Name___________________________ Date___________________
Activity
Blood Glucose Determination
Glucose is produced in the body by the breakdown of starch to hexoses (glucose, fructose, and
galactose) by enzymes in the salivary glands, pancreas, and intestines. Although most monosaccharides
are absorbed in the small intestines, some will pass to the portal circulation and store in the liver. In the
liver, glucose is stored in glycogen through the process known as Glycogenesis.
Objectives: at the end of the activity, the learner can:
1. Explain the clinical significance of carbohydrate metabolism
2. Describe the principles of glucose determination
3. State the average blood glucose values
Laboratory Methods:
There are three general types of methods used for the determination of blood glucose:
1. oxidation methods
2. aromatic amine methods
3. enzymatic methods
In general, the oxidation methods give slightly higher results 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. Example:
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 reduce 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 such as glutathione, uric acid, ascorbic acid, and creatinine will also
react in these oxidation methods. These interfering substances may be removed from the sample by
selecting the protein-free filtrate upon which the analysis is carried out.
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 condensing
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 oxidation tests and yield results closer to 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)
Reaction 2.1 glucose + O2 gluconic acid + H2O2
The amount of peroxide formed is measured in one of several ways. First, 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).
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.
Reaction 2.3 glucose + ATP G6P + ADP
Reaction 2.4 G6P + NADP 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 to
measure the overall "control" of the person with diabetes over several months. Glycosylated
hemoglobin determinations help determine whether the diabetic has kept his blood glucose at a
reasonable level during the preceding three to four months.
SPECIMEN COLLECTION
1. Either serum or plasma may be used.
2. Plasma or serum samples without preservatives should be separated from the cells or clot within a
half-hour of being drawn.
3. Glucose in separated unhemolyzed serum is generally stable up to eight hours at 25°C and up to 72
hours at 4°C.5
4. Glycolysis can be inhibited by collecting the specimen in sodium fluoride. Glucose in a sodium
fluoride-oxalate mixture is reported to be stable up to 24 hours at 25°C.5
MATERIALS PROVIDED
1. Glucose reagent
2. Glucose standard (100 mg/dl).
MATERIALS REQUIRED BUT NOT PROVIDED
1. Accurate pipetting devices.
2. Timer.
3. Test tubes and rack.
4. Spectrophotometer capable of reading at 340 nm.
5. Heating block or water bath (37°C).
PROCEDURE (AUTOMATED)
Refer to specific instrument application instructions.
PROCEDURE (MANUAL)
1. Appropriately label tubes: reagent blank, standard, control, sample, etc.
2. Pipette 1.0 ml of Reagent into all tubes.
3. Add 10 uL of sample to respective tubes. Mix well. Incubate all tubes at 37°C for five (5) minutes.
4. After incubation, zero the spectrophotometer with the reagent blank at 340 nm.
5. Read and record the absorbance of all tubes.
6. Interpret the result
Calculation:
C = 100 x A sample mg/dL
A standard
Questions:
1. What is hyperglycemia?, hypoglycemia?
_____________________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
_______________________________________________________________________
2. Enumerate and describe the hormones that regulate blood sugar levels
_____________________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
_______________________________________________________________________
3. Compare the types of Diabetes mellitus
_____________________________________________________________________________________
______________________________________________________________________________
_______________________________________________________________________
______________________________________________________________________________
