TITLE: Estimation of Blood Glucose Using Glucose Oxidase AIMS AND OBJECTIVES: To understand the importance of measuring blood

od glucose level. To understand the principles of enzymatic estimation of glucose. Enzymatic method yields maximum specificity for glucose estimation. Glucose can be measured by its reaction with glucose oxidase, in which gluconic acid and hydrogen peroxide are formed. Hydrogen peroxide than reacts with an oxygen acceptor, such as ortho-dianisidine, phenylamine-phenazone or any other chromogenic oxygen acceptors, in a reaction catalysed by peroxidase to form a colour. One of the advantages of enzymatic method is its specificity. This enables the estimation of glucose even in a complex mixture. Thus, this method is widely used in the field of clinical chemistry and for food analysis. In clinical chemistry, the enzymatic analysis of glucose in blood and urine has been modified as dipstick test. However in the present practical, the conventional enzymatic method shall be used. METHOD: 1. SAMPLES TEST: a. The following substances are added in the tube. i. 1.8 ml sodium sulphate-zink sulphate ii. 0.1 ml blood iii. 0.1 ml NaOH (0.1M) b. Mix carefully. c. d. Centrifuge at 3000 RPM for 5 min. Transfer the supernatant into new test tube.

INTRODUCTION

Note: The glucose concentration in the supernatant is 1/20 of the concentration in the blood sample.

2.

BLANK & STANDARD: a. b. Glucose standards 12 mg/dl are provided. Preparing concentration glucose standard point. CONCENTRATION (mg/dL) GLUCOSE STANDARD (mL) M1V1 : M2V2 WATER (mL) H2O = 2.0 ml 0.5 mL = 1.5 mL H2O = 2.0 ml 1.0 mL = 1.0 mL H2O = 2.0 ml 1.5 mL = 0.5 mL H2O = 2.0 ml 2.0 mL = 0 mL 2.0 mL 2.0 2.0 2.0 2.0 2.0 TOTAL mL PER TUBE

TUBES

3.0

(12)V1: 3.0 (2.0 ) V1 : 0.5 mL M1V1 : M2V2

6.0

(12)V1: 6.0 (2.0 ) V1 : 1.0 mL M1V1 : M2V2

9.0

(12)V1: 9.0 (2.0 ) V1 : 1.5 mL M1V1 : M2V2

12.0

(12)V1: 12.0 (2.0 ) V1 : 2.0 mL

BLANK

c.

Then, add 5 ml ortho-tolidine mixture where consisting; i. 1% ortho-tolidine 12.5 mg (500 units) glucose oxidase in 100 ml phosphate buffer, pH 7.0. MINUTE ADDED ORTHO-TOLIDINE 2 4 6 8 10 12 iv. Mix quickly. v. The time is staggered every 2 minutes when adding the ortho-tolidine solution as shown table above. vi. Incubate the tubes at room temperature for exactly 10 min. vii. Then, measure at absorbance 625nm. Notes: 2 ORTHO-TOLIDINE mL 5.0 5.0 5.0 5.0 5.0 5.0 TOTAL mL PER TUBE 7.0 7.0 7.0 7.0 7.0 7.0 ii. 4mg (100 units) peroxidase iii.

TUBES BLANK 1 2 3 4 SAMPLE

Ensure that each tubes absorbance is read exactly after 10 min. If the absorbance reading for the sample is too high, dilute the supernatant which was obtained earlier, (2x) with water and repeat the subsequent steps.

RESULTS: GLUCOSE CONCENTRATION (mg/dL) 0 3.0 6.0 9.0 12.0 SAMPLE DISCUSSION: On this experiment, blood glucose is estimated by using enzymes glucose oxidase. Where Ortho-toluidine is use as chemical for exploited to quantitative carbohydrates molecules to form Schiff bases with aromatic amines. Ortho-tolidine in a hot acidic solution will yield a colored compound with absorbance maxima at 625 nm. ABSORBANCE READING (nm) 0.000 0.181 0.120 0.006 0.005 0.045

GLUCOSE OXIDASE Glucose + O2 + H2O H2O2 + Reduced chromogen

Glucose Oxidase

Gluconic Acid + H2O2 Oxidized chromogen + H2O

Peroxidase

Glucose oxidase is the most specific enzyme reacting with only D-glucose and glucose oxidase converts D-glucose to gluconic acid. Added Mutarose to the reaction can facilitate the conversion of D-glucose to D-Glucose. Oxygen is consumed and hydrogen peroxide is produced. The reaction is measured based on rate of disappearance of oxygen. Spectrophotometer is used for read absorbance, where it proportional to the amount of glucose presents in the sample. However, on this experiment, the absorbance reading for concentration reading is not expected as well and it fully incorrect. Because the absorbance reading supposedly from the lower thru high value. But, on another hand for comparison with other group shown that the results reading are correct and accordingly as table below.

GLUCOSE CONCENTRATION (mg/dL) 0 3.0 6.0 9.0 12.0 The problems that can be come across are shown in table below;

ABSORBANCE READING (nm) 0.000 0.056 0.160 0.225 0.225

CAUSES 1.

DESCRPTION Pipetting sample or chemical solution inappropriate. Taken wrong sample reading. Unclean Cuvettes. Mislabeling or not label sample. Chemical are already expired. Improper making the chemical solution. Increase levels of uric acid, bilirubin, and ascorbic acid can cause falsely decreased values as a result of these substances being oxidized by peroxidase, which prevents the oxidation and detection of the chromogen. Galactose, an aldohexose, and mannose, an aldopentose, will also react with Orthotoluidine and produce a colored compound that can interfere with the reaction.

HUMAN ERROR

2. 3. 4. 1. 2. 1.

MATERIAL

SUBSTANCES

1. CHEMICAL

CALCULATION: Calculation concentration of blood glucose is not valid because the absorbance reading is to low to plot at the graph. But, as for example, the calculation shown; o 8.8 mg/dL (From plot graph) X 20 (times dilution) = 176 mg/dl If converted to unit mmol/L o 176 mg / dL X 1 dL/100 mL X 1000 mL/ L X 1 mmol/180 mg (gmw glucose :180) = 9.7 mmol/L

QUESTION: 1.What are the advantages of glucose oxidase method in comparison to various other methods of glucose analysis? 1. ADVANTAGES OF GLUCOSE OXIDASE This method most specific enzymatic than other method such as; reduction of cupric to cuprous salts, and reduction of ferricyanide to ferrocyanide method. 2. The reaction can be monitored polarographically either by measuring the rate of disappearance of oxygen. 2.Discuss the principles of the enzymatic assay method. PRINCIPLES OF THE ENZYMATIC ASSAY 4

1. 2. 3.

Glucose oxidase reacts specifically to D-glucose, where it converts to gluconic acid. The hydrogen peroxide is used to oxidize a dye compound. Two commonly used chromogens are 3-methyl-2-benzothiazolinone hydrazone and N,Ndimethylaniline. The principle reaction as shown below; Glucose + O2 + H2O
Glucose Oxidase

4.

Gluconic Acid + H2O2 Oxidized chromogen + H2O

H2O2 + Reduced chromogen

Peroxidase

CONCLUSION: 1. 2. The blood glucose level is not applicable on this test because value is too small. However, on the experiment the glucose oxidase method is more specific test for determination the glucose than other method. 3. Glucose test is useful in monitoring of hyperglycemia or hypoglycemia thru patient.