1. Medical Biochemistry Department
Determination of plasma glucose concentration By
All Staff Members of
Medical Biochemistry Department
ILO of the current topic:By the end of this topic, the student will be able to:
Estimate blood glucose concentration and perform oral glucose tolerance test.

2. Colorimetric estimation of blood glucose

3. Medical importance of blood glucose determination
Diagnosis and care of diabetic patients
Assess the effectiveness of proper insulin dose
Reveals individual patterns of blood glucose changes
Allows for quick response to high blood sugar

4. Diagnostic criteria of diabetes
Fasting blood glucose (FBG)
Normal fasting glucose ……. …….<100 mg/dl
Impaired fasting glucose (IFG) ……. 100–125 mg/dl
Provisional diagnosis of diabetes …….. ≥126 mg/dl

5. Diagnostic criteria of diabetes
Postprandial blood glucose (2hpp)
Normal 2-hours postprandial glucose ……...<140 mg/dl
Impaired glucose tolerance (IGT)…..……….140–199 mg/dl
Provisional diagnosis of diabetes ………...≥200 mg/dl

6. Aim of practical session
The aim of this practical session is to:
1. estimate blood glucose concentration by glucose oxidase reaction .
2. Perform oral glucose tolerance test.
3. Interpretate the resulting data whether it shows normoglycemic or hyperglycemic or hypoglycemic results.

7. colorimetric estimation of blood glucose by glucose oxidase reaction

8. Principle of the glucose oxidase reaction
Glucose is oxidized by glucose oxidase to form gluconic acid with liberation of hydrogen peroxide.
Glucose + O2 + H2O glucose oxidase Gluconic acid + H2O2

9. red colored product Principle of the glucose oxidase reaction
Hydrogen peroxide is dissociated to water and oxygen atom by a peroxidase enzyme.
The liberated oxygen is captured by a chromogen
( a mixture of 4-amino antipyrine & phenol) which is converted to a red violet complex.
H2O2 + Phenol + 4-amino antipyrine peroxidase
red colored product

10. Principle of glucose oxidase reaction
A colorimeter can be then used to measure the intensity of the color produced; the more glucose present the darker the color.

11. Activity 1 Materials required
Using a standard glucose solution to calculate the concentration of an unknown one.
Materials required
Clean dry pipettes (1ml) & test tubes.
glucose reagent
Known glucose solution(Standard)
Unknown glucose solution
Distilled water
A pump & goggles if available for lab safety.

12. for the first task Today
Remember that pipeting is very important

13. 1-Clean

14. Always use your dry index finger to close its opening
Always on measurement keep it in front of your eyes
Deliver gradually

16. For the Standard sample
Step 1:Label 3 dry test tubes B
For the Blank T
For the test sample S
For the Standard sample

17. Step 2: Practical Sample Standard Blank ___ 0.5ml Distilled water
Determine the glucose concentration in the provided sample as follows:
Sample
Standard
Blank
___
0.5ml
Distilled water
Glucose standard
Serum/plasma
1 ml
glucose reagent

18. Step 3: Pipette 0.5ml of the given Test sample-Standard-DW into their corresponding dry test tubes
0.9
0.5ml

19. Step 4: for all ; Pipette 1ml of the glucose reagent into the previous 3 test tubes.

20. B S T B
Step 5:Mix the contents of the tubes and let stand for 20 minutes at room temperature.

21. Step 6:Record the absorbance of both test & standard against the blank at 510 nm.

22. Calculate the test concentration by the following equation:
Concentration of blood glucose ( mg %) =
Absorbance of Test
X Concentration of standard
Absorbance of standard
Remember
the check List

23. Oral Glucose Tolerence Test
OGTT

24. Definition :
The administration of glucose to determine how quickly it is cleared from the blood

25. Indications of oral GTT
OGTT
Diagnosis of impaired glucose tolerance
Diagnosis of gestational diabetes
Diagnosis of alimentary glucosuria
Diagnosis of renal glucosuria

26. Precautions for OGTT

27. Carbohydrates restriction
AVOID
smoking
Eating from midnight
Drinking coffee
Carbohydrates restriction
Severe excercise

28. Conditions which prevent doing OGTT
Certain medicines
Alcohol drinking
Heart stroke
emotional stress
Low carbohydrate diet
Vomiting
Fever and infection
Recent surgery

29. Requirement of OGTT

30. How the OGTT is done : 1- Begin in the morning ( at 7-8 am ).
2- The subject sits quietly throughout the test.
3- A fasting blood sample (time 0) is drawn on fluoride-oxalate.
4 -fasting urine sample is collected.

31. How the OGTT is done :
5-75 g anhydrous glucose is dissolved in ml water and is ingested orally within 5 min .
6- Blood is drawn at intervals of 30 min for measurement of glucose.

32. How the OGTT is done : 8-In case of gastational case the test
7- urine samples also collected (to test for glucose ) every 30 min.
8-In case of gastational case the test
is extended for 3 hours(100 g glucose)

33. Interpretation of OGTT curve in normal & diabetic patients

35. Special OGTT Results Flat response Gestational diabetes
Renal Glucosuria
Alimentary
Glucosuria

36. Renal glucosuria
The urine sample corresponding to the peak of the curve contains glucose
The plasma glucose level is still below the normal renal threshold < 180 mg

37. Causes: Renal glucosuria Genetically inherited low renal threshold
Late pregnancy
Tubular reabsorption defect e.g. Fanconi syndrome

38. Alimentary (intestinal) glucosuria
A sharp rise in plasma glucose with early peak values exceeding the renal threshold and associated with glucosuria.
The 2-hours post prandial level is much below the fasting level

39. Alimentary (intestinal) glucosuria
This is due to rapid glucose absorption followed by a burst of insulin production which over-compensate, resulting in hypoglycemia.
Causes:
Some healthy individuals.
Severe liver cirrhosis.
Gastrectomy.
Thyrotoxicosis.

40. Causes: Flat response in OGTT
Plasma glucose levels fail to rise significantly after an oral glucose load.
Causes:
Insulinomia: over production of insulin.
Intestinal malabsorption syndrome.
Some hormonal defeciencies e.g. hypopituitrism, hypothyroidism

41. Gestational diabetes
Any degree of glucose intolerance with onset or first recognition during pregnancy
The preferred diagnostic test is the 100 gram 3 hour OGTT.

42. Gestational diabetes Diagnostic OGTT Values : Fasting 95mg/dl
1 hour after glucose load
180mg/dl
2 hours after glucose load
155mg/dl
3 hours after glucose load
140mg/dl
If two or more values are above the criteria, gestational diabetes is diagnosed

43. Causes of hyperglycemia
1-Diabetes mellitus : Type 1 & type 2.
2- High epinephrine : severe emotional stress, pheochromocytoma.
3- A rare glucagon secreting tumor.
4- High cotisole : Cushing syndrome or high ATCH.
5- Intestinal gluosuria.
6- Gestational diabetes.

44. Causes of hypoglycemia
1-Neonatal hypoglycemia: in RDS and pre-eclampsia .
2-. Infantile hypoglycemia:
a) Glycogen storage diseases in liver.
b) Impaired gluconeogenesis. (deficiency of glucose 6 phosphatase or fructose 1,6 bisphosphatase).
c) fructose induced hypoglycemia. (deficiency of aldolase B).
d) Sever classic galactosemia
e) Deficient fatty acid oxidation (Carnitine deficiency)

45. Causes of hypoglycemia
3- Adult hypoglycemia:
a) Over dose of insulin or oral hypoglycemic drugs.
b) Insulinoma.
c) Hypofunction of suprarenal cortex.
d) Chronic alcoholism.
e) Post prandial hypoglycemia due to sever over shooting of insulin.

46. Practice sheet
A male patient estimated his fasting plasma glucose twice(2 weeks between the two assays).The results were 120mg% and 122mg%. This patient was diagnosed as:
1) Diabetic
2) Pre diabetic
3) Non-diabetic

47. Draw the curve and comment on the results
This patient was subjected to an OGTT and the following results were obtained:
Fasting
1/2hour
1hour
11/2hour
2hour
Plasma
Glucose%
100
140
170
165
160
Urine
Glucose
Draw the curve and comment on the results

48. Draw the curve and comment on the results
Case 2
A 50 years female patient was subjected to an OGTT and the following results were obtained:
2 hours
1 1l2 hour
1 hour
1l2 hour
Fasting
220
240
260
190
145
Plasma
Glucose% + ++
------
Urine
glucose
Draw the curve and comment on the results