Glucose Analytical methods
Plan

Reference methods

Specimen collection

Lab Methods

o
Hexokinase
o
Glucose oxidase
o
Glucose dehydrogenase
Point of care methods
o
Glucose meters
o
dipsticks
The definitive method for measuring glucose concentration is the isotope dilution mass
spectrometry method and it is against this that all other methods have been evaluated.
In most labs plasma and serum are used for glucose determinations and for self monitoring whole
blood is used. Glucose levels can also be measured in CSF and urine samples. Glycolysis in
erythrocytes progresses once blood has been taken and this can decrease glucose concentrations by
about 5% in one hour. This rate may be increased in the presence of bacterial contamination. The
addition of sodium fluoride or sodium iodoacetate can inhibit glycolysis by preventing the action of
endolase by binding Magnesium ions required for its function. Oxalate is also added to prevent
coagulation.
Hexose kinase method
These methods are based on a coupled enzyme that uses Hexose kinase and glucose 6 phosphate
dehydrogenase.
hexokinase
Glucose +ATP
G6PD
Glucose 6 Phosphate
6 Phosphogluconate
NAD
NADH
The amount of reduced NADH produce is directly proportional to the amount of glucose in the
sample and is measured by the increase in absorbance at 340nm. A reference method involving a
deproteinisation step is available and is highly accurate and precise but is too time consuming for a
normal laboratory to undertake. The alternative approach is to apply the reaction directly to serum
or plasma and use a specimen blank to correct for interfering substances that absorb at 340nm.
Interference in the form of haemolysed samples have phosphate esters and enzymes that alter the
results and also drugs, bilirubin and lipaemic can cause interference. Absorbances can be calculated
at completion of the reaction (end point) or a fixed point after the initial reaction. Calibrators are
used to calculate the glucose concentration, and also to check the linearity of the method.
Absorbance in a reagent blank is measured as these coenzymes could affect the absorbance. There
are also hexokinase procedures where coloured products are produced and this enables absorbance
measurements in the visible range.
Glucose oxidase method
Glucose+H2O+O2
Glucose
oxidase
Gluconic acid +2H2O2
Peroxidase
o-Dianisidine+H2O2
oxidised o-Dianisidine +H2O
o-Dianisidine is a chromogenic oxygen acceptor which results in the formation of a coloured
compound that can be measured. Glucose oxidase is highly specific for the β-D-glucose form (α:β is
36:64). Complete reaction requires mutarotation of the α to β form. Some commercial preparations
of glucose oxidase contain an enzyme, mutarotase that accelerates this reaction, otherwise
extended incubation time allows spontaneous conversion. The peroxidase part of the reaction can
be inhibited (competitively) by various substances eg. Bilirubin, haemoglobin, ascorbic acid,
producing lower values. Calibrators and unknowns should be analysed simultaneously under
conditions where the rate of oxidation is proportional to the glucose concentration. This method
can be used for CSF measurement but not for urine as this contains substances that interfere with
the peroxidase.
Some instruments use an oxygen electrode which measures the rate of oxygen consumption (1st
reaction). This eliminates peroxidase interferences. To prevent H2O2 reacting to produce oxygen, it
is removed by reacting with alcohol or hydrogen and iodide ions. This method should not be used
for whole blood as this consumes oxygen.
Glucose dehydrogenase method
Glucose
Glucose
dehydrogenas
D-Glucono-δ-lactone
e
NAD
NADH
The amount of NADH produced is directly proportional to the glucose concentration. Mutarotase is
added to shorten the time to reach equilibrium. No interference shown with common agents.
Point of case testing
Portable meters allow close point of care monitoring of blood glucose levels. They use whole
blood and predominantly the glucose oxidase or hexokinase methods. In many strips a dye is
coloured by glucose oxidase-peroxidase chromogenic reaction. The meters use reflectance
photometry or electrochemistry to measure the rate of reaction or the final concentration of the
products. Reflectance photometry measures the amount of light reflected from a test pad containing
reagent. In electrochemical systems the enzymatic reaction in an electrode incorporated on the test
strip produces a flow of electrons. The current which is directly proportional to the amount of
glucose in the sample is converted to a digital readout. Blood glucose concentrations are about 1015% lower than serum or plasma but the meters can be calibrated to report plasma glucose values
when the specimen is whole blood.
This method has the advantages that small sample size is used, no liquid reagents are required and
there is improved stability during storage. However, several factors can affect the accuracy and
viability of the results from these meters;
-variability in performance among users, training issues
- Haematocrit, anaemia can increase the values falsely and polycythaemia can decrease the values.
-Defective reagent strips.
-Temperature/humidity
-High triglyceride concentrations
-Not accurate at high and low concentrations
- Inaccurate values with intravascular depletion.
For measurement of glucose in urine often sticks are used for this. The monitoring in this manner
lacks sensitivity and specificity and provides no information about blood glucose level below the
renal threshold. The paper tests strips all use glucose specific enzyme glucose oxidase in a
chromogenic assay. The filter paper is impregnated with glucose oxidase, peroxidase and the dye otolidine. False positive results can occur by contamination with H2O2 or bleach. False negatives can
result from reducing substances being present such as ketones or ascorbic acid.