Different Enzymatic Methods for Glucose

Glucose is measured in whole blood, plasma, serum or cerebrospinal fluid (CSF). Historically, blood glucose values were given in terms of whole blood, but most laboratories now measure and report plasma or serum glucose levels. Because red blood cells (erythrocytes) have a higher concentration of protein (e.g., hemoglobin) than serum, serum has a higher water content and consequently more dissolved glucose than does whole blood. Current procedures that measure glucose use enzymatic methodology.

Enzymatic Reactions Method for Glucose

Three enzymatic approaches to the measurement of glucose have been explored. Enzymatic methods are specific for glucose.

1. The hexokinase method is the reference method for glucose. The method involves two coupled reactions:

[Glucose + ATP] ------hexokinase, Mg2+------- [G6PO4 + ADP]

[G6PO4 + NADP+] -------G6PD------- [6-phosphogluconate + NADPH+ H+]

The increase in absorbance of NADPH at 340 nm is measured as directly proportional to glucose. The hexokinase reaction may also be coupled to an indicator reaction and measured through the development of a colored product.

2. The glucose oxidase method is specific for beta-D-glucose. The initial reaction:

[β-D-Glucose + O2] ------glucose oxidase------[gluconic acid + H2O2]

may be coupled with a peroxidase indicator reaction:

[H2O2 + reduced chromagen] ------peroxidase------ [oxidized chromogen + H2O]

or may be assessed by measuring oxygen consumption, using an oxygen electrode.

3. The glucose dehydrogenase method involves the measurement of NADH production:

Glucose + NAD+ --------------------------D-gluconolactone + NADH + H+

Acceptable Specimens:

Glucose in serum, plasma, and cerebrospinal fluid (CSF) may be measured by these methods. Glucose levels in serum or plasma are 10% to 15% higher than those in whole blood. Serum or plasma must be separated within 1 hour to prevent degradation by glycolysis. Glucose is stable for 24 hours in whole blood when preserved with sodium fluoride.

Reference Ranges:
Serum or plasma (fasting)= 74–100 mg/dL
CSF=  60% of serum or plasma level