PHR375 Practical Biochemistry

1. Pharos University in Alexandria Faculty of Dentistry PHR375 Practical Biochemistry

2. Practical “Week 3” Oral Glucose Tolerance Test &Serum Glucose

3. Glucose • Glucose is the primary energy source for the human body.

4. Source of glucose in the blood Dietary carbohydrates: After digestion a breakdown of carbohydrates in the diet, glucose is derived by its absorption a state of post absorptive hyperglycemia is realized. Glycogenolysis: Free blood glucose can be derived from the liver stores of glycogen. Gluconeogenesis: Blood glucose is derived through its endogenous synthesis from non-carbohydrate sources e.g. from amino acids, lactate and glycerol. The glucose level in blood is maintained within a fairly narrow range under diverse conditions (feeding, prolonged fasting or server exercise) by regulatory hormones such as insulin, glucagons or epinephrine.

5. Glucose Normal Value: 60-110 mg/dL serum. This value is increased in uncontrolled diabetes mellitus, hyperthyroidism or hyperpituitrism. While it is decreased in hyperinsulinism and hypopituitrism.

6. Oral Glucose Tolerance Test (OGTT) The ability of the body to utilize glucose is ascertained by measuring its glucose tolerance. The glucose load is most often given orally (75 g for adults or 100 g for pregnants), therefore the common test is technically an oral glucose tolerance test (OGTT) The intravenous glucose tolerance test (IGTT) is used only when the patient cannot administer glucose orally. In this test, a glucose load (0.5 g/kg body weight) is injected intravenously and blood samples are collected every 10 minutes for 1 hour.

7. What is the Glucose tolerance test ?? • During a glucose tolerance test, which may be used to diagnose diabetes mellitus, a fasted patient takes a 75 gm oral dose of glucose. Blood glucose levels are then measured over the following 2 hours. • Glycemic greater than or equal to 200 mg/dL at 2 hours or greater than or equal to 126 mg/dL fasting is diagnostic for diabetes mellitus.

8. Preparation of the patient for OGTT • The patient is instructed to eat normally and not to restrict carbohydrate intake in the days or weeks before the test. • The test should not be done during an illness, as results may not reflect the patient's glucose metabolism when healthy. • A full adult dose should not be given to a person weighing less than 43 kg otherwise exaggerated glucose may produce a false positive result. • Usually the OGTT should begin in the morning (7:00am-8:00am) as glucose tolerance exhibits a diurnal rhythm with a significant decrease in the afternoon.

9. Procedure for OGTT • The patient should have been fasting for the previous 8-14 hours. • A zero time (fasting) blood sample is drawn. • The patient is then given a glucose load solution to drink in a dose equal to 1.75 g/kg of body weight, to a maximum dose of 75g. The glucose load should be drunk within 5 minutes. • Following the glucose load, blood samples are drawn at different time intervals (30, 60, 90 and 120 minutes). For simple diabetes screening, the zero sample and the 2 hr sample could be the only collected samples. • Blood samples are centrifuged and serum is separated for glucose determination.

10. Procedure for OGTT

11. Interpretation of OGTT results • The resultant curve is known as oral glucose tolerance curve which has the following features in: • Normal individuals: • Fasting blood glucose is 60-110 mg/dl . • A gradual increase to a peak is reached after one hour. This peak amounts to 110-140 mg/dl (always below the renal sugar threshold). • A gradual decrease to fasting level is started after the peak is reached due to release of insulin from -cells. • All urine samples are free from glucose. • The total duration of the curve is about 2 hours (The fasting level is reached again after 2 hours from start of the test).

12. Interpretation of OGTT results • After taking the oral glucose dose, absorption occurs rapidly and the blood glucose concentration increases. • This stimulates the secretion of insulin for better utilization of the absorbed glucose. After 30-60 minutes (at the maximal value of the curve), the rate of absorption of the glucose is equal to its rate of utilization. this value (at the ascending limb of the curve) the rate of absorption is greater than the rate of utilization while after this value (at the descending limb of the curve) the rate of utilization is greater than the rate of absorption. At 120 minutes the serum glucose level drops below the fasting level due to excess production of insulin. This temporally existing hypoglycemia is beneficial in switching off the secretion of insulin after which the fasting level is regained and then maintained.

13. Interpretation of OGTT results Mild diabetes mellitus: The fasting glucose level is higher than normal (but still below the RST). After oral glucose administration the blood glucose level rises and exceeds the RST (glycosuria is accompanied). The decline in the curve is slower than normal and by its end, it is maintained at a level higher than the fasting level but lower than RST (no glycosuria). Severe diabetes mellitus: The fasting glucose level is higher than normal and may exceed the RST. After oral glucose administration the blood glucose level rises to a very high level. The decline in the curve is slower than normal and its end is maintained at a high level which is above the RST and is much higher than the fasting level. Glycosuria is present during and after the test.

14. Determination of glucose in serum In diabetes mellitus, there is mild to severe deficiency of insulin with corresponding decrease in rate of glucose utilization, i.e. decreased tolerance to glucose. Enzymatic methods for estimation of glucose: Glucose Oxidase method This is the most frequently used method in commercial kits for the determination of blood glucose level. It is specific, accurate and the absorbance of the product measure in the visible range (you don't need a spectrophotometer only use a colorimeter).

15. Enzymatic methods for estimation of glucose: Glucose + 2H2O + O2Gluconic acid + 2H2O2 Peroxidase H2O2 + phenol + 4-amino- antipyrinequinonemine (Colorless)(Colored) +H2O References: Tinder P. (1969) Ann. Clin Biochem, 6,24. Sample: Serum or Plasma Normal Values: 60- 100 mg/dL Glucose Oxidase

16. Enzymatic methods for estimation of glucose: Reagents:

17. Enzymatic methods for estimation of glucose: Stability: The reagents are stable up to the expiry date specified when stored at 2-8 0C

18. Practical Work In a clean dry test tube, add 10μl of distilled water, or standard glucose or serum sample, then add 1 ml working reagent (N.B. volumes are withdrawn by automatic pipettes using the proper disposable tips as instructed in the lab.)

19. Practical Work The working reagent contains: • Glucose oxidase. • Peroxidase.

20. Practical Work Mix the content of each tube and incubate for 10 min at 37°C using a thermostatically controlled water bath. Remove test tubes from the water bath, cool to room temperature and measure the absorbance of the final pink color of the test and standard against blank at max 500 nmN.B. the color is stable for 2 hours. Calculate the BSL (mg/dl) in the provided serum samples using the absorbance reading of standard glucose and applying the following equation: • Ctest= Cstd Atest / Astd

21. Practical Work Comment on the case (normal, mild diabetic or severe Diabetic).

23. Precautions • Use gloves while handling serum samples. • After terminating the practical procedure, gloves and test tubes are disposed of in the biohazard waste.

24. Thank You