Oral Glucose Tolerance Test (OGTT) Biochemistry Practical 1- Batch 17

Glucose Tolerance Test Aim: To understand the significance of glucose tolerance test in clinical diagnosis Objectives: At the end of this practical you should be able to: Use the spectrophotometer to assay glucose in the plasma / serum. Interpret the results of glucose tolerance tests.

Instrument - SPECTROPHOTOMETER

Colorimeter / Spectrophotometer Colorimeter - Instrument to quantify the intensity of the color. Intensity of the color proportional to the amount of light absorbed and the concentration of the substance. Uses a std solution (known concentration).

THEORY Optical density (absorption) of the substance of a colored solution is directly proportional to the concentration of the substance.

Glucose tolerance test Definition - test done to assess the ability of the body to tolerate extra glucose taken orally hence known as oral glucose tolerance test (OGTT).

Procedure for GTT 1. Patient was asked to come after over night fasting. 2. Fasting blood sample (T0) collected. 3. Patient was given 75 g of glucose in 150ml of water orally and blood samples were collected at intervals of 1 hr (T1), 2 hr (T2) and 2½ (T2½) hr along with this urine sample was also collected. 4. Blood and urine samples were used for determination of glucose.

Steps involved in practical 1. Determination of Blood Glucose- Spectrophotometric method - Glucose Oxidase Method. 2. Determination of Urine sugar- Benedict’s method.

Principle - Blood glucose Glucose Oxidase- peroxidase-chromogen sequence (Glucose Oxidase Method) Glucose oxidase Glucose + O2 →→→→→ Gluconic Acid + H2O2 peroxidase 2H2O2 + phenol + 4-amino antipyrine →→→→→ quinoneimine + 4H2O quinoneimine measured at 505 nm.

Principle - Urine glucose Benedict’s Method Glucose + Benedict’s reagent Reddish brown precipitate Reduction

Materials provided Serum samples (X and Y) taken at intervals of 0, 1, 2 and 2½ hours. Urine samples (X and Y) and boiling tubes. 1.5 ml plastic cuvettes (for taking spectrophotometer readings). Spectrophotometer. Glucose oxidase reagent - Combined Enzyme-Color Reagent Solution.

Method Reagent Blank (B) (1 test tube) Standard (S) (1 test tube) Test (T) - T0,T1,T2, T2½ (4 test tubes) Dist. water 100 L (0.1 ml) --- Standard 100 mg/dL Plasma Glucose oxidase reagent 1 ml (1000 L) Incubation – 20 min room temperature, O.D reading – 505 nm

Calculation Concentration of glucose (mg/dl) = O.D reading of the test _________________ x Concentration O.D reading of the standard of std glucose Multiplied by 100 to express in mg/dl.

INDICATIONS FOR DOING GTT Fasting blood glucose is 110–125 mg% ORAL GLUCOSE TOLERANCE TEST (OGTT) Test done to assess the ability of the body to tolerate extra glucose taken. Preparation of the patient Patient should take adequate carbohydrate for at least 3 days before the test. The patient should fast the previous night (10–16 hrs.) Just before the test and while the test, the patient should refrain from exercise, eating, drinking, smoking, diuretics, oral hypoglycemic agents / insulin.

PROCEDURE FOR OGTT After overnight fast, venous blood sample is withdrawn for plasma glucose estimation. A urine specimen is also collected to test for the presence of glucose. The patient is administered 75 gms glucose in 300 ml of water, which the patient has to drink within 3-5 minutes. Blood and urine samples are collected for 2 hrs at 30 min interval, after the ingestion of glucose.

Benedict’s Test

INTERPRETATION OF RESULTS Normal Impaired Diabetes Glucose tolerance Fasting plasma Glucose (mg/dL) < 110 110-125  126 2 hrs after glucose load (mg/dL) < 140  140 but < 200  200

Features of a normal GTT curve: Benedict’s Test Features of a normal GTT curve: Fasting level within normal (70-110mg/dL). Peak value at 1 hour less than “Renal threshold value” (180mg/dL). After 2 hours comes back to fasting value. No sugar in the urine at any time.

Features of Impaired Glucose Tolerance: Impaired GTT Curve Fasting Benedict’s Test Features of Impaired Glucose Tolerance: 1. Fasting level above normal (115mg/dL). 2. Peak value at 1 hour more than renal thresh hold 190mg/dL. 3. After 2 hours does not come back to fasting value. 4. Sugar in the urine seen after the plasma value cross the renal thresh hold value. 5. Such patients may become diabetic after few years.

Renal Diabetes Patient GTT Curve Benedict’s Test Normal Blood glucose concentration, But excretion of Glucose in the Urine. Renal Diabetes Patient GTT Curve Benedict’s Test Features of Renal Glycosuria: 1. Fasting level within normal (70-110mg/dL) 2. Peak value at 1 hour less than “Renal threshold value”. 3. After 2 hours comes back to fasting value. 4. But sugar is seen in the urine

GLYCOSURIA In a normal person there is no sugar in the urine. If there is sugar in the urine it is a pathological condition.--- “GLYCOSURIA”. There are two types of glycosuria: 1. Hyperglycemic glycosuria. 2. Renal glycosuria (Renal Diabetes).

Hyperglycemic glycosuria: if the blood glucose level goes above the renal threshold which is 180mg/dL the glucose is seen in urine. In normal person the whole glucose reabsorbed in the renal tubules. Emotional glycosuria [Transient] -- stress, pain. Endocrine glycosuria ---Diabetes Mellitus, Hyperthyroidism, Cushing syndrome.

Renal glycosuria: The blood sugar levels are within normal limit, less than the renal thresh hold [180mg/dL] but still glucose is present in urine. Types: Hereditary renal glycosuria --- due to absence of Carrier protein. 2. Acquired renal glycosuria --- due to kidney problems and due to heavy metal poisoning. 3. Lowered renal threshold ---normal renal threshold is 180mg/dL.

Laboratory test to diagnose glycosuria: Benedict’s test: 0.5 ml urine + 5ml Benedicts test, boil and cool. Blue colour of Benedicts --- “NORMAL PERSON”. Green precipitate to red precipitate -- “GLYCOSURIA”.

Questions What is GTT? How is GTT done? What are the features of a normal GTT? What are the features of (a) Impaired glucose tolerance. 4. What is meant by glycosuria? What are the types of glycosuria? What is meant by polyuria? 7. What is the renal threshold value of glucose. 8. What are the causes of renal glycosuria.

GTT Procedure & Clinical application & Data interpretation. Normal value of Blood Glucose as per WHO criteria (Fasting & PP). Sodium Fluride Mode of action. Clinical significance of “Glycosylated hemoglobin” (HbA1c) & Fructosamine. Types of Diabetes: Type-1, Type-2, Juvenile DM, Renal Diabetes. Mode of action of Insulin & Glucagon. Insulin Resistance. Polyuria, melituria / glycosuria, proteinuria. Complications of uncontrolled DM: Neuropathy, Nephropathy & Retinopathy. Steate a few clinical conditions in which “hyperglycemia” can be observed. What is the “Renal Threshold Value” of glucose. What is “Advanced Glycation End Products (AGEPs)” and its clinical significance. Name a few oral hypoglycemic agents. Relevance of Metformin in G6PD deficiency.