Determination of serum glucose

determination of serum glucose is very important for human body.

• Method of determination of serum glucose
• Principle
• Normal value
• Sample material
• Reagent
• Stability of the reagent
• Preparation of glucose reagent
• Procedure
• Calculation

Method of determination of serum glucose

Glucose oxidase method

Principle OF determination of serum glucose :-

• The aldehyde group of glucose is oxidized by glucose oxidase to give gluconic acid and hydrogen peroxide. The overall reaction is- glucose + H2O + O2 → gluconic acid + H₂O₂
• The hydrogen peroxide is broken down to water and oxygen by per-oxidase _H2O2 → H2O+ O2 ,The oxygen reacts with 4-aminophenazone in the presence of phenol to form a pink colored compound and intensity of which can be determined at 530 nm. On colorimeter.

Normal value glucose (fasting):-

Serum glucose (fasting): 70-110 mg/dl,

Postprandial (PP): Up to 140 mg/dl.

Random :- 130 mg/ dl

Sample material:-

Fluoride plasma or serum collected within 30 minutes of blood collection.

Reagent:-

1. Buffer enzyme :-

This reagent is prepared by mixing following constituents in 100 ml of phosphate buffer, (M/10, pH 7.0).

a) glucose oxidase (sigma)- 650 unit

b) peroxidase (sigma) – 500 unit

c) 4-Aminophenazone – 20mg

d) sodium azide – 30 mg

2. phenol reagent : ready to use – 100mg/dl

3. glucose standard – 100 mg/dl

stability of reagent:-

Reagent 1 & 2 are stable at 2-8°C for six months.
Reagent 3 is stable for one year when refrigerated.

Preparation of glucose reagent:-

Mix 2 parts of buffer/enzymes reagent and 1 part of phenol reagent to get glucose reagent. It is preferable to prepare the reagent fresh daily. Otherwise it is stable
at 2-8°C in an amber colored bottle for 2 weeks. (A pink coloration of the reagent due to aging does not affect the test results if a standard & blank is put up with each batch of the determination). Bring the reagent to room temperature before use.

Procedure:-

• first of all take three clean and sterilize test tube.
• add glucose reagent 1 ml in each tube.
• add serum only in the marking of test .
• add standard in standard tube only
• then add distilled water only tube blank
• Mix and keep at 37°C for 15 minutes or at room temperature for 30 minutes. Measure the intensities of the color at 530 nm (green filter).

Calculation:-

Glucose :- OD. OF TEST / OD. OF STANDARD X 100

OD = OPTICAL density