Biochemistry and metabolism

Experiment 7

Principle

Glucose is a monosaccharide. It is central molecule in carbohydrate metabolism. Stored as glycogen in liver and skeletal muscle.

D-Glucose occurs widely in plants and animals. It is an essential component of carbohydrate metabolism and occurs frequently in the free form along with D-fructose and sucrose. However, the more important forms are those of di- (lactose, maltose, sucrose), tri-, oligo- and polysaccharides (dextrins, starch, cellulose). It is present in significant quantities in honey, wine and beer, and a range of solid foodstuffs such as bread and pastries, chocolate and candies. Measurement of D-glucose is extremely important in biochemistry and clinical analysis, as well as in food analysis; it is mostly determined along with other carbohydrates.

For glucose estimation from any material, blood is collect in vial.

Glucose oxidase (GOD) oxidizes the specific substrate β-D- glucose to gluconic acid and hydrogen peroxide (H2O2) is liberated. Peroxidase (POD) enzyme acts on hydrogen peroxide to liberate nascent oxygen (O2), then nascent oxygen couples with 4- amino antipyrine/4-amino Phenazone and phenol to form red quinoneimine dye .

Procedure

Sample preparation

Fresh, clear and unheamolysed serum sample can be used. Although plasma sample prepared using EDTA, Oxalate or flouride can also be used.

Experiment

1. Take 3 fresh test tubes and label them as Blank, Standard and Test.

2. Now add 1 ml of Glucose reagent (enzyme) in all test tubes.

3. In Blank add 10 μl of D.W.

4. In Standard add 10 μl of Glucose standard and in Test add 10 μl of serum/plasma sample.

5. Mix & keep those tubes for incubation at 37 ͦC for 10 min or at room temperature for 30 min.

6. Measure the intensity of color at 505 nm filter (Green filter) using Spectrophotometer.

Calculation

Concentration of Glucose = O.D. of Test-O.D. of Blank × Concentration of Std.

O.D. of Std.- O.D. of Blank

Reaction type : End point

Standard Conconcentration : 100 mg/dl

Linearity is up to 500 mg/dl, If sample value is >500mg/dl, dilute the sample 1:2 with distilled water & repeat assay

Normal Range

BLOOD :

• Random Blood Sugar : 80-140 mg/dl

• Fasting Blood Sugar : 70 - 110 mg/dl

• Post Parendial Blood Sugar : <140 mg/dl

• Urine : Absent

Reference Material

Protocol by amrita lab

https://www.zmchdahod.org/pdf/college/07_ESTIMATION%20_OF_GLUCOSE_BY_GOD-POD-18-12-2018.pdf

Questions

1. Glucose oxidase reagent is a mixture of -------?

2. Define hyperglycemia and hypoglycemia?

3. What are the symptoms of hyperglycemia and hypoglycemia?

4. Blood is collected in EDTA bottle because ?

5. Enlist other methods for estimating glucose in serum and plasma samples.