Introduction
- For all practical purposes, glucose is the only sugar that is present in the blood.
- Glucose is absorbed by the body cells and is the major source of cellulose energy.
Normal values of blood glucose:
- Normal fasting level = 60-90mg/100ml of blood
- Glucose level half an hour (after meal) (post prandial) = 120-150mg/100ml of blood
- In normal healthy individuals the peak glucose level (at any time of the day) = 60-110 mg/100ml of blood is considered normal.
- normal HbA1c levels and range
Clinical significance of blood sugar level:
- Blood glucose level increases in diabetes mellitus, acute stress, hyperthyroidism and chronic liver disease.
- Blood glucose level decreases in Addison’s disease, hypothyroidism and cancer of the pancreas.
- The increase in the blood glucose level is called hyperglycemia and decrease in blood glucose level as hypoglycemia.
- People suffering from diabetes mellitus need to get their blood glucose tested frequently.
Methods used to measure blood glucose level
- Although a number of methods are used for glucose determination, commonly used two methods are discussed here.
- These can be grouped into two categories- chemical and enzymatic.
- Chemical method
- Folin-Wu method
- Ortho-Toluidine method
- Enzymatic method
- GOD-POD method. (Glucose oxidase method)
Chemical method to estimate blood glucose:
- Folin-Wu method:
- It is based on the principle that glucose when heated with an alkaline copper solution, reduces cupric ions to cuprous ions.
- The cuprous ions are then measured photometrically (colorimetrically) by adding phosphomolybdic acid which gets reduced to molybdenum blue.
- In this method, whole blood is used and the blood glucose value is determined by the intensity of blue color.
- Ortho-Toluidine method:
- This is an ideal manual method used for its rapidity, sensitivity, accuracy, and relative simplicity.
- It is based on the principle that the aldose sugar i.e. glucose on condensation with ortho-toluidine in glacial acetic acid gives a green colour that can be measured spectrophotometrically.
- Procedure: O-toluidine method is performed on plasma or serum.
- To a trichloroacetic acid filtrate of blood add O-toluidine dissolved in glacial acetic acid.
- The mixture is heated to 100oC for about 10 minutes.
- The mixture gives a stable green color.
- Measure the density photometrically.
Enzymatic method- GOD-POD method to estimate blood glucose:
- Enzymatic methods provide maximum degree of glucose specificity, hence are very good in estimating true blood glucose.
- For this method, only blood plasma or serum is used.
- The glucose remains stable for 24 hours at 2-8oC if serum or plasma is prepared within 30 minutes after collection.
- The enzyme peroxidase catalyzes the following reaction. The hydrogen peroxide formed reacts with phenol and 4 amino-phenazone to a red-violet dye as indicator.
- The intensity of the color formed is measured colorimetrically (or spectrophotometrically) which is directly proportional to the blood glucose level.
- Glucose + O2 + H2O ——(glucose oxiadase)———–> Gluconic acid + H2O2
- H2O2 + phenol + 4 aminophenazone ——(Hydrogen peroxidase)—–> Quinoneimine + 4H2O
- This test is not influenced by the pressure of uric acid, ascorbic acid, anticoagulants or bilirubin in blood.
Required reagents:
- Enzyme reagent:
- It is ready for use reagent that consists of-
- glucose oxidase, peroxidase, phenol, 4-amino phenazone phosphate buffer and stabilize.
- Standard:
- It is also ready, for use and consists of glucose conc. 100mg/100ml.
Procedure to estimate blood glucose:
- Take 3 test-tubes and mark them as blank, sample and standard. Add the following contents:
- Blank – 2ml of sample + 2ml of enzyme reagent
- Test or sample – 2ml of sample + 2ml of enzyme reagent
- Standard- 2ml of standard + 2ml of enzyme reagent
- Mix well and incubate all test-tubes for 10 minutes at 20-25oC.
- Measure the absorbance of the standard and the sample against the reagent blank at 500-546onm colorimetrically.