Estimation of Blood Glucose

EXPERIMENT NO - 12 Aim: To estimate blood sugar level in a given blood sample. (Image of a "Folins sugar tube") Apparatus: Folins sugar tube Pipettes graduated ... 2 ml, 5 ml. Flasks Photoelectric calorimeter Chemical reagents: Alkaline copper sulphate solution Phosphomolybadic acid Sodium tungstate 10% Sulphuric acid 2/3 N Benzoic acid solution Stock glucose solution Standard glucose solution No. 1 Standard glucose solution No. 2 Fluoride oxalate solution. Practical Biochemistry & Clinical Pathology Blood Analysis (Quantitative) Principle: In this method protein free filtrate is obtained [Folin-Wu filtrate] so that 10 ml of filtrate corresponds to 1 ml of blood sample. Protein free filtrate is obtained by precipitating the proteins of blood by tungstic acid. Then this protein free filtrate containing glucose is heated with alkaline copper sulphate solution. Thus glucose reduces copper sulphate to form equivalent quantity of cuprous oxide. This cuprous oxide formed is reduced with phosphomolybdic acid to produce corresponding equivalent quantity of molybdenum blue. The molybdenum blue gives intense blue colour, the intensity of which is directly proportional to cuprous oxide which corresponds to the amount of glucose present in given sample of "folin wu" filtrate. Reaction: C₆H₁₂O₆ + CU⁺⁺ → CU⁺ + Oxidation products of glucose. (glucose, cupric, cuprous) CU⁺ Na₂PO₄. 12 MoO₃ → CU⁺⁺ + 12 MoO (cuprous, cupric, molybdenum blue) The blue colour obtained with test blood sample is compared with standard solution by similar procedure and by using photoelectric calorimeter. The optical density of test and standard is measured and concentration of glucose in blood can be calculated using colorimetric principle. Clinical significance: The chief end product of carbohydrate digestion absorbed in the blood is monosaccharide like glucose and others like fructose galactose. etc. glucose metabolism supplies ... Major amount of energy for body activities Reserve fat depots Tissue glycolipids Amino acids Thus it appears that glucose metabolism plays a central role in carbohydrate metabolism which is closely associated with metabolism of protein and fat. Availability of glucose from various aietary sources and its management/utilisation is regulated by hormones of islets of langerhans. This can be explained schematically as follows ... (Diagram showing Glucose (Excess) being converted by β cells of Langerhans and Insulin into Glycogen (stored), and Glucose being used for Energy, Fat depots, and Tissue repair.) Thus blood sugar level is maintained and it is normally and expressed as Fasting-Post meal. Hyperglycemia i.e. increase in blood sugar level is a real characteristic sign of "diabetes mellitus". Excess free glucose appears in blood due to lack of insulin or in functioning of β cells to secrete insulin. In diabetes mellitus high values for fasting blood sugar are obtained and vary from normal to 200 mg/100 ml and over according to severity of condition. Increase in blood sugar level above 500 mg/100 ml indicates increasing possibility of coma. Hyperactivity of the thyroid, pituitary adrenal glands which includes sates of emotion stress increases blood sugar level about 150 mg%. Practical Biochemistry & Clinical Pathology Blood Analysis (Quantitative) Similar increase in blood sugar level is observed in convulsions and in terminal stages of many diseases. A moderate increase in blood sugar level i.e. hyperglycemia can occur in sepsis and number of infectious diseases. Increase in blood sugar level is also found in some intracranial diseases such as meningitis, encephalitis, tumours and haemorrhages. Method used : Folin Wu (modified) Procedure: Wash clean, label three folin-wu tubes as ... Unknown ... "U", Standard I - Std I, Standard II - Std II To the folin wu tube labelled as "U" take 2 ml of "Folin wu filtrate" In a folin wu tube labelled as "Std I" take 1 ml of standard sugar solution I (0.1 mg sugar) In a folin wu tube labelled as "Std II" take 1 ml of standard sugar solution II (0.2 mg sugar) To all above tubes add 1 ml of alkaline copper sulphate solution. Keep the tubes in boiling water bath for 6 to 8 minutes. Remove from the water bath and add 1 ml of phosphomolyddic acid to all tubes. Keep the tubes again in boiling water bath for 2 minutes and after 2 minutes cool to room temperature. Add 25 ml of distill water to each tube mix well and record. Compare the optical densities by using photoelectric calorimeter by using tube filter 420 mµ. Calculations: By using photoelectric calorimetry principle Concentration of glucose in given blood sample (Cu) = (Optical density of unknown blood glucose sample Eu / Optical density of standard glucose Es) × Concentration of std glucose (CS) Now as 1 ml of standard sugar solution is used for experiment it is fact that 1 ml of standard sugar solution = 0.1 mg of sugar i.e. Cs concentration of standard glucose = 0.1 mg of sugar. Therefore, Concentration of glucose in given blood sample CU = (Eu/Es) × CS = (Eu/Es) × 0.1 mg = "x" (suppose) Now "x" mg of sugar is present in 1 ml of folin wu filtrate of given blood sample i.e. in 0.1 ml blood. Note: (1 ml of folin wu filtrate = 0.1 ml of blood) ∴ To determine mg percentage of sugar If 0.1 ml blood = "x" mg of sugar ∴ 100 ml blood = (100 × x) / 0.1 ml blood Substitute value of "x" ... Practical Biochemistry & Clinical Pathology Blood Analysis (Quantitative) We get Cu = (100/0.1) × (Eu/Es) × 0.1 Concentration of unknown glucose in given blood sample = (Eu/Es) × 100 mg percent sugar. [Boxed Formula] Concentration of glucose in given blood sample CU = (Optical density of unknown blood sample Eu / Optical density of standard sugar solution) × 100 Report: Patients name Blood sugar (estimated) Blood sugar (Normal) Date: Signature: Preparation of reagents: Sodium tungstate: 10 % w/v (Na₂ WO₄ 2H₂O) dissolve 10 gm of sodium tungstate in 100 ml of distill water. Sulphuric acid: 2/3 N of sulphuric acid dissolve 3.5 gm in 100 ml of distill water. Alkaline copper sulphate solution: [A] (a) Anhydrous sodium carbonate ... 40 gms (b) Tartaric acid ... 7.5 gms (c) Dissolve in distill water ... 400 ml [B] (a) Copper sulphate crystalline ... 4.5 gm (b) Dissolve in distill water ... 200 ml Add solution 'B' to solution "A" with constant stirring and make the final volume with distill water to 1000 ml (1 litre) Phosphomolybdic acid reagent: (a) Molybdic acid ... 70 gm (b) Sodium tungstate AR ... 10 gm (c) Sodium hydroxide 10% ... 400 ml (d) Distill water ... 400 ml Boil for 30 minutes to remove ammonia. Add phosphoric acid (specific gravity - 1.75) - 250 ml. Add distill water to make 1 litre final volume. Benzoic acid solution: Benzoic acid ... 2.5 gms Distill water ... 1000 ml (1 litre) Boil to dissolve Stock glucose solution (1 gm/100 ml) Glucose (AR) ... 1 gm Benzoic acid solution ... 100 ml Standard glucose solution No. 1 (10 mg/100 ml) Stock glucose solution ... 1 ml Benzoic acid solution ... 100 ml Standard glucose solution No. 2 (20 mg/100 ml) Stock glucose solution ... 2 ml