扬州大学：《生物化学 Biochemistry》课程教学课件（讲稿）Experiments for Bichemistry 2_Experiment3,4,5

Experiment1GOD-PODMethod forThe Estimation ofBloodGlucose

GOD-POD Method for The Estimation of Blood Glucose Experiment 1

1Introduction: Glucose is the major carbohydrate present in the blood.: It serves as a principal fuel for all the body tissues including the brainIt undergoes a series of chemical reactions to produce energy.·Accurate and precise measurement of blood glucose level is of greatimportance in the diagnosis and management of diabetes and otherdisorders of carbohydrate metabolism, hyperglycemia, andhypoglycemia

1 Introduction • Glucose is the major carbohydrate present in the blood. • It serves as a principal fuel for all the body tissues including the brain. It undergoes a series of chemical reactions to produce energy. • Accurate and precise measurement of blood glucose level is of great importance in the diagnosis and management of diabetes and other disorders of carbohydrate metabolism, hyperglycemia, and hypoglycemia

Glucose-oxidase Peroxidase (GOD POD) method: The most common method of glucose estimationEstimationofBloodGlucoseByGOD-PODMethod＃

Glucose-oxidase Peroxidase (GOD POD) method • The most common method of glucose estimation

2 Principle> In the presence of atmospheric oxygen, glucose present in thespecimen is oxidized by the enzyme glucose oxidase (GOD) togluconic acid and hydrogen peroxide (H,O2).> H,O, couples with 4-aminoantipyrine and phenol in presence ofperoxidase (POD) to form red-colored quinoneimine dye, which ismeasured colorimetrically at 540 nm> The intensity of the color is directly proportional to the concentrationofglucosepresentinthespecimenGlucoseOxidase(GOD)Glucose+HzO+O2Gluconicacid + HzO2Peroxidase (POD)Red quinoneimine dye +H20HzOz+Phenol+4-aminoantipyrine

2 Principle  In the presence of atmospheric oxygen, glucose present in the specimen is oxidized by the enzyme glucose oxidase (GOD) to gluconic acid and hydrogen peroxide (H2O2).  H2O2 couples with 4-aminoantipyrine and phenol in presence of peroxidase (POD) to form red-colored quinoneimine dye, which is measured colorimetrically at 540 nm.  The intensity of the color is directly proportional to the concentration of glucose present in the specimen

3Reagents>Glucose standard>GOD-PODreagent:VEnzyme reagent mixture containing glucose oxidase (GOD), peroxidase(POD), 4-aminoantipyrine, phenol, and phosphate buffer (pH-7.0)some stabilizers and activators

3 Reagents Glucose standard GOD-POD reagent: Enzyme reagent mixture containing glucose oxidase (GOD), peroxidase (POD), 4-aminoantipyrine, phenol, and phosphate buffer (pH≈7.0), some stabilizers and activators

4Instruments>Test tubes>Pipettes, disposable tips, rack>Water bath>Colorimeter

4 Instruments Test tubes Pipettes, disposable tips, rack Water bath Colorimeter

5Procedure5.1 Label three clean, dry test tubes as Blank (B), Standard (S), and Test (T)5.2 Pipette as follows:BlankTestStandard1 mL1 mL1 mLGOD-PODReagent10 uLDistilledwater10 μuLGlucose standard一10 μLSample-5.3 Mix well and incubate at 37°C for 10 min. Or, at 25°C for 30 min5.4 Measure the ODs4o of the standard and test sample against the blank

5 Procedure Blank Standard Test GOD-POD Reagent 1 mL 1 mL 1 mL Distilled water 10 µL – – Glucose standard – 10 µL – Sample – – 10 µL 5.1 Label three clean, dry test tubes as Blank (B), Standard (S), and Test (T). 5.2 Pipette as follows: 5.3 Mix well and incubate at 37℃ for 10 min. Or, at 25℃ for 30 min. 5.4 Measure the OD540 of the standard and test sample against the blank

6 Calculation: Calculate the concentration of blood glucose in the specimen using thefollowing formula:AbsorbanceofTestX 100Conc.ofGlucoseinthespecimen(mg/dl)=AbsorbanceofStandard

6 Calculation • Calculate the concentration of blood glucose in the specimen using the following formula:

Experiment2TriglyceridesGPO/PAPmethod: Triglycerides (TAGs) are a form of fatty esters. They are produced in theliver by binding glycerol and other fatty acids, transported by VLDL andLDL and act as a storage source for energy.· Increased levels are found in hyperlipidemias, diabetes, nephroticsyndrome, hypothyroidismVIncreased levels arerisk factor for arteriosclerotic coronary diseaseandperipheralvasculardiseaseVDecreased levels are found in malnutrition and hyperthyroidism。 The kit uses GPO/PAP method to determine TAGs in serum/plasma

Triglycerides GPO / PAP method • Triglycerides (TAGs) are a form of fatty esters. They are produced in the liver by binding glycerol and other fatty acids, transported by VLDL and LDL and act as a storage source for energy. • Increased levels are found in hyperlipidemias, diabetes, nephrotic syndrome, hypothyroidism. Increased levels are risk factor for arteriosclerotic coronary disease and peripheral vascular disease. Decreased levels are found in malnutrition and hyperthyroidism. • The kit uses GPO/PAP method to determine TAGs in serum/plasma Experiment 2

1 Principle· Serum triglycerides are hydrolyzed to glycerol and free fatty acids bylipoproteinlipaseIn the presence of ATP and glycerol kinase (GK), the glycerol isconverted to glycerol-3-phosphate, which then is oxidized by glycerolphosphate oxidase (GPO) to yield hydrogen peroxide.The oxidative condensation of 4-Chlorophenol and (4-AAP)4-aminophenazone in the presence of Peroxidase (POD)and hydrogenperoxide produces a rose colored dye which is measured at 550 nmThe intensity of the color formed is directly proportional to thetriglycerides concentration in the sample

1 Principle • Serum triglycerides are hydrolyzed to glycerol and free fatty acids by lipoprotein lipase. • In the presence of ATP and glycerol kinase (GK), the glycerol is converted to glycerol-3-phosphate, which then is oxidized by glycerol phosphate oxidase (GPO) to yield hydrogen peroxide. • The oxidative condensation of 4-Chlorophenol and (4-AAP) 4- aminophenazone in the presence of Peroxidase (POD) and hydrogen peroxide produces a rose colored dye which is measured at 550 nm. • The intensity of the color formed is directly proportional to the triglycerides concentration in the sample