3. Pharmacology-III: (Marks-30) Estimation of glucose in blood in normal condition and after administration of insulin; biological assay of digitalis, histamine and insulin; microbiological assay of antibiotics and vitamins; spectrophotometric estimation of blood pigments; toxicity test of the drugs like, phenobarbitone, nikethamide, some antineoplastic drugs, pilocarpine, etc.

1. Laboratory Manual
Course: 410
Subject: Practical - Pharmacology-III
Prepared By
Md. Imran Nur Manik
M.Pharm
Department of Pharmacy
University of Rajshahi
Rajshahi-6205, Bangladesh
Available at
Essential Pharma documents
www.pharmacydocs.blogspot.com

2. 1
Sl. No. Date Name of the experiment Page No.
01 19.09.11
Estimation of glucose concentration in blood after oral
administration of glucose.
2–8
02 20.09.11
Estimation of glucose concentration in blood at fasting
condition.
9–13

3. 2
Experiment No. 01 Date: 19.09.11
Name of the experiment: Estimation of glucose concentration in blood after oral
administration of glucose.
Introduction:
Glucose is the primary source of energy for the body's cells. The mean normal blood
glucose level in humans is about 80–120 mg/dl (4.4–6.1 mmol/L). The human body naturally
tightly regulates glucose levels in blood as a part of metabolic homeostasis.
Sugar levels in blood outside the normal range may be an indicator of a medical
condition.
Hypoglycemia: Hypoglycemia is a condition is which blood sugar level is present below
the normal, that is, below 80 mg/dL. Symptoms may include lethargy, impaired mental
functioning, irritability, shaking, twitching, sweating and loss of consciousness, weakness in
arm and leg muscles. Hypoglycemic symptoms are relieved by the administration of glucose.
Hyperglycemia: It is a condition in which blood sugar level increases above the normal
i.e. 120 mg /dL. Long-term hyperglycemia causes many of the long-term health problems
associated with diabetes, including eye, kidney, heart disease and nerve damage. When the
blood sugar level exceeds the renal threshold (180 mg /dL) sugar appears in urine.
Diabetes mellitus: Diabetes is a metabolic disorder characterized by hyperglycemia due
to deficiency of insulin. This is a disease in which body does not properly control the amount
of sugar in blood. As a result the level of sugar in the blood becomes too high, that is, 7 m
mol/L in fasting condition and after 2 hours of diet is more that 11.1 m mol/L.
Classification: There are two main types of diabetes mellitus.
1. Type – 1: Insulin dependent diabetes mellitus (IDDM) or juvenile onset diabetes.
2. Type – 2: Non-insulin dependent diabetes mellitus (NIDDM) or maturity onset
diabetes.
Symptoms of Diabetes Mellitus:
i. Hyperglycemia
ii. Polyuria
iii. Polyphagia
iv. Polydipsia
v. Weight loss
vi. Weakness
vii. Itching
viii. Increased frequency of infections
ix. Fasting blood glucose is higher than 126 mg/100ml.
Md.
Imran
Nur
Manik

4. 3
Risk factors of diabetes: Diabetes mellitus can causes serious hyperglycemia and if it
left untreated, it can result in:
i. Retinopathies iv. Nephropathy
ii. Glaucoma v. Cardiovascular complication
iii. Neuropathies vi. Increased incidence of toxemia of pregnancy
Units:
The international standard way of measuring glucose levels in blood are in terms of a
molar concentration, measured in mmol/L. In the United States, mass concentration is
measured in mg/dL.
Since the molecular weight of glucose C6H12O6 is about 180 g/mol, for the measurement
of glucose, the difference between the two scales is a factor of 18, so that 1 mmol/L of
glucose is equivalent to 18 mg/dL.
Glucose level in blood at different stages:
Normal range = 80 – 120 mg / 100 (4.4–6.1 mmol/L) ml
Post prandial glucose level in blood:
72–126 mg/dl (4–7 mmol/L): Normal
> 126–180 mg/dl (7–10 mmol/L)): Suggestive of diabetes
> 180 mg/dl 10 mmol/ L): Almost certainly diabetes
Fasting blood glucose level:
65–110 mg/dl 3.6–6.1 mmol/L): Normal
> 110–160 mg/dl (6.1–9 mmol/L): Suggestive of diabetes
> 180 mg/dl (10 mmol/L): Almost certainly diabetes
Measurement of glucose level in blood:
Glucose is measured in whole blood, plasma or serum. Historically, blood glucose values
were given in terms of whole blood, but most laboratories now measure and report the serum
glucose levels. Because red blood cells (erythrocytes) have a higher concentration of protein
(e.g., hemoglobin) than serum, serum has a higher water content and consequently more
dissolved glucose than does whole blood.
Measurement techniques:
Two major methods have been used to measure glucose.
1. The first one is a chemical method exploiting the nonspecific reducing property of
glucose in a reaction with an indicator substance that changes color when reduced. Since
other blood compounds also have reducing properties (e.g., urea), this technique can produce
erroneous readings in some situations (5 to 15 mg/dl has been reported). In this method,
different oxidizing agent (e.g., Fehling solution, Arsenic molybdate, Alkaline ferricyanide
etc.) are used.
Md.
Imran
Nur
Manik

5. 4
2. The more recent technique is the enzymatic method in which specific enzymes to
glucose are used. The two most common employed enzymes are glucose oxidase and
hexokinase. This method is less susceptible to give erroneous reading.
Blood glucose laboratory tests:
1. Fasting blood sugar test (FBS)
2. Urine glucose test
3. Two-hour postprandial blood sugar test (2-h PPBS)
4. Oral glucose tolerance test (OGTT)
5. Intravenous glucose tolerance test (IVGTT)
6. Glycosylated hemoglobin (HbA1C)
7. Self-monitoring of glucose level via patient testing
Oral glucose tolerance test: A screening test for diabetes mellitus, in which plasma
glucose levels are measured after the patient consumes an oral glucose load. Plasma glucose
levels between 140199 mg/dl suggest impaired glucose tolerance and plasma glucose levels
when exceed 200 mg/dl after 2 hours of drinking a 75 g glucose load suggest diabetes
mellitus.
It is regarded as the gold standard of clinical tests of the insulin / glucose control system,
but is difficult to administer, requiring much time and repeated blood tests.
Different factors affecting the glucose level in experiment:
1. Collection of blood in clot tubes for analysis permits the metabolism of glucose in the
sample by blood cells until separated by centrifugation. As a result glucose concentration in
sample may reduce.
2. Higher than normal amounts of white or red blood cell counts can lead to excessive
glycolysis in the sample, with substantial reduction of glucose level if the sample is not
processed quickly.
3. Ambient temperature at which the blood sample is kept prior to centrifuging and
separation of plasma/serum also affects glucose levels.
4. The glucose level in sample may also be reduced due to the use of normal glass
centrifuge tube. This loss of glucose can be prevented by using Fluoride tubes (i.e., gray-
top) since fluoride inhibits glycolysis.
5. Error rates for blood glucose measurements systems vary, depending on laboratories
and on the methods used. Spectrophotometry techniques can be biased by color changes in
cell (from airborne or finger borne contamination) or interference (e.g., tinting contaminants)
with light source or the light sensor.
So, these factors must be carefully considered during the experiment to find out accurate
result.
Md.
Imran
Nur
Manik

6. 5
Principle:
The glucose of blood sample determination is based on the reaction of glucose with
Fehling solution. Fehling solution is mild oxidizing agent and easily oxidizes the reducing
sugar such as glucose, present in blood. Blood also contain a small glucoronate which may
give rise to Cu2O formation but here mainly blood glucose is considered.
Fehling solution contain Cu2+
ions and is prepared by adding Fehling’s-A solution
containing CuSO4 to Fehling’s-B solution containing NaOH and Rochelle salt (Na-K
tartrate).
During oxidation to aldehyde to acid, the Cu2+
ions reduced to Cu+
ions which are
precipitated as red color of Cu2O.
R–CHO + 2Cu2+
+ 3OH–
 R–COO–
+ 2Cu+
+ 2H2O
2Cu2+
+ 2OH–
 Cu2O  + H2O
The color of the blood sample (after reduction with Fehling solution) is adjusted with
Arsenic molybdate to form a color complex.
Arsenic molybdate + Cu2O  Color complex (soluble)
The absorbance of color complex is measured by spectrophotometer at 520 nm
wavelength (max). By plotting the absorbance value of blood glucose sample on the
calibration curve, we can easily calculate the blood glucose concentration.
Reagents:
i. Arsenic molybdate
ii. 10% Na-tungstate
iii. Anticoagulant (Na-oxalate)
iv. Fehling solution (A:B = 25:1)
v. Glucose
vi. 2/3 N H2SO4
Md.
Imran
Nur
Manik

7. 6
Function of the reagents:
i. Arsenic molybdate: It is used to form complex with the solution so that, it can give
proper absorbance at maximum wavelength.
ii. Na-tungstate: It is used to precipitate the proteins and cell contents present in the
blood.
iii. Anticoagulant Na-oxalate: It is used to prevent the clotting of blood within the test
tube.
iv. Fehling solution: It consists of two components: Fehling’s A a copper sulphate
solution and Fehling’s B a solution of potassium sodium tartrate and sodium hydroxide. It
is used to form color complex with the solution so that, it can give proper absorbance at
maximum wavelength.
v. Sulfuric acid: It is used to precipitate the proteins and cell contents present in the
blood as we find the clear supernatant.
Apparatus:
i. Test tube
ii. Graduated pipette
iii. Water bath
iv. Volumetric flask
v. Measuring cylinder
vi. Spectrophotometer
vii. Centrifuge tube
viii. Centrifuge machine
Preparation of standard glucose solution (Stock solution):
0.1 gm of glucose was taken in 100 ml volumetric flask and volume was adjusted to 100
ml by dissolving glucose and adding distilled water. Therefore, the concentration is 1 mg/ml
or 1000 µg/ml.
Preparation of 20 µg/ml, 40 µg/ml, 60 µg/ml, 80 µg/ml, 100 µg/ml solution:
5 volumetric flasks were taken, washed and marked respectively with concentration as
above. Then 2 ml, 4 ml, 6 ml, 8 ml and 10 ml of stock solution were taken in corresponding
volumetric flask and adjusted to mark with distilled water.
Preparation of sample solution:
i. 3 ml of blood sample was withdrawn into a test tube containing 1 ml Na-oxalate
anticoagulant.
ii. 0.5 ml blood, 0.5 ml Na-tungstate, 0.5 ml 2/3N H2SO4 and 8.5 ml distilled water were
taken in a centrifuge tube and was centrifuged for 5 min at 400 rpm.
iii. Then 0.5 ml of supernatant serum was taken in another test tube and 0.5 ml Fehling
solution was added in it and mixed well.
Md.
Imran
Nur
Manik

8. 7
Procedure
i. 7 test tubes were taken and labeled as blank, 20 µg/ml, 40 µg/ml, 60 µg/ml, 80 µg/ml,
100 µg/ml and sample.
ii. 0.5 ml distilled water (for blank), 0.5 ml of each standard solution and 0.5 ml
supernatant fluid were taken by 1 ml pipette into corresponding test tubes and 0.5 ml Fehling
solution was added in each test tube.
iii. Then test tubes were heated in water bath for 30 minutes and cooled.
iv. 0.5 ml Arsenic molybdate and 8.5 ml distilled water were added in each test tube and
mixed well.
v. The absorbance was taken in spectrophotometer (UV) at 520 nm wavelength for all
solution.
vi. Finally a standard point calibration curve for glucose was obtained by plotting
absorbance versus concentration. Then the concentration of supplied sample was calculated
from standard curve for respective absorbance.
Data:
For glucose concentration in blood after oral administration of glucose:
Concentration of solution (g/ml) Absorbance (max = 520 nm)
Blank
20
40
60
80
100
Sample
Calculation:
Concentration of sample from curve is = µg/ml
Dilution factor:
0.5 ml blood is taken into 10 ml of solution
 Dilution factor =
ml
ml
5.0
10
= 20
Actual concentration of glucose in blood = µg/ml
Md.
Imran
Nur
Manik

9. 8
Result:
After experiment from the calibration curve it was found that, the concentration of blood
glucose after oral administration of glucose was µg/ml.
Comment:
Md.
Imran
Nur
Manik

10. 9
Experiment No. 02 Date: 20.09.11
Name of the experiment: Estimation of glucose concentration in blood at fasting
condition.
Introduction:
Glucose is the primary source of energy for the body's cells and blood lipids (in the form
of fats and oils) are primarily a compact energy store. Glucose is transported from the
intestines or liver to body cells via the bloodstream and is made available for cell absorption
via the hormone insulin, produced by the body primarily in the pancreas.
The mean normal blood glucose level in blood in humans is about 80–120 mg / 100 ml
(4.4–6.1 mmol/L). However, this level fluctuates throughout the day. Glucose levels are
usually lowest in the morning, before the first meal of the day (termed the fasting level) and
rise after meals for an hour or two.
The human body naturally tightly regulates glucose levels in blood as a part of metabolic
homeostasis. Sugar levels in blood outside the normal range may be an indicator of a medical
condition.
Measurement of fasting glucose level in blood:
The fasting glucose level in blood is a much poorer screening test because of the high
variability of the experimental conditions such as the carbohydrate content of the last meal
and the energy expenditure between the last meal and the measurement.
The fasting glucose level in blood is measured after a fast of 8 hours, is the most
commonly used indication of overall glucose homeostasis, largely because disturbing events
such as food intake are avoided. Abnormalities in these test results are due to problems in the
multiple control mechanism of glucose regulation.
Conditions affecting glucose levels are shown in the table below:
Table: Causes of abnormal glucose levels in blood.
Persistent
hyperglycemia
Transient
hyperglycemia
Persistent
hypoglycemia
Transient
hypoglycemia
Diabetes mellitus Pheochromocytoma Insulinoma
Acute alcohol
ingestion
Adrenal cortical
hyperactivity
Cushing's syndrome
Severe liver disease
Adrenal cortical
insufficiency
Addison's disease
Drugs: salicylates,
antituberculosis
agents
Hyperthyroidism Acute stress reaction Hypopituitarism Severe liver disease
Acromegaly Shock Galactosemia
Several glycogen
storage diseases
Obesity Convulsions
Ectopic insulin
production from
tumors
Hereditary fructose
intolerance
Md.
Imran
Nur
Manik

11. 10
Principle:
The simplest plasma or blood test for glucose used in establishing the diagnosis of
diabetes is fasting blood glucose test. The blood sample is drawn from person in morning
prior to breakfast (usually overnight fast).
The normal fasting plasma glucose is usually set in between 3.6 mmol/L (65 mg/dl) and
6.1 mmol/L (110 mg/dl). The diagnosis of diabetes may be confirmed in patient with two or
more fasting plasma glucose level that are elevated above 7.8 mmol/L (140 mg/dl).
The glucose of blood sample determination is based on the reaction of glucose with
Fehling solution. Fehling solution is mild oxidizing agent and easily oxidizes the reducing
sugar such as glucose, present in blood. Blood also contain a small glucoronate which may
give rise to Cu2O formation but here mainly blood glucose is considered.
Fehling solution contain Cu2+
ions and is prepared by adding Fehling’s-A solution
containing CuSO4 to Fehling’s-B solution containing NaOH and Rochelle salt (Na-K
tartrate).
During oxidation to aldehyde to acid, the Cu2+
ions reduced to Cu+
ions which are
precipitated as red color of Cu2O.
R–CHO + 2Cu2+
+ 3OH–
 R–COO–
+ 2Cu+
+ 2H2O
2Cu2+
+ 2OH–
 Cu2O  + H2O
The color of the blood sample (after reduction with Fehling solution) is adjusted with
Arsenic molybdate to form a color complex.
Arsenic molybdate + Cu2O  Color complex (soluble)
The absorbance of color complex is measured by spectrophotometer at 520 nm
wavelength (max). By plotting the absorbance value of blood glucose sample on the
calibration curve, we can easily calculate the blood glucose concentration.
Md.
Imran
Nur
Manik

12. 11
Reagents:
i. Arsenic molybdate
ii. 10% Na-tungstate
iii. Anticoagulant (Na-oxalate)
iv. Fehling solution (A:B = 25:1)
v. Glucose
vi. 2/3 N H2SO4
Function of the reagents:
i. Arsenic molybdate: It is used to form complex with the solution so that, it can give
proper absorbance at maximum wavelength.
ii. Na-tungstate: It is used to precipitate the proteins and cell contents present in the
blood.
iii. Anticoagulant Na-oxalate: It is used to prevent the clotting of blood within the test
tube.
iv. Fehling solution: It consists of two components: Fehling’s A a copper sulphate
solution and Fehling’s B a solution of potassium sodium tartrate and sodium hydroxide. It
is used to form color complex with the solution so that, it can give proper absorbance at
maximum wavelength.
v. Sulfuric acid: It is used to precipitate the proteins and cell contents present in the
blood as we find the clear supernatant.
Apparatus:
i. Test tube
ii. Graduated pipette
iii. Water bath
iv. Volumetric flask
v. Measuring cylinder
vi. Spectrophotometer
vii. Centrifuge tube
viii. Centrifuge machine
Preparation of standard glucose solution (Stock solution):
0.2 gm of glucose was taken in 100 ml volumetric flask and volume was adjusted to 100
ml by dissolving glucose and adding distilled water. Therefore, the concentration is 1 mg/ml
or 1000 µg/ml.
Preparation of 20 µg/ml, 40 µg/ml, 60 µg/ml, 80 µg/ml, 100 µg/ml solution:
5 volumetric flasks were taken and washed and marked respectively with concentration as
above. Then 2 ml, 4 ml, 6 ml, 8 ml and 10 ml of stock solution were taken in corresponding
volumetric flask and adjusted to mark with distilled water.
Md.
Imran
Nur
Manik

13. 12
Preparation of sample solution:
i. 3 ml of blood sample was withdrawn into a test tube containing 1 ml Na-oxalate
anticoagulant.
ii. 0.5 ml blood, 0.5 ml Na-tungstate, 0.5 ml 2/3N H2SO4 and 8.5 ml distilled water were
taken in a centrifuge tube and was centrifuged for 5 min at 400 rpm.
iii. Then 0.5 ml of supernatant serum was taken in another test tube and 0.5 ml Fehling
solution was added in it and mixed well.
Procedure
i. 7 test tubes were taken and labeled as blank, 20 µg/ml, 40 µg/ml, 60 µg/ml, 80 µg/ml,
100 µg/ml and sample.
ii. 0.5 ml distilled water (for blank), 0.5 ml of each standard solution and 0.5 ml
supernatant fluid were taken by 1 ml pipette into corresponding test tubes and 0.5 ml Fehling
solution was added in each test tube.
iii. Then test tubes were heated in water bath for 30 minutes and cooled.
iv. 0.5 ml Arsenic molybdate and 8.5 ml distilled water were added in each test tube and
mixed well.
v. The absorbance was taken in spectrophotometer (UV) at 520 nm wavelength for all
solution.
vi. Finally a standard point calibration curve for glucose was obtained by plotting
absorbance versus concentration. Then the concentration of supplied sample was calculated
from standard curve for respective absorbance.
Data:
For glucose concentration in blood at fasting condition:
Concentration of solution (g/ml) Absorbance (max = 520 nm)
Blank
20
40
60
80
100
Sample
Md.
Imran
Nur
Manik

14. 13
Calculation:
Concentration of sample from curve is = µg/ml
Dilution factor:
0.5 ml blood is taken into 10 ml of solution
 Dilution factor =
ml
ml
5.0
10
= 20
Actual concentration of glucose in blood = µg/ml
Result:
After experiment from the calibration curve it was found that, the concentration of blood
glucose at fasting condition was µg/ml.
Comment:
Md.
Imran
Nur
Manik