5: Glucose Tolerance Test and Determination of Blood Glucose

Introduction

Glucose is the major energy source for the body processes. The energy that is necessary for much of the molecular synthesis and metabolic processes of the cells is provided by the metabolism of glucose. Therefore, the level of circulating glucose is of vital interesting. Fasting blood glucose level above or below the normal range usually indicate disease. Blood glucose determinations are, therefore used for diagnosis and follow-up of abnormalities of carbohydrate metabolism.

âOral Glucose Tolerance Test (OGTT)

OGTT is a test including series of measurement of blood glucose before and after glucose administration. These values are useful to evaluate individuals and establish values for normal and disease state. The recommended procedures are:

1. A fasting blood sample should be collected after 10-16 hours fast.

2. A glucose dose of 50g is prepared in flavored water (such as lemon juice) at a concentration no greater than 25g/dl, and should be drunk in about 5 minutes.

3. Blood samples should be collected at 30 minutes intervals for 2 hours.

âDetermination of glucose

Blood glucose concentration is measured using a modified glucose oxidase method. The oxidation of sample glucose is catalyzed by glucose oxidase to form hydrogen peroxide and gluconate. The hydrogenperoxide is then determined by the peroxidase oxidative coupling of N,N-diethylaniline ( N,N-DEA ) with 4-amino-antipyrine ( 4-AAP ).

Glucose oxidase

D-Glucose + O₂+ H₂O D-Gluconic acid + H₂O₂

Peroxidase

2 H₂O₂ + 4-Aminoantipyrine + N,N-Diethylaniline

Purple dye (detect at 553 nm )

II. Materials and Methods :

A. Materials :

1. Phosphate buffer ( 50 mM ) (can be stored at room temperature for certain period of time )

Weigh 6.8 gm KH₂PO_{4 ,} bring up with 900 ml of distilled H₂O ( dH₂O ), adjust pH to 7.0 with approximately 53 ml, 1M NaOH or 6 ml 10 M NaOH. Bring up to 1 liter with dH₂O.

2. Combined reagent ( 100 ml ): make fresh each day and store refrigerated until use.

Glucose oxidase 0.01963 g (50,000 U / 0.37 g solid )

Peroxidase ( Horseradish ) 0.00333 g (50,000 U / 0.264 g solid )

Aminoantipyrine 0.062 g

Sodium azide 0.1 g

N,N-Diethylaniline 50 ml

Transfer the above reagents to a 100 ml volumetric flask and dilute to volume with phosphate buffer.

3. Glucose standard solution :

Transfer 1 g of glucose into a 100 ml volumetric flask and diluted to volume with aqueous 0.2% benzoic acid solution. From this stock solution, prepare 50, 100, 150, 200, 300 mg / dl with 0.2% benzoic acid. Store refrigerated.

B. Equipment

1. Spectrophotometer

2. Cuvettes

3. Borosilicate glass tubes

C. Procedure:

1. Pipet 20ml of blood sample ( plasma or serum ), or standards, or 0.2% benzoic acid to 10 ml test tubes.

吸取20ml之血液檢體 (血漿或血清), 標準品, 及0.2％苯甲酸 到10ml之測試管中。

2. Add 5 ml combined reagent to each tube. Invert with parafilm to mix and record the time.

每根測試管中加入5ml之combined reagent. 以石蠟膜封住管口倒置混合並紀錄時間

3. Let stand at room temperature for 30 minutes, Read the absorbance against the combined reagent blank at 553 nm.

於室溫靜置30分鐘, 以combined reagent當作空白對照值, 記錄波長553 nm下的吸光值

IV. Reference :

1. Kabasakalian, P., Kalliney, S. and Westcett, A. ( 1974 ) Enzymatic blood glucose determination by colorimetry of N,N-Diethylaniline-4-Aminoantipyrine. Clin. Chem. 20 , 606-607.

2. National diabetes data group (1979) Classification and diagnosis of diabetes mellius and other categories of glucose intolerance. Diabetes 28 , 1039-1057.