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Anglia Ruskin University
Department of Life Sciences

Metabolism and its Control (MOD002803)
The Glucose Tolerance Test

Introduction: The normal plasma glucose levels in humans is around 3.6 – 6.0 mmol/L. Diabetes mellitus is a condition where the glucose levels in blood are raised as the body cannot utilise it appropriately. This may be due to a lack of insulin, or because of resistance to the insulin in circulation. The diagnosis of diabetes is made using an oral glucose tolerance test (OGTT). For the OGTT samples for blood glucose are normally collected into fluoride-oxalate tubes. The fluoride salt, normally NaF, inhibits glycolysis by the cells in the sample and the oxalate, generally potassium oxalate K2C2O4, acts as an anti-coagulant. There are three main methods for the analysis of glucose, each one employing an enzymatic reaction; the glucose oxidase method, the hexokinase method and the glucose dehydrogenase method. The latter is not commonly used in a clinical laboratory setting. This practical will utilise the glucose oxidase method:

Glucose oxidase
Glucose + 2H2O + O2 ((((((( Gluconic acid + H2O2

Peroxidase
H2O2 ((((((( H20 + O2

Reduced chromogen (colourless) ((((((( oxidised chromogen (coloured)

Glucose is the limiting factor and the rate of production of the coloured product is proportional to oxygen production and therefore glucose concentration. The absorbance of the coloured product will be proportional to concentration of the product provided that the Beer-Lambert law is obeyed.

Specialist Rooms – Dav204/5
Specialist Equipment- adjustable pipettes, calibrated dispenser, spectrophotometers, thermostatically controlled waterbaths

Reagents:

• Standard glucose concentration 100mM for construction of a standard curve. • “Patient samples” for analysis from time T=0 and T=2hrs • Glucose oxidase reagent (CAUTION harmful, irritant). This contains glucose oxidase, peroxidase...
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