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CHAPTER - 4

Materials and Methods


Institute of Pharmaceutical Technology, SPMVV, Tirupati 72

Materials

i) Drugs

Phenytoin (Anglo French Drugs and Industries Ltd., Bangalore, India), Carbamazepine

(Intas Pharmaceuticals, Ahmedabad, 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 Spectrphotometric 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-08/2009,

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 phenytoin

concentration.

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

Phenytoin group: Received phenytoin (20 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 phenytoin (20 mg/Kg).

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

daily for 45 days along with phenytoin (20 mg/Kg).

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

with phenytoin (20 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 phenytoin (20 mg/Kg).

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

days along with phenytoin (20 mg/Kg).

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

days with phenytoin (20 mg/Kg).



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

along with phenytoin (20 mg/Kg).

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


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


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

phenytoin (20 mg/Kg).

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


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


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

Phenytoin (Thaakur and Puspha, 2007; Vohora, et al., 2000; Al-Humayyd, 1997), Vit

C (Alsaif, 2009; Siamak, 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; Narasimhanaidu, et al., 2005; Raza, et al., 2003).

The animals (six out of twelve) were subjected to the following behavioural tests on 0,

15th, 30th and 45th 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. Brain homogenates were in addition subjected to the estimation of

Ach E activity.

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

above protocol for 45 days. On 45th day, three hours after the last dose of phenytoin

and antioxidant supplementation the animals were subjected to maximal electro shock

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

in phenytoin alone treated group and groups subjected to combination of phenytoin

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

orbital plexus under light ether anaesthesia for the estimation of serum phenytoin

level.



Work Plan

Selection of antioxidants

Investigation of influence of selected antioxidants on the adverse effects of Phenytoin

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

Phenytoin (20 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



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

2.1Assessment 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 Assessment of Regional brain Ach

E activity

2.4 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 phenytoin 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

(0.2mM) 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 0.1ml 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, 0.5ml 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 1ml of methanol, vortexed for

30s. 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 (Blios, 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 0.5m3 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, 1ml (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 90oC, 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 Estimation of Acetylcholine Esterase activity in brain regions

Principle: Acetylthiocholine iodide was used as a synthetic substrate for the assay of

Ach E, replacing the natural substrate acetylcholine (Ach). This enzyme hydrolyses

the substrate to yield acetate and thiocholine. The free thiol group of thiocholine reacts

with DTNB (Ellman's reagent) included in the assay mixture, producing the yellow 4-

nitrothiolate anion. The release of this yellow anion is measured at 412 nm. The assay



was performed at 30°C in 0.1 M sodium phosphate buffer with 10 mM DTNB and

12.5 mM acetylthiocholine iodide.

Reagents

1. 1 M NaCl: 5.845 g /100 ml water

2. 1 M MgCl2: 20.33 g/100 ml water

3. 0.5 M Tris-HCl, pH 7.5

4. 0.2 M EDTA: 7.44 g of EDTA was dissolved in 50 ml water and 16 ml of 1 N

NaOH was added to bring to neutral pH and was made up to 100 ml with water and

diluted to 1 in 100 to get 2mM solution.

5. Cocktail: 13 ml of reagent 1, 2.0 ml of reagent 2, 10 ml of reagent 3 and 10 ml of

reagent 4 were mixed.

7. 0.1 M Acetyl thiocholine chloride: 20.17 mg/ml of Acetyl thiocholine chloride was

dissolved in glass distilled water and preserved frozen in amber colour bottle.

8. 1mM DTNB: 0.396 mg/ml of DTNB was dissolved in glass distilled water.

9. Reaction mixture: 10.5 ml of Cocktail is mixed with 3 ml of DTNB (to be prepared

fresh) and 6.5 ml water.

Procedure: About 20-50 mg of rat brain homogenate preparation gives linear rate of

reaction up to 10-15 min. To 2 ml of reaction mixture, 0.1 M acetylthiocholine 30 µl,

enzyme preparation 100 µl and water 870 µl (volume of enzyme preparation could be

varied according to convenience) were added. A substrate blank without enzyme is



necessary since acetylthiocholine always has 2-5% of free –SH groups (depending on

the batch number and make) and it is unstable.

Calculation: AChE activity is defined as the number of micromoles (µmol) of

acetylthiocholine iodide hydrolyzed per minute (min) per milligram (mg) of protein.

Specific activity of AChE (µmol/min) = OD per min

4.43

OD – Optical Density

This, when divided by protein, gives specific activity. The specific activity of AChE is

expressed in µmol/min/mg of protein (Hestrin, 1949; Ellman, et al., 1961).

2.4 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 5m

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 60oC 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. Hemotoxylin 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 >1350U/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.

ALT

LDH



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

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.

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


MDH

AST



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

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.

ALP



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 (Gomall, 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.

(Doumasa, et al., 1971).

Reagent Composition

Albumin reagent

Succinate buffer (pH- 4.2) 75 mmol/l

Bromocresol green 0.14 g/l

Albumin Standard

Albumin Standard concentration 3 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 532nm (Chatterjee and Sil, 2006;

Mate, 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

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 -- --

Glucose Oxidase

Peroxidase



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 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 > 180U/l

Cholesterol oxidase > 200U/l

Peroxidase > 1000U/l

Cholesterol standard solution 200 mg/dl

Cholesterol esterase

Cholesterol Oxidase

Peroxidase



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

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

Glycerol kinase

Glycerol-3-phosphate oxidase

Peroxidase



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).

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 phenytoin concentration by HPLC method

i) Chromatographic conditions

Mobile phase consisting of methanol: water: glacial acetic acid (67: 33: 1 v/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 230 nm and sensitivity was set at 0.001

a.u.f.s.

ii) Preparation of standard graph

a) Standard solutions

Stock solution of 100 µg/ml of phenytoin 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 phenytoin

was used.

b) Plasma extraction procedure

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

carbamazepine 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 20µl was injected onto C18 column. The retention times were

4.49 min. and 5.15 min. for phenytoin and carbamazepine 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 were processed

with graphpad instat software.

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