Indira Gandhi Institute of Pharmaceutical Sciences

(Approved by: A.I.C.T.E. & P.C.I, New Delhi, Affiliated to

BHUBANESWAR
B.P.U.T & O.S.B.P, Bhubaneswar)

PHARMACOLOGY-||

B.PHARM

PRACTICAL NOTEBOOK

NAME

REGD NO

SEM
 CERTIFICATE

Name:

Semester:
Roll No:
Registration No:
Course:

Institution
This is certified to be the bonafied work of the student in the
Laboratory during the
academic year

No. of practicals certified out of in the

subject

Teacher In charge Principal

Examiner
 Index
Experiments Page no.
1. Dose calculation in pharmacological experiments 1-6
|2. Antiallergic activity by mast cell stabilization assay 7-8
|3. Study of anti-ulcer activity of a drug using pylorus ligand (SHAY) rat mnodel and 9-10
NSAIDS induced ulcer model.
4. |Study of effect of drugs on gastrointestinal motility 11-12
5
Effect of agonist and antagonists on guinea pig ileum 13-14
6 Estimation of serum biochemical parameters by using semi- autoanal yser 15-21
|7. Effect of saline purgative on frog intestine 22-23
Insulin hypoglycemic effect in rabbit 24-25
9. Test for pyrogens (rabbit nmethod) 26
10.|Determination of acute oral toxicity (LD50) of a drug from a given data 27-28
11Determination of acute skin irritation / corrosion of a test substance 29
12. Determination of acute eye irritation / corrosion of a test substance 30
13 Calculation of pharmacokinetic parameters from a given data 31-32
14. Biostatistics methods in experimental pharmacology( student's t test, ANOVA) 33
15. Biostatistics methods in experimental pharmacology (Chi square test, Wilcoxon 34-35
|Signed Rank test)
 EXPERIMENT 1

Dosage calculation and stock solution preparation in experimental

animals’ studies
Dosage calculation and stock solution preparation in preclinical studies, involving
the use of experimental animals is important in screening and development of new drugs.
Experimental animals have been of very important tools in the history of non-human
research models. Dosage calculation and stock solution preparation based on dosage
rationale formula are prerequisites to drug administration in experimental animals.

2. Vehicle of choice, drugs dissolution and volume selection rationale

A vehicle is any substance that acts as a medium in which a drug is administered. Vehicle,
which is an essential consideration in all animal research should be biologically inert, have
no toxic effects on the animals and not also influence the results obtained for the compound
under investigation.

Example of suitable vehicles for animal research include; water, normal saline (0.9%
sodium chloride), 50% polyethylene glycol, 5 to 10% Tween 80, 0.25% methylcellulose or
carboxymethylcellulose.

According to the OECD’s (organization of economic corporation and development’s)

guidelines, dosage of drug (mg) should be constituted in an appropriate volume not usually
exceeding 10 ml/kg (1 ml/100g) body weight of experimental animals (mice and rats) for
non-aqueous solvent in oral route of administration. However in the case of aqueous
solvents, 20 ml/kg (2 ml/100g) body weight can be considered.

Final dilution volume should not exceed 20 ml/kg.

Based on 10 ml/kg volume selection, required dose volume for a 100 g rat can be calculated
as follows:
3. Dosage calculation and preparation of stock solution of crude plant extract for
experimental animals: With reference to table 1 above, stock solutions and doses of a plant
extract (With selected doses, 200 mg/kg and 400 mg/kg) for a rat weighing 120 g be
calculated as follows;

1 ml of dissolved plant extract from a given stock solution (960 mg/48 ml = 20 mg/ml) is
the required dose (from selected dose of 200 mg/kg) for a rat weighing 100 g. However,
1.2 ml from the same stock solution is the required volume for a rat weighing 120 g (which
is meant to receive 24 mg of the plant extract). Having successfully prepared a stock
solution (960 mg/48 ml = 20 mg/ml) for a selected dose of 200 mg/kg, stock solution of the
same plant extract with a higher selected dose (400 mg/kg) can be easily be prepared by
dissolving 960 mg of plant extract with half the volume (24 ml) used in the previous stock
(960 mg/48 ml), thereby yielding a higher concentration (960 mg/24 ml = 40 mg/ml) which
is twice the concentration of the formal stock’ as shown in table 2 below. In this case
animals with similar body weight from two different selected dose categories (200 mg/kg
and 400 mg/kg respectively) will receive the same volume, but different concentrations.

4. Dosage calculation and preparation of stock solution of a reference drug (example:

Sylimarin) for experimental animals. Sylimarin used in animal model of screening for
agents with hepatoprotective, nephroprotective and anti-oxidant properties at standard
doses ranging between 25 mg/kg to 200 mg/kg body weight.
Conclusion:
 EXPERIMENT -2

ANTI-ALLERGIC ACTIVITY BY MAST CELL STABILIZATION ASSAY

AIM: To screen the anti-allergic activity of the drugs.

PRINCIPLE: In allergic diseases mast cells plays an important role by defending the
antigens. IgE antibodies formed in response to antigen antibody complex attaches to the
surface receptors of mast cells and rises calcium influx leading to degranulation of mast
cells which releases some pro-inflammatory mediators such as histamine and eicosanoids.

REQUIREMENTS:

Animals: Guinea pigs of 400-600 g of either sex, Albino rats of 175-200 g of either sex

Drugs : Histamine dihydrochloride aerosol (0.2% w/v)

Chlorpheniramine maleate (2 mg/kg, s.c.)
Disodium chromoglycate (50 mg/kg. i.p.)
Reagents: Saline solution (0.9%)
RPMI 1640 buffer medium (PH 7.2-7.4)
egg albumin (100 μg/mL)
toluidine blue solution (1%)
Instruments: Microscope with 10X magnification lens.

PROCEDURE: Evaluation of bronchoconstriction in guinea pigs by using histamine

aerosol. Selected guinea pigs are divided into two groups consisting of 3 animals in each.
Animals have to be fasted overnight. Group-1 receives normal saline. Group-2 animals
receive Chlorpheniramine maleate (2 mg/kg, S.C.). Before the drug treatment animals
should be exposed to histamine aerosol (0.2%) in histamine chamber. Then determine the
end point i.e. pre-convulsion dyspnea (PCD) is the time of time of exposure of histamine
aerosol to onset of dyspnea that leads to convulsion. As early as PCD is observed the
animals should be removed from chamber and placed in fresh air and time of onset of PCD
is to be noted on day zero. Then animals have to treat with drug after 24 hours. After 1 hr
of drug administration once again animals are exposed to histamine aerosol and PCD is
determined. Percentage (%) of protection offered by the drug can be calculated by the below
formula.

Percentage (%) protection= (1-T1/T2) ×100

Where T1= mean value of PCD before drug administration, T2= mean value of PCD after
drug administration.

Mast cell stabilization activity

Albino rats of either sex are divided into two groups consisting of 3 animals in each. Group-
1 receives normal saline, and Group-2 receives Disodium chromoglycate (50 mg/kg. i.p.)
for 3 days. Inject 10 ml/kg of 0.9% saline into peritoneal cavity on 4th day to each animal.
Massage the peritoneal region of the animal gently for 5 min, then collect the peritoneal
fluid and transfer to the test tube which is carrying 7-10 ml of RPMI buffer. Centrifuge the
fluid for 400-500 RPM. Discard the supernatant and wash the pellets of mast cells twice
with same buffer by centrifugation. Add egg albumin to the above cell suspension and
incubate at 37oC for 10 min. Later the suspension has to stain with 1% toluidine blue
solution and observe the slide under microscope for calculating number of granulated and
degranulated mast cells in each group (total 100 cells are having to be counted from
different visual areas).

CONCLUSION

Pre-treatment of animals with standard drugs stabilizes mast cell membrane and generates
nitric oxide as defensive mechanism that inhibits the release of chemokines, which are
responsible for vasoconstriction.
 EXPERIMENT -3

STUDY OF ANTI-ULCER ACTIVITY OF A DRUG USING PYLORUS LIGAND

(SHAY) RAT MODEL

AIM:
To study the anti-ulcer activity of a drug using pyloric ligand (SHAY) rat model.

PRINCIPLE:
Peptic ulcer is one of the most prevalent gastrointestinal disorders. The aim of the present
study is to demonstrate the antiulcer activity of drugs using pylorus ligand (SHAY) rat
model. This was first demonstrated by Shay in 1945. Ligation of rat pylorus results gastric
acid accumulation in the fore-stomach leads to acute gastric ulcers. This procedure is used
to screen the drugs for their anti-secretary and antiulcer activity.

REQUIREMENTS
Animals : Albino Wistar rats of 150-200 g are selected for the study.
Drugs : Ether (anesthetic), Ranitidine 20 mg/kg, p.o, 0.9% normal saline
Reagents : 0.1 N NaOH, Phenolphthalein, Topfer’s reagent,
Instruments : Dissecting microscope, Burette, PH meter, Surgical instruments.

PROCEDURE

Animals are to be divided into two groups consisting of 3 animals in each group. Saline is
to be administered to control group and Ranitidine (20 mg/kg, P.O.) to other group. Animals
have to be fasted for one day with free access to water. 30 min prior to ligation process, the
drug (Ranitidine) should be given. Under light ether anesthesia a midline abdominal
incision is made and pylorus will be ligated with proper care and the wound is closed. Then
rats are to be placed individually in separate cages without food and water during this period
and allowed them to recover. Sacrifice the animals by decapitation after 4 hours and open
the stomach and collect the stomach contents in a centrifuge tube. Determine the PHPH
meter. Open the greater curvature of the stomach and clean the part with saline. Under 10X
magnification lens ulcers are to be observed and ulcer index is calculated by using the
formula given below.

Ulcer index= (U1 + U2+ U3) × 10-1

U1 = Average of number of ulcers per animal
U2 = Average of severity score
U3 = Percentage (%) of animals with ulcer
Intensity of ulcers with scoring: 0 – normal coloration; 0.5 – red coloration; 1 – spot
ulcer; 1.5 – hemorrhagic stress; 2 – deep ulcer; 3 – perforations.
Ulcer score: 1 mm (exact) = 1; 1-2 mm = 2; >2 mm = 3; >3 mm = 4

Analysis of stomach contents:

Measure total volume of gastric content and centrifuge at 1000 rpm for 10 minutes. Pipette
out one ml of supernatant liquid of the centrifuged content and dilute with 10 ml distilled
water. Titrate the liquid against 0.01N NaOH using Topfer’s reagent as indicator, till the
end point (appearance of orange colour). The volume of NaOH used is to be noted to
estimate free acidity. Titration has to be continued till the appearance of pink colour and
the volume of NaOH run down is to be noted to calculate total acidity.

Calculation of acidity is given below:

CONCLUSION

Comparison of ulcer index between study groups estimates the potency of antiulcer activity
of test drug. Decrease in volume of gastric contents, free and total acidity determines anti-
secretory activity of test drug and rise in PH evaluates acid neutralizing action of test drug
 Experiment - 4
EFFECT OF DRUGS ON GASTRO INTESTINAL MOTILITY

AIM : To study the effect of drugs on gastro-intestinal motility.

PRINCIPLE: Intestinal motility is regulated by the enteric nervous system of the gut
(Auerbach’s and Meissner’s plexuses) and the activity of this system can be modified by
autonomic nervous system. Hence effect of sympathomimetic and parasympathomimetic
drugs on intestinal motility can be studied by using isolated piece of intestine.
Parasympathomimetic drugs stimulate enteric neurons to release acetylcholine at
neuromuscular junctions and enhance muscle tone and rhythmicity of intestine.
Sympathomimetic drugs acts on alpha and beta receptors and releases adrenaline which in
turn prevents release of acetylcholine and inhibits muscle tone and rhythmicity.

Guinea pig ileum is advantageous for assay purposes as it produces steady baseline for
studying effects of drugs. Rabbit intestine (ileum, deuodenum, jejunum) usually jejunum is
used for the effects of pendular movements. In the present study rabbit ileum is selected for
estimating the effects of selected drugs on intestinal motility.

REQUIREMENTS:
Animals: Medium sized rabbit
Drugs: Adrenaline/Acetylcholine- 10 ug/ml,
Atropine sulphate- 100 ug/ml,
isoproterenol/isoprenaline 10 ug/ml,
Propranolol- 1 mg/ml, Phenylephrine- 10 ug/ml,
phentolamine- 0.1 ug/ml.

Solutions: Tyrode solution

Apparatus used: Kymograph, Dissecting board, Dissecting instruments, scissors,

petriplates, Syringe, Frontal writing lever, water bath with temperature controlling unit,
organ bath with aeration tube.

PROCEDURE

The procedure adopted for the study is the modified Finkleman method developed by
Walker and Scott. Select a medium sized rabbit for the study. Fast the animal for 24 hours
prior to experiment as food in gut results in messy dissection and flushing of gut contents
may damage the intestine. Before sacrificing the rabbit, prepare Tyrodes Ringer solution
and place about 250 ml of this solution in an ice cold flask. Sacrifice the animal by cervical
decapitation without use of anesthetic as it may affect the gut motility. Shave the abdomen
of the animal and vacuum the surface to remove adhered fur. Make a midline incision
through the skin and abdominal muscles. Locate ileum and a part of ileum was taken 10 cm
away from ileocaecal valve. An optimal length of tissue (5-6 cms) is cut carefully and tie
the thread to antimesenteric border on both sides and place them in the Tyrode solution.

Record the rhythmic activity of the ileum by using frontal writing lever and kymograph.
Suspend the tissue in organ bath of Tyrode solution (100 ml) at 370c with adequate oxygen
supply (mixture of 95% O2 and 5% of CO2). Tie one end of the thread of the tissue to fixed
point inside the organ bath and the other end to the lever for recording contractions on the
kymograph. Stabilise the tissue in the solution to the conditions for about 30 minutes.
Ensure the lever should be placed horizontally and record the normal contractions followed
by effects of drugs on muscles.

After recording normal contractions inject the drugs one by one and observe for force of
contraction and tone, frequency of contractions before and after drug administration. Inject
0.1 ml of drugs in the succession order in the organ bath and the responses are recorded.
After noting the effect of every drug, drain the muscle bath and refill with fresh warm
Tyrode solution (100 ml). Take the control reading before and after each drug response.
Maintain wash out period for 15-20 minutes for change of every drug and check the next
drug response only the when the tone and amplitude returned to original value
approximately. The drug and dose name should be mentioned in the recording after taking
response of each drug.
Order of adding drugs.
1. 0.1 ml of Ach, 1.0 ml of Ach, 0.1 ml of NE, 1.0 ml of NE (Ach increases contraction
and NE relaxes the tissue)
2. 1.0 ml of phenylephrine, 1.0 ml of isoproterenol (phenylephrine causes contraction by
inhibiting adenylate cyclase- alpha adrenergic agonists; isoproterenol causes relaxation by
showing beta agonist action)
3. 1.0 ml of phenatolamine (alpha adrenergic blocker). Wait for 2 minutes then proceed
for adding 1.0 ml of phenylephrine.
5. 1.0 ml of propranolol (beta adrenergic blocker). Wait for 2 minutes then proceed for
adding 1.0 ml of isoproterenol
6. 1.0 ml of atropine- wait for 3 minutes- add 1.0 ml of Ach- (to check for
parasympatholytic activity of atropine)

CONCLUSION

The effect of drugs on intestinal motility can be easily interpreted by the responses taken
on kymograph.
 EXPERIMENT 5
Effect of Certain Agonists and Antagonists on Isolated Guinea-pig Ileum

Guinea pig ileum is a smooth muscle receive dual nerve supply from autonomic nervous
system of both sympathetic and parasympathetic. Parasympathetic tone is dominant in
ileum which Ach cause contraction by stimulating M3-R
Why Guinea pig ileum is used?

1. No myogenic contraction so any drug induce contraction can be seen.

2. Very sensitive to histamine unlike the rabbit due to the presence of histaminase
enzyme.
Types of receptors present in guinea-pig ileum

➢ Cholinergic receptors.
➢ Serotonergic or tryptaminergic receptors (5-HT1→5-HT7).
➢ Histaminergic receptors.
➢ Adrenergic receptors.
Types of receptors in guinea-pig ileum:

1- Cholinergic receptors:

Receptor Agonist Antagonist

Nicotinic neuronal
Dil. Nicotine Conc. Nicotine
receptor (Nn- R)
muscarinic receptor
Ach Atropine
(M3 -R)

2- Serotonergic or tryptaminergic (5-HT ) receptors :

▪ Serotonin = 5-HT(5-hydroxytryptamine).
▪ 5-HT2A receptor found in smooth muscle and bronchi .

▪ Receptor Agonist Antagonist

-Methysergide &
ketanserin (selective)
5-HT2A (Gq11) serotonin
-cyproheptadine (non
selective block both 5-
HT2A &5-HT receptors)
3- Histaminergic receptors:

• Histamine is an autacoids naturally occurring in the body.

Receptor distribution Action Agonist Antagonist
Smooth
Mepyramine
muscles contraction Histamine
H1 cyproheptadine
Gastric Histamine Cimetidine
mucosa Gastric acid secretion
H2 Ranitidine
Decrease release of
H3 Presynaptic Histamine Histamine -

4- Adrenergic receptors:

Receptor Agonist Antagonist

α1 Adrenaline phentolamine

α2 Adrenaline -

Conclusion:
 EXPERIMENT 6

Estimation of serum biochemical parameters by using semi- autoanalyser

BILIRUBIN

• Bilirubin (hematoidin) is the yellow breakdown product of normal heme catabolism.

Bilirubin is excreted in bile and urine, and elevated levels may indicate certain diseases. It
is responsible for the the background straw-yellow color of urine (via its reduced
breakdown product, urobilin – the more obvious but variable bright yellow colour of urine
is due to thiochrome, a breakdown product of thiamine), the brown color of feces (via its
conversion to stercobilin), and the yellow discoloration in jaundice. • Bilirubin consists of
an open chain of four pyrrole-like rings (tetrapyrrole) a porphyrin ring. 3

TYPES OF BILIRUBIN

Direct bilirubin (conjugated bilirubin-BC) -In the liver, bilirubin is conjugated with
glucuronic acid by the enzyme glucuronyl transferase, making it soluble in water. Much of
it goes into the bile and thus out into the small intestine. However, 95% of the secreted
bilirubin is reabsorbed by the intestines (Terminal Ileum) and reaches the liver by portal
circulation and then resecreted by the liver into the small intestine. This process is known
as enterohepatic circulation. The remaining 5%-> large intestine->urobilinogen->
stercobilin-> feces.

Unconjugated or indirect bilirubin (BU) - Insoluble in water.

Total bilirubin ("TBIL") – It measures both BC and BU.

Total and direct bilirubin levels can be measured from the blood, but indirect bilirubin is
calculated from the total and direct bilirubin.

TOTAL BILIRUBIN = INDIRECT BILIRUBIN + DIRECT BILIRUBIN

NORMAL LEVELS

• Direct bilirubin: Less than 0.4 mg/dL or 7 μmol/L

• Total bilirubin: less than 1.5 mg/dL or less than 26 μmol/L 5

MEASUREMENT METHODS Originally the Van den Bergh reaction was used for a
qualitative estimate of bilirubin. Total bilirubin is now often measured by the 2,5-
dichlorophenyldiazonium (DPD) method.

URINE TESTS Urine bilirubin may also be clinically significant. Bilirubin is not normally
detectable in the urine of healthy people. If the blood level of conjugated bilirubin becomes
elevated, e.g. due to liver disease, excess conjugated bilirubin is excreted in the urine,
indicating a pathological process.

Unconjugated bilirubin is not water-soluble and so is not excreted in the urine. Testing urine
for both bilirubin and urobilinogen can help differentiate obstructive liver disease from
other causes of jaundice.

INCREASES Total bilirubin is elevated in obstructive condition of the bile duct, hepatitis,
cirrhosis, in hemolytic disorders and several inherited enzyme deficiency.

Indirect bilirubin is elevated by prehepatic causes such as hemolytic disorder or liver

diseases resulting in impaired entry, transport or conjugation within the liver. Monitoring
of indirect bilirubin in neonates is of special importance as it the indirect (or free) bilirubin
bound to be albumin that is able to cross the BBB more easily, increasing the danger of
cerebral damage.

HYPERBILIRUBINEMIA

Hyperbilirubinemia results from a higher-than-normal level of bilirubin in the blood.

1. Mild rises in bilirubin may be caused by the following:

• Hemolysis or increased breakdown of red blood cells.

• Gilbert's syndrome – a genetic disorder of bilirubin metabolism that can result in mild
jaundice, found in about 5% of the population

• Rotor syndrome- non-itching jaundice, with rise of bilirubin in the patient's serum,
mainly of the conjugated type.

2. Moderaterise in bilirubin may be caused by:

• Pharmaceutical drugs-(especially antipsychotic, some sex hormones,
Sulfonamides are contraindicated in infants less than 2 months old as the increase
unconjugated bilirubin leading tokernicterus.
• Hepatitis (levels may be moderate or high) • Chemotherapy • Biliary stricture
(benign or malignant)
3. Very high levels of bilirubin may be caused by:

• Neonatal hyperbilirubinaemia- where the newborn's liver is not able to properly process
the bilirubin causing jaundice unusually large bile duct obstruction, e.g. stone in common
bile duct, tumour obstructing common bile duct etc.

• Severe liver failure with cirrhosis(e.g. primary biliary cirrhosis)

DEMO ESTIMATION - by ERBA kit METHODOLOGY-

PRINCIPLE: Bilirubin reacts with diazotized sulphanilic acid in acidic medium to form
pink colored azobilirubin with absorbance directly proportional to bilirubin concentration.
Direct bilirubin, being water soluble directly reacts in acidic medium. However indirect or
unconjugated bilirubin is solubilized using a surfactant than it reacts similar to direct
bilirubin.

SAMPLE: Unhaemolysed serum or plasma. Avoid hemolysis as it causes falsely low

results. Sample should be protected from bright light as bilirubin is photo labile. Samples
may be stored refrigerated for 3 days or frozen for 1 month. REAGENT COMPOSITION
Surfactant 1.00% HCL 100 mmol/L Sulphanilic acid 5 mmol/L REAGENT 2: DIRECT
BILIRUBIN REAGENT Sulphanilic acid 10 mmol/L HCL 100 mmol/L REAGENT 3:
SODIUM NITRITE REAGENT Sodium nitrite 144 mmol/L REAGENT 1: TOTAL
BILIRUBIN REAGENT

Conclusion:

ASSAY PROCEDURE TOTAL BILIRUBIN / DIRECT BILIRUBIN Pipette into test

tubes marked Blank Standard Test Working reagent 500 μl 500 μl 500 μl Distilled water
25 μl - - Standard/calibrator - 25 μl - Test - - 25 μl

GLUCOSE

Glucose (C6H12O6, also known as D-glucose, dextrose, or grape sugar) is a simple

monosaccharide found in plants. It is one of the three dietary monosaccharides, along with
fructose and galactose, that are absorbed directly into the bloodstream during digestion. An
important carbohydrate in biology, cells use it as a secondary source of energy and a
metabolic intermediate. Glucose is one of the main products of photosynthesis and fuels for
cellular respiration. Glucose exists in several different molecular structures, but all of these
structures can be divided into two families of mirror-images (stereoisomers). Only one set
of these isomers exists in nature, those derived from the "particular chiral form" of glucose,
denoted D-glucose. The chemical D-glucose is sometimes referred to as dextrose. Glucose
is a major source of energy for most cells of the body; insulin facilitates glucose entry into
the cells.

INCREASES Due to diabetes mellitus, in patients receiving glucose containing fluids

intravenously, during severe stress and cerebro vascular accidents.

DECREASES On insulin administration, as a result of insulinoma, inborn errors of

carbohydrate metabolism or on fasting.

FUNCTION
• ANALYTE IN MEDICAL BLOOD TEST Glucose is a common medical analyte
measured in blood samples. Eating or fasting prior to taking a blood sample has an effect
on the result. A high fasting glucose blood sugar level may be a sign of prediabetes or
diabetes mellitus.

• ENERGY SOURCE Glucose is a ubiquitous fuel in biology. Use of glucose may be by

either aerobic respiration, anaerobic respiration, or fermentation. Glucose is the human
body's key source of energy, through aerobic respiration, providing approximately 3.75
kilocalories (16 kilojoules) of food energy per gram. Breakdown of carbohydrates (e.g.
starch) yields mono- and disaccharides, most of which is glucose. Glucose is a primary
source of energy for the brain, and hence its availability influences psychological processes.
When glucose is low, psychological processes requiring mental effort (e.g., self-control,
effortful decisionmaking) are impaired. Use of glucose as an energy source in cells is via
aerobic respiration or anaerobic respiration.

NORMAL VALUES Fasting Value Post Prandial Category of a person Minimum Value
Maximum Value 2 hours after consuming glucose Normal 70 100 Less than 140 Early
Diabetes 101 126 140 to 200 Established Diabetes More than 126 - More than 200.

ESTIMATION OF GLUCOSE Enzymatic methods for glucose determination are classified

into three groups: 1. Methods with glucose oxidase, 2. Methods with hexokinase, 3.
Methods with glucose dehydrogenase.

PRINCIPLE Glucose oxidase (GOD) converts glucose to gluconic acid. Hydrogen peroxide
formed in this reaction, in presence of peroxidase (POD) oxidatively couples with
4aminoantipyrine and phenol to produce red quinoneimine dye. This dye has absorbance
maximum at 505 nm (500- 550 nm). The intensity of the colour complex is directly
proportional to the concentration of glucose in sample. Principle: (Trinder’s method ) -D-
glucose Mutarotase -D-glucose +H2O+O2 -D-glucose Glucose oxidase H2O2+ 4-
aminophenazone+phenol The intensity of the color concentration in the sample. formed D-
gluconic acid+H2O2 Peroxidase is Quinonemine +4 H2O proportional to the glucose

DEMO ESTIMATION - by ERBA kit ASSAY PROCEDURE Pipette into tubes marked
Working reagent Distilled water Standard Test Blank 1000ul 10ul --- Standard 1000ul -
10ul -- Test 1000ul --10ul

SGOT

Aspartate transaminase (AST) also called aspartate aminotransferase is commonly known

as SGOT(AspAT/ASAT/AAT) or serum glutamic oxaloacetic transaminase (SGOT), is a
pyridoxial phosphate (PLP)dependent transaminase enzyme. AST catalyses the reversible
transfer of an α- amino group between aspartate and glutamate and, as such, in an important
enzyme in amino acid metabolism. Transaminase or aminotransferase is an enzyme that
catalyses a type of reaction between an amino acid and a α–keto acid. An amino acid
contains an amine (NH2) group. A keto acid contains a keto (=O) group. In transamination,
the NH2 group on one molecule is exchanged with the =O group on the other molecule.
The amino acid becomes a keto acid, and the keto acid becomes an amino acid. AST is
found in liver, heart,skeletal muscle, kidney, brain and red blood cells, and it is commonly
measured clinically as a marker for liver health.

CLINICAL SIGNIFICANCE SGOT is important in the clinical diagnosis of human

disease. AST is associated with liver parenchymal cells, heart, skeletal muscle, kidney,
brain, red blood cell are released from cells as a part of cell injury that occurs in myocardial
infraction, hepatitis, acute pancreatitis, acute haemolytic anaemia, severe burns, acute renal
disease, musculoskeletal disease and trauma. Assay of these enzyme activities in blood
serum can be used both in diagnosis and in monitoring the progress of a patient during
treatment. AST was defined as biochemical marker for diagnosis of acute myocardial
infraction earlier. AST is commonly measured clinically as a part of diagnostic liver
function test to determine liver health such as liver cancer, liver cirrhosis.

INCREASES: Increased levels are associated with liver diseases or damage, myocardial
infraction, muscular dystrophy.

DECREASES: Decreased levels are observed in patients undergoing renal dialysis and
those with B6 deficiency. Monitoring the change in the levels over a period of time is
beneficial to the physician evaluating myocardial infraction or following chronic or
resolving hepatitis. FUNCTIONS Aspartate transaminase catalyses the interconversion of
aspartate and α-ketoglutarate to oxaloacetate and glutamate. Reaction catalysed by
aspartate aminotransferase. Aspartate(Asp) + α-ketoglutarate ↔ oxaloacetate + glutamate

REFERENCE VALUES • Female-6-34 IU/L • Male-8-40 IU/L

ESTIMATION OF SGOT LEVEL IN SERUM PRINCIPLE AST (Transaminase enzyme)

catalyses the following reaction. L-Aspartate +2- Oxaloglutarate Oxaloacetate+ L-
glutamate In this present method salts is used which selectively reacts with oxaloacetate to
produce a colour complex that is measured photomertically.

DEMO ESTIMATION - by ERBA kit METHOD AST L-Aspartate + 2-Oxaloglutarte

Oxaloacetate + L-Glutamate MDH Oxaloacetate + NADH Malate +NAD LDH Sample
pyruvate + NADH L-Lactate +NAD AST : Aspartate aminotransferase MDH : Malate
dehydrogenase LDH : Lactate dehydrogenase SAMPLE Unhaemolysed serum or
Heparinised plasma. According to the IFCC expert panel on enzymes, AST is stable for 3
days at 4oc.
ASSAY PROCEDURE Allow the working reagent to attain 37oc before performing the
test. Pipette Volumes Working reagent 1000ul Test 100ul.

SGPT

Alanine transaminase or ALT is a transaminase enzyme. It is also called serum glutamic-

pyruvic transaminase (SGPT), or alanine amino transaminase (ALAT). ALT is found in
plasma and in various bodily tissues, but is most commonly associated with the liver. An
alanine aminotransferase (ALT) test is often part of an initial screening for liver disease.
Normally, ALT is found inside liver cells. But if the liver is inflamed or injured, ALT is
released into the bloodstream. In a normally healthy individual, the level of SGPT is
measurable in the blood. When there is acute liver damage, the level of SGPT tends to rise
dramatically. ALT is present in high concentration in the liver and to a lesser extent in
kidney, heart, skeletal muscle, pancreas, spleen and lungs. The next stage of the liver test
for SGPT is to understand the underlying cause of the liver damage. The liver could be
damaged by an infectious disease such as mononucleosis or hepatitis. This damage is
generally temporary and heals after the patient has recovered from the condition. The level
of SGPT is also elevated in an individual who is suffering from bile related problems. There
are many different medications that are likely to cause an elevation in the level of SGPT in
the blood. This elevation tends to be temporary and gets reversed as the patient’s body
absorbs the medication or passes it out of the system in the urine. When a drug overdose
has occurred, the patient may suffer from liver damage which, apart from causing an
elevation in the level of ALT also causes other typical symptoms of liver damage. The liver
test for SGPT is diagnostically relevant and can be used with other tests such as the ALT
or SGOT test. These tests can confirm whether the elevation in the level of SGPT is related
to liver damage or related to bile duct problems. The liver test for SGPT is almost never
conducted in isolation. Significantly elevated levels of ALT (SGPT) often suggest the
existence of other medical problems such as viral hepatitis, diabetes, congestive heart
failure, liver damage, bile duct problems, infectious mononucleosis, or myopathy. For this
reason, ALT is commonly used as a way of screening for liver problems. Elevated ALT
may also be caused by dietary choline deficiency. However, elevated levels of ALT do not
automatically mean that medical problems exist. Fluctuation of ALT levels is normal over
the course of the day, and ALT levels can also increase in response to strenuous physical
exercise.

INCREASES

Increases levels are generally a result of primary liver diseases such as cirrhosis
,carcinoma,viral or toxic hepatitis and obstructive jaundice.

DECREASES
Decreased levels may be observed in renal dialysis patients and those with vitamin B6
deficiency.

REFFERENCE VALUES Female ≤ 34 IU/L Male ≤ 45 IU/L FUNCTION It catalyzes the

transfer of an amino group from L-alanine to α-ketoglutarate, the products of this reversible
transamination reaction being pyruvate and L-glutamate. L-glutamate + pyruvate ֖ α-
ketoglutarate + L-alanine ALT (and all transaminases) require the coenzyme pyridoxal
phosphate, which is converted into pyridoxamine in the first phase of the reaction, when an
amino acid is converted into a keto acid. ESTIMATION OF SGPT PRINCIPLE ALT
(GPT) catalyze the transfer of amino groups from specific amino acids to ketoglutaric acid
yielding glutamic acid and oxaloacetic or pyruvic acid respectively. These ketoacids are
then determined colorimetrically after their reaction with 2,4dinitrophenylhydrazine
(DNP). L-Alanine + α – Ketoglutarate ↔ Pyruvate + L-Glutamate Pyruvate + NADH + H+
↔ L – Lactate + NAD+

DEMO ESTIMATION - by ERBA kit METHODS ALT(Alanine aminotransferase) L-

Alanine + 2- Oxoglutarate Pyruvate + L-Glutamate LDH(Lactate dehydrogenase) L –
Lactate + NAD+ Pyruvate + NADH REAGENT RECONSTITUTION Allow the reagent
bottle and Aqua-4 to attain room temperature (15-30oc). Add the amount of Aqua-4
indicated on the label to contents of each vial. Swirl to dissolve ,do not shake vigorously.

SAMPLE Unhemolysed serum or heparinised plasma. Anticoagulant such as Heparin or

EDTA are suitable. ALT is stable for 3 days at 2-8oc.

ASSAY PROCEDURE Pipette Volumes Working reagent 1000ul Test 100ul 20

Conclusion:
 EXPERIMENT - 7

EFFECT OF SALINE PURGATIVE ON FROG INTESTINE

AIM: To study the effect of saline purgative on frog intestine.

PRINCIPLE: Saline purgatives are the salts comprising of highly charged ions and do not
crosses cell membrane freely. They remain inside the lumen and retain water through
osmotic forces. They increase the volume of the contents of the bowel, stretch the colon
and produces normal stimulus for contraction of the muscle that leads to defecation. The
aim of the present study is to examine the effect of saline purgative on frog intestine.

REQUIREMENTS
Animal : Frog
Reagents : 0.9% to 0.45% of saline (hypotonic),
27% Magnesium sulphate (hypertonic),
Frogs Ringer solution (isotonic)

Instruments used: Frog’s board, pithing needle, dissecting instruments, needle with
thread, tuberculin syringe with needle.

PROCEDURE: Pith the frog and place it on a dissecting board. Expose the abdominal
cavity and carefully trace the small intestine. Make the small intestine into three
compartments by tying threads of different colours in such a way that no fluid can move
from one compartment to the other. Inject 0.2 ml of each hypotonic solution into first
compartment, 0.2 ml of hypertonic solution to second compartment and 0.2 ml of isotonic
solution into third compartment. Wait for 20 minutes and the observations are to be
recorded.
 Fig: Representation of Saline purgatives

CONCLUSION:

Hypotonic solution causes the fluid to move from lumen into circulation by process osmosis
thereby shrinks the tissue. Hypertonic solution moves the fluid from cells into the lumen
and swells the tissue and isotonic solution did not shows any fluid movement across the
intestinal membrane.
 EXPERIMENT-8

STUDY THE INSULIN HYPOGLYCEMIC EFFECT IN RABBIT

AIM : To study the insulin hypoglycemic effect in rabbit.

PRINCIPLE: Insulin is a peptide hormone produced by the beta cells of pancreas in

response to high glucose levels in the blood. Released insulin acts on the insulin receptors
on body cells and activates glucose transporters to absorb more glucose into the cells
thereby regulates carbohydrate, protein and fat metabolism in body cells. Reduced blood
glucose levels inhibit insulin release and stimulate alpha cells of pancreas to release
glucagon to maintain glucose levels in the blood by glycogenolysis and
gluconeogenesis. The aim of the present study is to evaluate the effect of insulin in rabbits
at different time intervals.

REQUIREMENTS
Animals : Healthy rabbits weighing 1800-3000 gms.
Drugs : 20 units of insulin preparation. One unit contains 0.04082 mg of insulin
Reagents : Normal saline, HCl, 0.5% phenol, 1.4-1.8% glycerin.

PROCEDURE

Select healthy rabbits weighing 1800-3000 gms for the study. They should be maintained
in uniform diet for 7 days. Fast the animals for 18 hrs with no access to water before starting
the procedure. Select three animals for the study and inject 1 unit/ml of insulin. Prepare
drug solution freshly. Weigh 20 units of insulin accurately and dissolve it in normal saline.
Acidify the solution by using HCl to pH 2.5. Add 0.5% of phenol as preservative and 1.4-
1.8% of glycerin and make the final volume to 20 units/ml of solution. Withdraw 2 ml of
blood from marginal ear vein of each rabbit and estimate blood glucose level by using
suitable biochemical method and the concentration of glucose can be noted down as initial
blood glucose level. Then inject insulin (1 unit/ml) to the animals and check the blood sugar
level up to 5 hours at the interval of 1 hour each and the determine blood glucose levels as
final blood sugar level and compared both initial and final blood glucose levels.
CONCLUSION

Mean percentage decrease of blood glucose levels at different time intervals determines
the effect of insulin.
 EXPERIMENT 9
Test for pyrogens (rabbit method)
Aim: Learn how to determine the pyrogen in parenteral preparations by injecting the sample
in rabbits for pyrogen testing.

Introduction: Pyrogen test is performed to check the presence or absence of pyrogens in all
aqueous parenterals. Rabbits are used to perform the test because their body temperature
increases when pyrogen is introduced by the parenteral route.

For this test, three healthy rabbits are selected each weighing at least 1.5 kg. No rabbit
should be selected if:

1. It has a normal temperature greater than 49.8°C.

2. It was used in a positive test during last two weeks or negative test during last two days.

Method for Pyrogen Test: The pyrogen testing is performed in an air-conditioned room.
The food and water is withheld to rabbit overnight. A clinical thermometer is inserted in
the rectum of each rabbit to a depth of not less than 7.5 cm. Two readings of the temperature
of rabbit in normal conditions should be taken at the interval of half an hour before start the
test and mean of the both should be calculated to determine the initial temperature. The
equipment, injectors and needles used in the test should be pyrogen-free. These should be
washed with water for injection and then heated at 260°C for two hours. The injection is
warmed to 38°C before injecting to the rabbits. 0.5 to 1.0 ml per kg dose should be injected
through the ear vein. Six reading of temperature is recorded at an interval of half an hour.

Pyrogen Test Results: The response of each rabbit is detected by the difference of initial
temperature and the highest temperature recorded. The response of all three rabbits gives
the sum of responses and can be concluded as:

i) If the sum of responses does not greater than 1.4°C and any of rabbit shows the
response less than 0.6° C, the product passes the test.
ii) I sum of responses is greater than 1.4 °C or any of rabbit shows the response 0.6
or greater, continue the test using 5 rabbits.
iii) If the test is done using 5 rabbits, then if the sum of responses of all 5 rabbits is
greater than 3.7°C and the individual response of not more than three rabbits is
greater than 0.6°C, the product passes the test.
Conclusions:
 EXPERIMENT 10

Determination of acute oral toxicity (LD50) of a drug from a given data:

Median lethal dose, LD50 is a measure of the lethal dose of a toxin dose required to kill
half the members of a tested population after a specified test duration. LD50figures are
frequently used as a general indicator of a substance's acute toxicity. A lower LD50 is
indicative of increased toxicity.
LD50 is usually determined by tests on animals such as laboratory mice.
The LD50 is usually expressed as the mass of substance administered per unit mass of test
subject, nanograms (suitable for botulinum), micrograms, or grams (suitable
for paracetamol) per kilogram.
LD50 is not the lethal dose for all subjects; some may be killed by much less, while others
survive doses far higher than the LD50. Measures such as "LD1" and "LD99" (dosage
required to kill 1% or 99%, respectively, of the test population) are occasionally used for
specific purposes.
Lethal dosage often varies depending on the method of administration, LD50 figures are
often qualified with the mode of administration, e.g., "LD50 i.v."

LD50 :
Substance Animal, Route LD50
g/kg

Water rat, oral 90,000 mg/kg 90

Sucrose (table sugar) rat, oral 29,700 mg/kg 29.7
Glucose (blood sugar) rat, oral 25,800 mg/kg 25.8
Vitamin C (ascorbic acid) rat, oral 11,900 mg/kg 11.9
Glyphosate (isopropylamine salt of) rat, oral 10,537 mg/kg 10.537
Lactose (milk sugar) rat, oral 10,000 mg/kg 10
Aspartame mice, oral 10,000 mg/kg 10
Urea rat, oral 8,471 mg/kg 8.471
Ethanol (Grain alcohol) rat, oral 7,060 mg/kg 7.06
Methanol human, oral 810 mg/kg 0.81
Sodium chloride (table salt) rat, oral 3,000 mg/kg 3
 LD50 :
Substance Animal, Route LD50
g/kg

Paracetamol (acetaminophen) mouse, oral 338 mg/kg 0.338

Ibuprofen rat, oral 636 mg/kg 0.636
Psilocybin (from magic
mouse, oral 280 mg/kg 0.280
mushrooms)
rat,
Ketamine 229 mg/kg 0.229
intraperitoneal
Aspirin (acetylsalicylic acid) rat, oral 200 mg/kg 0.2
Caffeine rat, oral 192 mg/kg 0.192

Conclusion:
 EXPERIMENT 11

Determination of acute skin irritation / corrosion of a test substance

Aim: To test skin irritation/corrosion by using rabbit

Skin irritation and skin corrosion refer to localized toxic effects resulting from a topical
exposure of the skin to a substance.

The Globally Harmonized System of Classification and Labeling of Chemicals (GHS)

defines skin irritation as “the production of reversible damage to the skin following the
application of a test substance for up to 4 hours” and defines skin corrosion as “the
production of irreversible damage to the skin; namely, visible necrosis through the
epidermis and into the dermis, following the application of a test substance for up to 4
hours”.

Structure of Skin: The skin is the largest human organ, but is more than just a protective
covering for the body. The skin is composed of multiple layers and serves many functions
important to survival. The outermost layer of the skin is called the epidermis. The epidermis
is made up of approximately four layers of epithelial cells called keratinocytes. The
epidermal keratinocytes and junctions between these cells form the barrier of the skin,
preventing substances from penetrating the skin, and water and electrolytes from leaking
out of the body. The deeper layer of the skin is called the dermis. The high content of
collagen and elastin in the dermis impart strength and elasticity to the skin. Blood vessels,
nerves, sweat glands, and hair roots are also found within the dermis. The innermost layer
of the skin, called the subcutaneous layer, is relatively thick and primarily composed of fat
cells. It is a source of insulation and physical protection for the body as well as a source of
energy for the cells.

Method of testing
A test substance is applied to the shaved bare skin about 6 cm2 of healthy young adult
albino rabbits and the area is covered with gauze (OECD Test Guideline (TG) 404; Acute
Dermal Irritation/Corrosion). The substance is removed after four hours and the rabbit’s
skin is observed at specific times for irritant responses for as many as 14 days. One animal
is usually tested first. The GHS reports that animal skin irritation and corrosion responses
are quite variable, so the document explains a range of responses for classification purposes.

Conclusion:
 EXPERIMENT 12

Determination of acute eye irritation / corrosion of a test substance

OBJECTIVES

1. Instill drugs carefully into the rabbit's eye by the pouch method without injuring the
cornea.
2. Study the effects of drugs on the rabbit's eye.
3. Record, analyze and interpret the observations obtained during the experiment .

Animals: Rabbits

Apparatus: Droppers, measuring scale, torch, cotton wool, calculator

Drugs & solutions:

1. Saline
2. Eserine salicylate 0.5%
3. Atropine sulphate 1.0%
4. Lignocaine 1.0%

PROCEDURE

Place the rabbit (No.1) on the table. Measure the diameter of both the pupils with the
help of a scale. Observe the condition of the conjunctiva (congested or not) and elicit the
corneal and light reflexes. Record your findings. In the left eye put one drop of saline
and in the right eye one drop of eserine. Use the pouch method for instilling the drops.
After adding the drops, the medial canthus should be pressed for 30 seconds. Record the
following parameters at one minute, 5minutes and ten minutes after instilling the drug and
saline. Parameters to be measured:
1. Diameter of the pupil
2. Light reflex
3. Corneal reflex
Record your observations in a tabular form. Repeat the same procedure for atropine, and
lignocaine on separate rabbits (Nos 2, & 3 ) .

Repeat the experiment in your free time using ExPharm v2 for Windows - A Computer
Assisted Learning (CAL) software programme with simulated experiments.

Conclusion:
 EXPERIMENT 13
Calculation of pharmacokinetic parameters from a given data

OBJECTIVES : 1. present data in a tabular form.

2. carry out basic statistical analysis of data.
3. interpret the results and draw conclusions.

I. Data Presentation:

An introductory class will be taken on data presentation (30 min) and students will be divided into
4 groups. Each group will be given simulated results (data) of an experiment and asked to devise a
table to display the data (30 min). Plenary will be held where each group will present their table.

Task A : Read the following situations and present the data in a tabular form :

1. In an experiment, 2 groups of 6 rats were injected with amphetamine and saline respectively and
food intake was measured for 2 hrs. The initial weight of rat food was 10 g for each rat. At the end
of 2 hrs, the remaining food was weighed and the following data were obtained:

Group I - Saline - 8.21, 5.3, 6.40, 7.584, 6.120, 7.2 g

Group II - Amphetamine - 9.5, 9.473, 9.24, 8.9, 9.90, 10 g

2. The effects of atropine (1.0 % solution) on the pupil size was studied in 8 rabbits. The pupil size
was measured before and after administration of saline in one eye and atropine in the other. The
data obtained were as follows.

Right eye Left eye

Saline(1drop)before 5, 6, 3, 4, 7, 5, 6, 5 mm Atropine(1 drop)before 6, 4,7,3,5, 6, 5, 6 mm

after 6, 5, 4, 4, 6, 6, 6, 6 mm after 7, 8, 9, 6, 8, 9, 6, 8 mm

3. The cardiostimulant activity of a plant extract (2 μg) was compared with that of epinephrine (2
μg) Twelve isolated frog heart preparations were used and heart rate was observed after
administration:

Preparation no. 1- 6: Plant extract - 91, 89, 83, 59, 81, 87 bpm

Basal heart rate- 58, 62, 54, 45, 75, 80 bpm

Epinephrine - 90, 85, 62, 60, 85, 91 bpm

Preparation no. 7-12:

Basal heart rate- 75, 70, 61, 52, 73, 75 bpm

4. The analgesic effect of morphine (3 mg/kg; sc) and aspirin (10 mg/kg; sc) were compared using 0.6%
acetic acid writhing. The data are as follows.

Saline (n=6) : 22, 25, 29, 30, 21, 32 writhings (in 15 min)

Aspirin (n=7) : 15, 18, 18, 20, 22, 17, 16 writhings (in 15 min)

Morphine(n=6) : 5, 8,11, 2, 10, 4 writhings (in 15 min)

Conclusion:
 EXPERIMENT 14

Biostatistics methods in experimental pharmacology (student’s t test, ANOVA)

Introduction to Biostatistics:
Introduction to Biostatistics It is the branch of statistics that deals with data relating to living
organisms. A Biostatistician would be involved with carrying out research, devising
experiments, and providing an in depth analysis of all results. This is a great opportunity to
make a difference because by carrying out this crucial research, a Biostatistician can make a
difference to health care and public health.

In experimental pharmacology we have to understand when to use and how to calculate and
interpret different measures of central tendency (mean, median and mode) and dispersion
(range, IR and standard deviation) We have to identify the types of error encountered in
statistical analysis, the role of sample size and implications for decision making. Describe basic
assumptions required for utilization of common statistical tests including the student’s t-test,
Paired t-test, chi square analysis, wilcoxon signed rank etc.

Student’s t-Test : Two types

1. Independent
2. Paired

X and Y are the two populations. The bar above it means sample mean.
The n 1 and n 2 are the sample sizes. Sp = pooled standard deviation
Analysis of Variance (ANOVA) Is a technique whereby the total variation present in a data
set is partitioned or segregated into several components.

For example, if four drug levels with their six possible combinations are to be compared, and
each comparison is made by using Alpha = .05,
-there is a 5% chance that each comparison will falsely be called significant.
So the recommended use of ANOVA protects the researcher against error inflation by first
asking if there are differences at all among means of the groups. Some basic concepts in
experimental designs are the minimum requirements to appreciate the approach of ANOVA in
estimating and testing the hypotheses about - population means or about - population variances.

It may be pointed out that when experiments are designed with the analysis in mind,researchers
can, before conducting experiments, identify those sources of variation that they consider
important and choose a design that will allow them to measure the extent of the contribution
of these sources to total variation.

Conclusion:
 EXPERIMENT 15

Biostatistics methods in experimental pharmacology (Chi square test, Wilcoxon Signed

Rank test)

Chi-square test

The Chi-square test is a non-parametric test of proportions. This test is not based
on any assumption or distribution of any variable. This test, though different,
follows a specific distribution known as Chi-square distribution, which is very
useful in research. It is most commonly used when data are in frequencies such as
number of responses in two or more categories. This test involves the calculations
of a quantity called Chi-square (x 2 ) from Greek letter ′Chi′(x) and pronounced as
′Kye.′ It was developed by Karl Pearson.

Applications

1. Test of proportion: This test is used to find the significance of difference in two or more
than two proportions.
2. Test of association: The test of association between two events in binomial or
multinomial samples is the most important application of the test in statistical methods.
It measures the probabilities of association between two discrete attributes. Two events
can often be studied for their association such as smoking and cancer, treatment and
outcome of disease, level of cholesterol and coronary heart disease. In these cases, there
are two possibilities, either they influence or affect each other or they do not. In other
words, you can say that they are dependent or independent of each other. Thus, the test
measures the probability (P) or relative frequency of association due to chance and also
if two events are associated or dependent on each other. Varieties used are generally
dichotomous e.g. improved / not improved. If data are not in that format, investigator
can transform data into dichotomous data by specifying above and below limit.
Multinomial sample is also useful to find out association between two discrete
attributes. For example, to test the association between numbers of cigarettes equal to
10, 11- 20, 21-30, and more than 30 smoked per day and the incidence of lung cancer.
Since, the table presents joint occurrence of two sets of events, the treatment and
outcome of disease, it is called contingency table (Con- together, tangle- to touch).

How to prepare 2 × 2 table

When there are only two samples, each divided into two classes, it is called as four cell or 2 ×
2 contingency table. In contingency table, we need to enter the actual number of subjects in
each category. We cannot enter fractions or percentage or mean. Most contingency tables have
two rows (two groups) and two columns (two possible outcomes). The top row usually
represents exposure to a risk factor or treatment, and bottom row is mainly for control. The
outcome is entered as column on the right side with the positive outcome as the first column
and the negative outcome as the second column. A particular subject or patient can be only in
one column but not in both. The following table explains it in more detail:
Even if sample size is small (< 30), this test is used by using Yates correction, but frequency
in each cell should not be less than 5. Though, Chi-square test tells an association between two
events or characters, it does not measure the strength of association. This is the limitation of
this test. It only indicates the probability (P) of occurrence of association by chance. Yate′s
correction is not applicable to tables larger than 2 X 2. When total number of items in 2 X 2
table is less than 40 or number in any cell is less than 5, Fischer′s test is more reliable than the
Chi-square test.

2) Wilcoxon-Matched-Pairs Signed-Ranks Test

This is a non-parametric test. This test is used when data are not normally distributed in a paired
design. It is also called Wilcoxon-Matched Pair test. It analyses only the difference between
the paired measurements for each subject. If P value is small, we can reject the idea that the
difference is coincidence and conclude that the populations have different medians.

Conclusion: