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SBP 3107

ENDOCRINOLOGY & REPRODUCTIVE PHYSIOLOGY


PRACTICAL 1
LABORATORY PROCEDURES
Assoc. Prof. Dr. Sabrina Sukardi

A. LABORATORY RULES
a. Attendance for laboratory sessions is compulsory.
b. Lab coat must be worn at all time while working in the laboratory.

B. GROUPING
a. A class representative/leader to be selected among the students to
coordinate class activities.
b. Students are grouped into 4 equal groups.
c. Each of 4 groups is subdivided into 3 sub-groups.
d. A sub-group leader is to be appointed among group members and he/she
is responsible for the safety of the equipments and other facilities to be
used in the experiments.

C. REPORT WRITING
a. Group report:
Each sub-group is required to submit a laboratory report.
b. Content of the report:
The laboratory report must contain the following section;
i.
Introduction:
a. Background of the study
b. Problem of the study
c. Objectives of the study
ii.

Materials and method:


a. Design of experiment
b. Materials used
- Experimental animals
- Specific techniques used

iii.

Results:
a. Tables of means, statistical analysis, etc
b. Graphs

iv.

Discussion:
a. Summary of the results
b. Literatures to support the findings

v.

Conclusion:
- Summary of the findings
1

vi.

References:
- List of literatures cited

c. Laboratory reports need to be submitted at the latest of two weeks after


completion of each laboratory class.

SBP 3107

ENDOCRINOLOGY & REPRODUCTIVE PHYSIOLOGY


PRACTICAL 2
ENDOCRINOLOGY

MEASUREMENT OF BLOOD GLUCOSE LEVEL IN WHOLE BLOOD


Assoc. Prof. Dr. Mohamad Aziz Dollah
2

Introduction:
Glucose is an essential fuel for neurons, erythrocytes and contracting skeletal
muscle cells. The higher centers of the brain are highly sensitive to even a short
term deficiency of glucose.
Glucose enters most of the cells via active process but it is necessary to
maintain adequate concentration of glucose in the blood. It is not surprising to see
that the levels of several hormones in the plasma are changed with the small
changes in blood glucose concentration. Normal value of blood glucose for normal
person is between 70-100 mg/ml (3.9-6.1 mmol/L). Two hours after meals, blood
glucose levels should be less than 140 mg/ml (7.8 mmol/L).
Blood glucose level has been used as one of the indicators to determine the
health status of an individual.

Its level can determine the functionality of blood

glucose regulating system as well as to diagnose some other related diseases.


There are many kits for home used available in the market for determine of
blood glucose. There is either for quantitative or qualitative determination of blood
glucose level. One of the kits which is called ACCU-CHECK- produced by Roche, will
be used for determination of blood glucose using whole blood sample.

This kit

provides a simple direct method for determination of blood glucose using only a
drop of whole blood.

Objectives of the experiment:


The objectives of this experiment is to determine the glucose level during
fasting and non fasting condition and to demonstrate the changes of the blood
glucose level following ingestion of glucose solution.

Glucose ingestion is to

stimulate the condition after meal. The effect of increasing the concentration of
ingested glucose also will be studied in this experiment.
Principle of the technique:
Each blood glucose ACCU-CHECK tm Test Strip contains the following reagents:
Potassium ferricyanide (43.7%), Glucose dehydrogenase (1.2%), buffer (24.7%),
stabilizer (19.4%) and non-active ingredient (11.0%).

When a drop of blood is applied to the curve at the edge of the ACCUCHECKtm test strip, the glucose dehydrogenase enzyme in the strip converts the
glucose in the blood sample to gluconolactone. This reaction creates a harmless
electrical current which is in proportional with the magnitude of the reaction. The
meter will interpret the current produces into the amount of glucose in the blood.
Students will use this kit to determine their blood glucose levels during
fasting and study the effect of glucose loading on the level of blood glucose during
fasting.

Materials:
1.
2.
3.
4.
5.

ACCU-CHECKtm Test Strip


ACCU-CHECKtm Blood Glucose Meter
Lancets
Alcohol
Glucose solution (0,10,20,40,80 gm/50ml)

Methodology:
A. Measurement of blood glucose
1. If you are using a new pack of ACCU-CHECKtm Test Strip, take out the
chip code included in the pack and insert it into the code chip slop in
the meter with meter turned off.
2. Switch on the glucose meter.
3. Insert a test strip to the glucose meter.
4. Clean your finger tip using alcohol and punch using lancet needle to
take a few drops of blood.
5. Touch the drop of blood to the curve at the edge of the test strip. Hold
finger to the curve of test strip and the yellow window of the strip is
completely filled up with blood (no yellow color is visible). Blood will
be drown into the strip automatically. Do not place the blood drop on
the top of the test strip.
6. Wait for about 30 seconds and take the reading.
7. Remove the test strip from the meter and discard the strip.
B. Blood glucose levels during fasting and after glucose ingestion
1. Divide the class into 8 groups equally and allocated the group to the
treatment as shown in table 1.
2. Choose 4 members from each group of 1-4 and asked them to fast
(without food) overnight but allowed to take plane water.
3. Choose 4 members from each group of 5-8 and asked them to take
normal meal two hours before class.
4

4. During the measurement of blood glucose, the fasting and non-fasting


members are required to rest and relax and do less physical activity.
5. Measure the blood glucose level as describe in A.
6. Ask the members to drink 10 ml of glucose solutions accordingly (0,
20, 40, 80 gm/50ml) as in the following table 1.
7. Measure the blood glucose levels at 30, 60 and 90 minutes after
drinking glucose solution.
8. Record the glucose level in the form provided.
9. Share the results with other groups to complete the form.
Table 1: Allocation of the students group to the experiment to study the effect of
fasting and drinking various concentration of glucose solution on blood glucose
level.
Concentration of glucose (gm/50ml)
0
20
40
80
G1
G2
G3
G4
G5
G6
G7
G8

Nutritional Status
Fasting
Normal Feeding
Data analysis:

1. Pooled the data of the blood glucose level from your group and others.
2. Calculate the means and standard deviation of the blood glucose level in
each time of sampling for each concentration of glucose given.

Table 2: Mean sd of blood glucose level (mmol/L) following of glucose solution


ingestion in fasting and normal meal students.
Concentration of glucose solution
Nutritional
Status

Time

Fasting

0
30
60
90

Total
Normal
feeding
Total

0
30
60
90

(gm/50ml)
20
40
G2
G3

G6
G7

0
G1

G5

80
G4

G8

Total

3. Do the statistical analysis to see the effects of different concentrations of


glucose ingestion on the level of blood glucose measured at different
times after drinking of glucose solution in fasted and non-fasted subject.
The statistical model is as follow:
Blood glucose = student + time + student*time + group + nutritional
status +
concentration of glucose solution + nutritional status*
concentration of glucose solution + error

Analysis of variance:

Source

df

Student

Time

Student*time

MS

Group

Nutritional status

Concentration of glucose solution

Nutritional status x glucose concentration

Error

24

Total

35

4. Compare the means.


5. Plot the graph of blood glucose vs time for each glucose solution taken up
and for each nutritional condition.
Data interpretation:
Interpret and discuss your findings accordingly.
6

Discussion:
The discussion will be at least on the following questions:
a. What is the normal glucose levels in the blood during fasting?
b. What is the effect of glucose ingestion during fasting and no fasting?
c. How does the glucose in blood change following glucose
infusion/injection?

Data Collection sheet


Blood glucose level following ingestion of different concentration of
glucose solution of fasting and normal feeding students
FS Fasting (G1-G4)
NF - Non fasting/with meal (G5-G8)

Grou
p

Subjec
t

Matric
No.

Glucose
ingestion
(gm)

Nutrition
al Status

FS

FS

FS

FS

20

Time after
ingestion
(min)
0
30
60
90
0
30
60
90
0
30
60
90
0
30
60

Blood
glucose
level
(mmol/L)

FS

20

FS

20

FS

40

FS

40

FS

40

FS

80

FS

80

FS

80

NF

NF

NF

NF

20

NF

20

90
0
30
60
90
0
30
60
90
0
30
60
90
0
30
60
90
0
30
60
90
0
30
60
90
0
30
60
90
0
30
60
90
0
30
60
90
0
30
60
90
0
30
60
90
0
30
60
90
0
30

NF

20

NF

40

NF

40

NF

40

NF

80

NF

80

NF

80

60
90
0
30
60
90
0
30
60
90
0
30
60
90
0
30
60
90

0
30
60
90
0
30
60
90
0
30
60
90

SBP 3107

ENDOCRINOLOGY & REPRODUCTIVE PHYSIOLOGY


PRACTICAL 3
ENDOCRINOLOGY
ROLE OF INSULIN IN GLUCOSE REGULATION
Assoc. Prof. Dr. Mohamad Aziz Dollah

Introduction:
Food taken up everyday consists of carbohydrate, protein, fat, minerals and
water.

The food is digested in the alimentary tract to be easily absorbed and

transported to the body.

Carbohydrate is digested to simple sugars such as

glucose; protein to amino acids; fat to fatty acids.

Thus, the blood leaving the

digestive tract is very rich in nutrients (glucose, amino acids, fatty acids, vitamins,
minerals) in which the level of nutrients have to be reduced to a physiological level
before the blood is circulated throughout the body.
Insulin, secreted by the beta cell of the islet of Langerhans in the pancreas,
regulates sugars level in the circulatory blood.

High level of sugars induces

secretion of insulin by its secretary cells. One hour after taken 50 gm of glucose by
mouth, the plasma insulin level increased from fasting level (16uU/ml) to a
maximum level (58uU/ml) whereas after 100gm of glucose, the rise was from 17
uU/ml to 158 uU/ml.

other nutrient that might induce insulin secretion is amino

acids such as leucine, arginine, lycine and valine.


10

Insulin causes rapid uptake, storage and use of glucose by almost all tissues
of the body especially by the liver, muscles and adipose tissue. In the liver, insulin
inhibits phophorylase, the enzyme that causes the breakdown of liver glycogen to
into glucose. Insulin also enhances the uptake of glucose from the blood in the liver
cells by increasing the activity of the enzyme glucokinase.

The activities of

glycogen forming enzymes such as phosphofructokinase and glycogen synthetase


are also increase to enhance the formation of glycogen in the liver. In the normal
resting muscle tissue, the cell membrane is not permeable to glucose but its
permeability of muscle cell membrane to glucose and possibly use up as energy
source rather than fatty acids.
Low level of insulin in the blood can cause the glucose level to reach very
high in the blood and if it persists for the long period, this condition is known as
diabetes mellitus.
Some drugs influence the secretion of insulin by the pancreas. Tolbutamide
for instance increases the secretion of insulin whereas thiazide drugs are known to
inhibit the secretion of insulin in response to glucose in vitro. In addition, diazoxide
together with chlorthiazide reduce the excessive serum insulin values in rat.
The importance of insulin in regulation of blood glucose level is studied in this
experiment.

Rats will be used as the model of experiment.

The condition of

diabetes mellitus will be induced by injection of streptozotocin (STZ) drug and the
effect can be observed within 3-4 days later. Insulin is then injected to see whether
the glucose levels in the blood change following infusion of glucose solution.

Reagents:
1.
2.
3.
4.

Streptozotocin (STZ)
Citrate buffer (ph 4.5)
Insulin
Glucose solution

Materials:
1.
2.
3.
4.

1ml syringe with needle


Rats
Rat cages with water and feeder
Rat feed
11

5. Weighing balance for rat


6. ACCU-CHECKtm Test Strips
7. ACCU-CHECKtm Blood Glucose Meter
Methodology:
Experimental Design
The role of insulin in glucose regulation will be studied on diabetic and normal
rats. In this experiment, 48 rats are divided equally according to body weight into
16 groups consisting 3 rats each. The treatments to be imposed to each group are
shown in Table 1.

Table 1: Experimental Design for study of insulin role in rat


Diabetic Status
Insulin
injection

Insulin
No Insulin

0
G1
G1

Normal

Diabetic

Glucose Solution

Glucose Solution

20
G2
G2

40
G3
G3

80
G4
G4

0
G5
G5

20
G6
G6

40
G7
G7

80
G8
G8

n = 3 rats
Experimental Animals:
1. Keep the rats in the cages according to their group in the animal house.
Feed and water to be given accordingly.
2. The experimental period lasts for about one week.
Treatments:
1. At day -4 (Tuesday), 0.2ml of STZ solution (50mg/kg bw) is injected
subcutaneously to all diabetic-induced rats.
2. At day -1 (Thursday), all the rats are fasted over night but water is given
as usual.
3. At day 0 (Friday), 0.2ml of glucose solution at various concentration is
injected subcutaneously to the respective groups.
4. 30 minutes after glucose injection, 0.2ml insulin (1 IU/kb) is injected
subcutaneously to the rats in normal insulin and diabetic insulin groups
while the no insulin groups are injected with the buffer only.
12

Measurements:
1. Blood glucose level is determined using glucose kit as describe in the
previous laboratory exercise.

The glucose level is measured at the

following schedule.
a. Just before glucose infusion
b. Just before insulin or buffer injection
c. 2 times at 30 minutes interval after insulin or buffer injection

2. Record the glucose level in the form provided.


3. Share the results with other groups to complete the form.
Data Analysis:
1. Pooled the data of the blood glucose level from your group and others.
2. Calculate the means and standard deviation of the blood glucose level in
each time of sampling for each concentration of glucose given.
3. Calculate the means and standard deviation of the glucose level.
Table 2: Meansd of blood glucose level (mmol/L) following of glucose solution
ingestion followed by insulin injection in normal and diabetic rats.
Insulin
injecti
on

Tim
e

Insulin

-30
0
30
60

Total
No
Insulin

-30
0
30
60

Total
Grand Total

Normal
Glucose Solution
20
40

80

Diabetic
Glucose Solution
20
40

Total
80

4. Do the statistical analysis to see the effects of different concentration of


glucose ingestion and followed by insulin injection on the level of blood
glucose measured at different times of normal and diabetic rats.
Statistical model of the experiment is as follow:

13

Blood glucose = rat + time + rat*time + diabetic status + concentration


of
glucose solution + injection + error
Analysis of variance:

Source

df

Rat

Time

Rat*Time

12

Diabetic Status

Concentration of Glucose Solution

MS

Injection

Error

37

Total

60

Results:
Plot the means of glucose level vs time of experiment.
Use time = 0 as time before glucose injection.
Discussion:
The discussion will be at least on the following questions:
a. What is the normal glucose levels in the blood of rats during fasting?
b. What is the effect of STZ injection on glucose levels in the blood?
c. How does the glucose in blood change following glucose
infusion/injection?
d. What is the effect if insulin injection on the blood glucose levels?
e. How does the insulin injection influence the blood glucose level?
f. How do you conclude this experiment.

14

Data collection sheet


Blood glucose level following ingestion of different concentration
of glucose solution of normal and diabetic rats
Group
(Stud)

Group
(Animal
)

Animal
No.

Diabetic
Status

Glucose
Concent.

Injection

Normal

Insulin

Normal

Insulin

Normal

Insulin

Normal

No
Insulin

Normal

No
Insulin

Normal

No
Insulin

Normal

20

Insulin

Normal

20

Insulin

Normal

20

Insulin

15

Time
(min)
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after

Glucose Level
(mg/ml)

Normal

20

No
Insulin

Normal

20

No
Insulin

Normal

20

No
Insulin

Normal

40

Insulin

Normal

40

Insulin

Normal

40

Insulin

Normal

40

No
Insulin

Normal

40

No
Insulin

Normal

40

No
Insulin

Normal

80

Insulin

Normal

80

Insulin

16

60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after

-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)

Normal

80

Insulin

Normal

80

No
Insulin

Normal

80

No
Insulin

Normal

80

No
Insulin

Diabetic

Insulin

Diabetic

Insulin

Diabetic

Insulin

Diabetic

No
Insulin

Diabetic

No
Insulin

Diabetic

No
Insulin

Diabetic

20

Insulin

10

11

17

30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after

-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before

12

13

14

Diabetic

20

Insulin

Diabetic

20

Insulin

Diabetic

20

No
Insulin

Diabetic

20

No
Insulin

Diabetic

20

No
Insulin

Diabetic

40

Insulin

Diabetic

40

Insulin

Diabetic

40

Insulin

Diabetic

40

No
Insulin

18

glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after

-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after

15

Diabetic

40

No
Insulin

Diabetic

40

No
Insulin

Diabetic

80

Insulin

Diabetic

80

Insulin

Diabetic

80

Insulin

Diabetic

80

No
Insulin

Diabetic

80

No
Insulin

Diabetic

80

No
Insulin

16

SBP 3107

-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after
-30 (before
glu)
0 (before ins)
30 min after
60 min after

ENDOCRINOLOGY & REPRODUCTIVE PHYSIOLOGY


PRACTICAL 4
ENDOCRINOLOGY
REGULATION OF HEART FUNCTION BY ADRENALIN
Assoc. Prof. Dr. Mohamad Aziz Dollah
19

Introduction:
Heart is an important organ of the body. It function as a pump to circulate
blood from the liver enriched in nutrients and from the lung enriched in oxygen, to
the organs throughout the body and circulate back the blood containing metabolic
by products of the cells to the execratory organs for excretion of these metabolic by
products before the blood is circulate back to the body.
The heart function is regulated by system many factors through nervous and
hormonal systems to meet the immediate or long term supply of the blood by the
organs which varies from time to time or from one physiological condition to others.
One of the hormones that involved in regulation of heart function is adrenalin
hormone.

It is secreted by adrenal gland in respond to external stimuli which

mainly related to stress. Adrenalin stimulates the heart function by increasing the
heart rate, force of contraction and volume of contraction with the purpose to
increase blood flow and supply to the organs. These effects will be studied in this
experiment using frog heart as a model.

Objectives of the experiment:


1. To study the characteristic of heart function under normal condition.
2. To study the effect of adrenalin on the heart function.
Methodology:
Follow the procedures in the manual of Power Lab for preparation of frog
heart, measurement of normal heart functions and manipulation of heart functions
using adrenaline.

20