Question

1.) A person is suspected of diabetes mellitus. He checks his blood glucose level by glucose oxidase method. After the experiment, OD of his blood sample was obtained as 0.08. Based on the glucose standard curve seen in the virtual lab, calculate his blood glucose level. Is he had diabetes mellitus? (Note: Normal blood glucose level in our body is 70-110mg/dl).

2.) Find out the role and chemical reactions of the following reagents in the blood glucose level determination by glucose oxidase method.

a) Sodium sulphate – Zinc sulphate solution b ) Glucose oxidase reagent c) Peroxidase

3.) A person did estimation of blood glucose level by glucose oxidase method by referring the virtual lab procedure. But he forgot to add ortho - toluidine reagent during glucose oxidase reagent preparation.

a) What is the observation of the result? b) Suggest three other chemicals used in place of ortho - toluidine?

Answer 1

1. The O.D 0.08 corresponds to the blood glucose less than 50 mg/dl which suggests that the person is suffering from low glucose. So it is case of either Hypoglycaemia.

Sodium sulphate is used in the reagent to decrease the solubility of oxygen in reagent.As this oxygen will again oxide the cuprous oxide. This may give false positive. While the zinc sulphate is used to remove protein from the sample that may hinder with the glucose estimation. The zinc sulphate precipitate protein by forming sulfhydryl compounds and zinc is removed as zinc hydroxide.

Glucose oxidase: In body the glucose is present in the foem of alpha and beta. So to test the whole glucose of the blood it is mandatory to convert all beta form glucose to the alpha . Glucose oxidase has property of mutarotating the glucose. So it is used in the test.

Peroxides are also used to determine glucose especially in urine samples as the samples as the samples are impregnated with iodine, and the hydrogen peroxide produced oxidizes iodide that intensify of brown color corresponding to glucose concentration in urine.

2. If there is no addition of ortho toludine, then there will be no development of intense green colour corresponding to the aldosugars like pentoses and hexoses. As there will be no color change in the sample we will not be able to determine the glucose correct concentration by colorimetry.

In place of O toludine we can use:

1. 2:6-dichlorophenol-indophenol: this dye gets reduced with ascorbic acid after which it has been studied as the oxygen acceptor in the glucose-oxidase reaction for the estimation of glucose.
2. Hexokinase: It is the most specific method, it convert glucose to glucose 6 phosphate and reduces NADPH to NADP that can be measured directly through spectrophoteometry.
3. Folin reagent: By folin wu method uses folin reagent which is lkaline copper solution which when react with reducing sugars like glucose result into insoluble cuprous oxide which is then allowed to react with phospharomolybdate to form molybdenum blue colored complex