Medical Biochemistry Department Determination of plasma glucose concentration By All Staff Members of Medical Biochemistry Department 2011-2012 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.

Colorimetric estimation of blood glucose

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

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

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

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.

colorimetric estimation of blood glucose by glucose oxidase reaction

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

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

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.

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.

for the first task Today Remember that pipeting is very important

1-Clean

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

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

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

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

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

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

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

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

Oral Glucose Tolerence Test OGTT

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

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

Precautions for OGTT

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

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

Requirement of OGTT

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.

How the OGTT is done : 5-75 g anhydrous glucose is dissolved in 250-300 ml water and is ingested orally within 5 min . 6- Blood is drawn at intervals of 30 min for measurement of glucose.

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)

Interpretation of OGTT curve in normal & diabetic patients

Special OGTT Results Flat response Gestational diabetes Renal Glucosuria Alimentary Glucosuria

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

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

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

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.

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

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.

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

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.

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)

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.

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

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

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