample, 25 µl of internal standard (100 µg/ml phenytoin

solution) was added and extracted with 1.7 ml of ethyl acetate, vortexed for 1 min and

centrifuged at 13,000 rpm for 8 min. The supernatant was evaporated to dryness and

the residue was reconstituted with 100 µl of mobile phase, vortexed for 1 min. and 20

µl was injected onto C18 column. The retention times were 3.673 min and 6.065 min

for carbamazepine and phenytoin respectively. The peak area obtained at different

concentrations of the drug was plotted against the concentrations of the drug (Garg, et

al., 1999; Chen, et al., 2001).

STATISTICAL ANALYSIS

The results were expressed as mean ± S.E.M (n=6). The statistical analysis was

performed by means of ANOVA followed by Tukey-Kramer's Multiple Comparison

Test. P value < 0.05 was considered as statistically significant. Data was processed

with Graph Pad Instat software.

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