Glucose Methods

Laboratory Methods:

Blood Glucose: There are three general types of methods used for the determination of
blood glucose: oxidation methods, aromatic amine methods, and enzymatic methods. In
general, the oxidation methods give results than are slightly higher than the other
methods because they also measure non-glucose reducing substances.
The oxidation methods for blood glucose are based on the reducing properties of
glucose. Copper reduction tests are among the oldest methods for glucose determination.
In a hot alkaline solution, glucose will reduce cupric salts to cuprous salts. The quantity
of cuprous salts produced is directly proportional to the glucose concentration. Other
procedures make use of the reduction of alkaline ferricyanide, which is yellow, to a
ferrocyanide, which is colorless. The decrease of yellow color is dependent upon the
glucose concentration.
Non-glucose reducing substances will also react in these oxidation methods. Non-
glucose reducing substances present in the blood include glutathione, uric acid, ascorbic
acid, and creatinine to name a few. These interfering substances may be removed from
the sample by selection of the protein-free filtrate upon which the analysis is carried out.
Protein-free filtrates involve the precipitation of protein from the specimen by various
acids. This procedure removes proteins which interfere with many chemical
determinations by causing turbidity, foaming, or by direct interference with color
reactions. Some of the protein-free filtrates, most notably the zinc sulfate-barium
hydroxide method of Nelson-Somogyi, give filtrates that are almost free of non-glucose
reducing substances. This filtrate gives values closer to the “true glucose”. Protein-free
filtrates are widely used in the clinical chemistry laboratory in many determinations other
than glucose.
The aromatic amine methods for glucose determination depend on the reaction of
various aromatic amines with glucose to form colored derivatives. The most widely used
procedure involves the condensation of the aldehyde group of glucose with the amino
group of o-toluidine to give a green chromogen. These tests are more specific for glucose
than are oxidation tests and yield results that are closer the “true glucose”.
Enzyme methods for glucose determination are the only methods that have
absolute specificity for glucose. The two enzymes most commonly used are glucose
oxidase and hexokinase.
Glucose oxidase catalyzes the oxidation of glucose to gluconic acid and hydrogen
peroxide (Reaction 2.1)

glu cos e
oxidase
Reaction 2.1 glucose + O2 gluconic acid + H2O2
 
The amount of peroxide formed is measured in one of several ways. Many use a second
enzyme, peroxidase, that catalyzes the oxidation of a chromogen (o-toluidine, o-
dianisidine) to form a colored product (Reaction 2.2).
 peroxidase
Reaction 2.2 H2O2 + chromogen   colored compound + H2O

Hexokinase catalyzes the phosphorylation of glucose in the presence of ATP

(Reaction 2.3). The glucose-6-phosphate formed is converted to 6-phosphogluconate by a
second enzyme, glucose-6-phosphate dehydrogenase (Reaction 2.4). NADPH is formed
in the reaction and can be measured at 340 nm.

hexokinase
Reaction 2.3 glucose + ATP Mg
  G6P + ADP
( II )

G6P
+
Reaction 2.4 G6P + NADP dehyrogense
 6-phosphogluconate + NADPH + H

Glycosylated Hemoglobin (Hemoglobin A1C): When the blood glucose level is high over
a period of time, hemoglobin becomes glycosylated. The glucose molecule binds
covalently to the terminal valine group of the beta chain. Once the hemoglobin molecule
has become glycosylated, it remains so over the life of the red cell (approximately 120
days). The quantitation of this glycosylated hemoglobin is used as measure of the overall
“control” of the diabetic over a period of several months. A blood glucose level only tells
the physician the patient’s glucose level at the time the specimen was obtained.
Glycosylated hemoglobin determinations are useful in determining whether the diabetic
has kept his blood glucose at a reasonable level during the preceding three to four
months.