Annex VI
Calculate the results from the following
expression:
Sample absorbance
- - - - - - - x Standard concentration
Standard absorbance
= Sample concentration
Constraints
With hyperlipidemic or slightly hemolysed
sera, as well as sera containing large amounts
of bilirubin, a serum blank can be prepared
by adding the same amount of serum in the
sample to a reagent consisting of the phenol
solution diluted with an identical volume of
water. This serum blank is measured against
the reagent alone and any absorbance obtained is subtracted from that provided by
the serum treated with the whole reagent.
With strongly hemolysed sera and sera containing large amounts of bilirubin, use of a
Somogyi filtrate prepared by 10: 1 dilution is
to be preferred; the filtrate volume to be used
is 10 times as high as that of serum employed
in the direct method. Standards must be
diluted to the same extent (10: 1) and added
in proportional amounts in order to ensure
constancy in the calculations. Acid precipitants (e.g. trichloroacetic acid) are inappropriate for use in the glucose oxidase
method.
Glucose oxidase is specific to P-D-glucose; however, dissolved glucose exists 36 %
as the (X form and 64 % as the P form. For the
glucose to be thoroughly oxidized, the a form
must be mutarotated to the P form. The rate
of this conversion depends on pH and temperature. Some commercially available glucose
oxidase preparations contain the enzyme
glucomutarotase, which accelerates the reaction.
193
Enzymatic processes for determining glucose should never be calibrated with freezedried serum. In fact, freeze-drying can cause
some glucose to bind to proteins and proteinbound glucose is unavailable for enzymatic
analysis under moderate conditions. By contrast, the strongly acidic conditions of the 0toluidine method ensure recovery of the
whole glucose.
The glucose oxidase method performs
quite well; however, preparing the reagents
can be tedious and laboratory personnel may
opt for using commercially available reagent
kits such as those from Biodynamics/Boehringer Mannheim Corp. (Indianapolis), Sclavo
(Wayne, NJ) or Worthington Biochemical
Corp. (Freehold, NJ).
Normal values and interpretation
The fasting concentration of glucose in serum
or plasma as determined by the abovedescribed methods typically ranges from 70
to 110 mg/dL (3.9-5.8 mmo1!L). The levels
provided by the earlier methods are about 5 %
higher. The glucose concentration in whole
blood is somewhat different. Although most
methods (enzymatic and o-toluidine) currently in use for this purpose analyse serum
or plasma, glucose values thus determined are
taken to be fasting blood sugar (FBS) values;
unless otherwise stated, however, the values
given in this and subsequent sections are
glucose concentrations in serum.
High fasting glucose levels (up to 500
mg/dL or 28 mmol!L) are typically found in
diabetic patients, depending on the severity
of their condition.
Hypoglycemia (low sugar levels in blood)
is most often the result of insulin overdose
during antidiabetic treatments.
Calculate the results from the following
expression:
Sample absorbance
- - - - - - - x Standard concentration
Standard absorbance
= Sample concentration
Constraints
With hyperlipidemic or slightly hemolysed
sera, as well as sera containing large amounts
of bilirubin, a serum blank can be prepared
by adding the same amount of serum in the
sample to a reagent consisting of the phenol
solution diluted with an identical volume of
water. This serum blank is measured against
the reagent alone and any absorbance obtained is subtracted from that provided by
the serum treated with the whole reagent.
With strongly hemolysed sera and sera containing large amounts of bilirubin, use of a
Somogyi filtrate prepared by 10: 1 dilution is
to be preferred; the filtrate volume to be used
is 10 times as high as that of serum employed
in the direct method. Standards must be
diluted to the same extent (10: 1) and added
in proportional amounts in order to ensure
constancy in the calculations. Acid precipitants (e.g. trichloroacetic acid) are inappropriate for use in the glucose oxidase
method.
Glucose oxidase is specific to P-D-glucose; however, dissolved glucose exists 36 %
as the (X form and 64 % as the P form. For the
glucose to be thoroughly oxidized, the a form
must be mutarotated to the P form. The rate
of this conversion depends on pH and temperature. Some commercially available glucose
oxidase preparations contain the enzyme
glucomutarotase, which accelerates the reaction.
193
Enzymatic processes for determining glucose should never be calibrated with freezedried serum. In fact, freeze-drying can cause
some glucose to bind to proteins and proteinbound glucose is unavailable for enzymatic
analysis under moderate conditions. By contrast, the strongly acidic conditions of the 0toluidine method ensure recovery of the
whole glucose.
The glucose oxidase method performs
quite well; however, preparing the reagents
can be tedious and laboratory personnel may
opt for using commercially available reagent
kits such as those from Biodynamics/Boehringer Mannheim Corp. (Indianapolis), Sclavo
(Wayne, NJ) or Worthington Biochemical
Corp. (Freehold, NJ).
Normal values and interpretation
The fasting concentration of glucose in serum
or plasma as determined by the abovedescribed methods typically ranges from 70
to 110 mg/dL (3.9-5.8 mmo1!L). The levels
provided by the earlier methods are about 5 %
higher. The glucose concentration in whole
blood is somewhat different. Although most
methods (enzymatic and o-toluidine) currently in use for this purpose analyse serum
or plasma, glucose values thus determined are
taken to be fasting blood sugar (FBS) values;
unless otherwise stated, however, the values
given in this and subsequent sections are
glucose concentrations in serum.
High fasting glucose levels (up to 500
mg/dL or 28 mmol!L) are typically found in
diabetic patients, depending on the severity
of their condition.
Hypoglycemia (low sugar levels in blood)
is most often the result of insulin overdose
during antidiabetic treatments.