• 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 100^oC 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 2-8^oC 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 20-25^oC.
• Measure the absorbance of the standard and the sample against the reagent blank at 500-546^onm colorimetrically.

Blood Glucose: normal value, clinical significance and methods of estimation

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