192
4 The Measurement Process in Chemistry
Annex VI
Determination of glucose in serum
learning objective:
To illustrate a determination routinely performed by clinical control laboratories.
Reference:
J.D. Bauer
Clinical Laboratory Methods
C. V. Mosby Co., St louis, Missouri, 1984.
Glucose oxidase method
Principle
Glucose is oxidized to glucuronic acid by the
enzyme glucose oxidase in the presence of
oxygen (air). The reaction releases hydrogen
peroxide, which, in the presence of another
enzyme, peroxidase, is decomposed, the oxygen thus formed oxidizing the chromogen,
which can then be measured colorimetricalIy. The amine o-dianisidine was used for
this purpose in earlier methods; however,
the phenol-amino antipyrine mixture is a
better chromogen as it gives a dark red
colour. The reaction is highly specific for
glucose, so it provides glucose contents very
close to the true values. High concentrations
of reductants - particularly ascorbic acid
and, to a lesser extent, uric acid - can interfere with the reaction by competing with
the chromogen for the oxygen released, thus
leading to underestimated results. Hemoglobin also interferes as it causes the hydrogen
peroxide to decompose prematurely, thereby
also giving rise to underestimated values.
This interference is seemingly less serious
with the phenol-antipyrine mixture than it
used to be with the former chromogen (0dianisidine). With strongly hemolysed sera
or whole blood, a Somogyi filtrate must be
used.
Reagents
1. Phosphate buffer, 0.1 mollL, pH 7.0
Dissolve 8.5 g anhydrous disodium hydrogen phosphate (Na2HP04) and 5.3 g
potassium dihydrogen phosphate (KH2 P04)
in about 8 dl water. Check solution
pH and adjust to 7.0 ± 0.1 by addition
of a small volume of 1 moUl NaOH
or HCI, as required. Finally, dilute to
1 litre.
2. Peroxidase reagent
Dissolve 175 mg (0.75 mmol) 4-aminoantipyrine (4-aminophenazone, Sigma
Chemical Co.) and 2 mg peroxidase
(Sigma Type II) in 5 dL of the phosphate
buffer. This solution is stable for about
four weeks if stored refrigerated.
3. Glucose oxidase reagent
Add 2 mL glucose oxidase stock solution
(Sigma Type V, 1000 unit/mL) to the peroxidase reagent. This solution is stable for
one week if stored in a refrigerator.
4. Phenol solution
Dissolve 2.0 g (21.3 mmol) phenol and 9 of
NaCI in water, and make to 1 litre. This
solution is stable for several months at
room temperature.
5. Standards
The standards used are the same as those
employed in the previous direct determination procedure.
Procedure and calculations
Add 50 ilL serum or standard to 2 mL of the
glucose oxidase reagent in a test tube and
mix. Next, add 2 mL of the phenol reagent,
stopper the tube and shake to facilitate
aeration. Heat the tube in a bath a 37°C for
15 min, allow to cool and read the absorbances of the sample and standards at 510 nm
against a reagent blank subjected to the same
treatment.
4 The Measurement Process in Chemistry
Annex VI
Determination of glucose in serum
learning objective:
To illustrate a determination routinely performed by clinical control laboratories.
Reference:
J.D. Bauer
Clinical Laboratory Methods
C. V. Mosby Co., St louis, Missouri, 1984.
Glucose oxidase method
Principle
Glucose is oxidized to glucuronic acid by the
enzyme glucose oxidase in the presence of
oxygen (air). The reaction releases hydrogen
peroxide, which, in the presence of another
enzyme, peroxidase, is decomposed, the oxygen thus formed oxidizing the chromogen,
which can then be measured colorimetricalIy. The amine o-dianisidine was used for
this purpose in earlier methods; however,
the phenol-amino antipyrine mixture is a
better chromogen as it gives a dark red
colour. The reaction is highly specific for
glucose, so it provides glucose contents very
close to the true values. High concentrations
of reductants - particularly ascorbic acid
and, to a lesser extent, uric acid - can interfere with the reaction by competing with
the chromogen for the oxygen released, thus
leading to underestimated results. Hemoglobin also interferes as it causes the hydrogen
peroxide to decompose prematurely, thereby
also giving rise to underestimated values.
This interference is seemingly less serious
with the phenol-antipyrine mixture than it
used to be with the former chromogen (0dianisidine). With strongly hemolysed sera
or whole blood, a Somogyi filtrate must be
used.
Reagents
1. Phosphate buffer, 0.1 mollL, pH 7.0
Dissolve 8.5 g anhydrous disodium hydrogen phosphate (Na2HP04) and 5.3 g
potassium dihydrogen phosphate (KH2 P04)
in about 8 dl water. Check solution
pH and adjust to 7.0 ± 0.1 by addition
of a small volume of 1 moUl NaOH
or HCI, as required. Finally, dilute to
1 litre.
2. Peroxidase reagent
Dissolve 175 mg (0.75 mmol) 4-aminoantipyrine (4-aminophenazone, Sigma
Chemical Co.) and 2 mg peroxidase
(Sigma Type II) in 5 dL of the phosphate
buffer. This solution is stable for about
four weeks if stored refrigerated.
3. Glucose oxidase reagent
Add 2 mL glucose oxidase stock solution
(Sigma Type V, 1000 unit/mL) to the peroxidase reagent. This solution is stable for
one week if stored in a refrigerator.
4. Phenol solution
Dissolve 2.0 g (21.3 mmol) phenol and 9 of
NaCI in water, and make to 1 litre. This
solution is stable for several months at
room temperature.
5. Standards
The standards used are the same as those
employed in the previous direct determination procedure.
Procedure and calculations
Add 50 ilL serum or standard to 2 mL of the
glucose oxidase reagent in a test tube and
mix. Next, add 2 mL of the phenol reagent,
stopper the tube and shake to facilitate
aeration. Heat the tube in a bath a 37°C for
15 min, allow to cool and read the absorbances of the sample and standards at 510 nm
against a reagent blank subjected to the same
treatment.