sbp 3107 endocrinology and reproductive physiology

SBP 3107 ENDOCRINOLOGY & REPRODUCTIVE PHYSIOLOGY

PRACTICAL 1

LABORATORY PROCEDURESAssoc. Prof. Dr. Sabrina Sukardi

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

B. GROUPINGa. 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 studyb. Problem of the studyc. Objectives of the study

ii. Materials and method:a. Design of experimentb. Materials used Experimental animals Specific techniques used

iii. Results:a. Tables of means, statistical analysis, etcb. Graphs

iv. Discussion:a. Summary of the resultsb. Literatures to support the findings

v. Conclusion: Summary of the findings

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.


PRACTICAL 2ENDOCRINOLOGY

MEASUREMENT OF BLOOD GLUCOSE LEVEL IN WHOLE BLOODAssoc. Prof. Dr. Mohamad Aziz Dollah

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-CHECKtm 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 ACCU-CHECKtm 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. ACCU-CHECKtm Test Strip2. ACCU-CHECKtm Blood Glucose Meter3. Lancets 4. Alcohol5. Glucose solution (0,10,20,40,80 gm/50ml)

Methodology:A. Measurement of blood glucose1. 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 ingestion1. 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. 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.

Nutritional StatusConcentration of glucose (gm/50ml)

0204080

FastingG1G2G3G4

Normal FeedingG5G6G7G8

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.

Nutritional StatusTimeConcentration of glucose solution (gm/50ml)Total

0204080

G1G2G3G4

Fasting

0

30

60

90

Total

Normal feedingG5G6G7G8

0

30

60

90

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:

SourcedfMSFStudent 1Time 2Student*time2Group1Nutritional status1Concentration of glucose solution2Nutritional status x glucose concentration2Error24Total35

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.

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 ofglucose solution of fasting and normal feeding students

FS Fasting (G1-G4)NF - Non fasting/with meal (G5-G8)

GroupSubjectMatric No.Nutritional StatusGlucose ingestion (gm)Time after ingestion (min)Blood glucose level (mmol/L)

11FS00

30

60

90

2FS00

30

60

90

3FS00

30

60

90

21FS200

30

60

90

2FS200

30

60

90

3FS200

30

60

90

31FS400

30

60

90

2FS400

30

60

90

3FS400

30

60

90

41FS800

30

60

90

2FS800

30

60

90

3FS800

30

60

90

51NF00

30

60

90

2NF00

30

60

90

3NF00

30

60

90

61NF200

30

60

90

2NF200

30

60

90

3NF200

30

60

90

71NF400

30

60

90

2NF400

30

60

90

3NF400

30

60

90

81NF800

30

60

90

2NF800

30

60

90

3NF800

30

60

90



ROLE OF INSULIN IN GLUCOSE REGULATIONAssoc. 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.

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. Streptozotocin (STZ)2. Citrate buffer (ph 4.5)3. Insulin4. Glucose solution

Materials:1. 1ml syringe with needle2. Rats3. Rat cages with water and feeder4. Rat feed5. Weighing balance for rat6. ACCU-CHECKtm Test Strips7. ACCU-CHECKtm Blood Glucose Meter

Methodology:Experimental DesignThe 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

Insulin injectionDiabetic Status

Normal Diabetic

Glucose Solution

Glucose Solution

02040800204080

Insulin G1G2G3G4G5G6G7G8

No InsulinG1G2G3G4G5G6G7G8

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.

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 infusionb. Just before insulin or buffer injectionc. 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 injectionTime Normal Diabetic Total

Glucose SolutionGlucose Solution

02040800204080

Insulin -30

0

30

60

Total

No Insulin -30

0

30

60

Total

Grand Total

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:

Blood glucose = rat + time + rat*time + diabetic status + concentration of glucose solution + injection + errorAnalysis of variance:

SourcedfMSFRat4Time3Rat*Time12Diabetic Status1Concentration of Glucose Solution2Injection1Error37Total60

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.

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 StatusGlucose Concent.InjectionTime (min)Glucose Level (mg/ml)

111Normal0Insulin-30 (before glu)

0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after

21Normal 0No Insulin-30 (before glu)

0 (before ins)

30 min after

60 min after

2Normal0No Insulin-30 (before glu)

0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after

2Normal 20Insulin-30 (before glu)

0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after

591Diabetic 0Insulin-30 (before glu)

0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after

101Diabetic 0No Insulin-30 (before glu)

0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after

7131Diabetic40Insulin-30 (before glu)

0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after


0 (before ins)

30 min after

60 min after



REGULATION OF HEART FUNCTION BY ADRENALINAssoc. Prof. Dr. Mohamad Aziz Dollah

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

sbp 3107 endocrinology and reproductive physiology

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