Peroxidase-Coupled Glucose Method

Excerpt

Glucose is the most abundant carbohydrate in peripheral circulation, and blood glucose is the most common analysis done in the clinical laboratory. The body actively maintains blood glucose levels between 80 to 120 mg/dl through metabolic processes such as gluconeogenesis, glycogenolysis, glycolysis, and glycogenesis. Gluconeogenesis is an anabolic pathway that synthesizes glucose from amino acids and other molecules, while glycogenolysis is a catabolic pathway involved in the breakdown of glycogen into glucose for energy use.

These pathways are activated when the body signals an increased demand for glucose and are mediated mainly by the hormone glucagon. Conversely, glycolysis is a catabolic process that breaks down glucose, and glycogenesis is an anabolic process involved in the buildup of glycogen from glucose molecules. These pathways are activated when the body needs to reduce blood glucose levels, such as after a heavy meal, and are mediated mainly by the hormone insulin. When the body's regulatory hormones fail to maintain glucose levels, high blood glucose levels (hyperglycemia) and low blood glucose levels (hypoglycemia) can occur.

In metabolic conditions such as diabetes mellitus, blood glucose is no longer maintained within normal limits, and thus patients need medications for glycemic control. The prevalence of diabetes in the U.S. is estimated to be 22.3 million adults, with over 90% of cases attributed to type II diabetes. Diabetes is a significant cause of morbidity and mortality worldwide; however, hyperglycemia and hypoglycemia are seen in several other medical conditions. Accurate measurements of blood glucose are essential in the screening, diagnosing, and monitoring patients with a wide variety of metabolic conditions.

Glucose oxidase is one of the most widely used enzymes for glucose detection because of its ability to reduce oxygen to hydrogen peroxide (H2O2) while also oxidizing glucose to gluconic acid. At neutral pH, glucose exists in two forms, α-D-glucose and β-D-glucose. As the β-D-glucose is consumed in the reaction, the equilibrium of the two isomers of glucose is maintained by shifting production towards β-D-glucose, which allows glucose oxidase to act on all the glucose in the solution.

One way of measuring blood glucose in the laboratory is by using the glucose oxidase-peroxidase (GOD-POD) method. The principle of the GOD-POD reaction is as follows: glucose is oxidized to gluconic acid while oxygen is simultaneously reduced to hydrogen peroxide by the enzyme glucose oxidase. Hydrogen peroxide is then split to form water and nascent oxygen by the enzyme peroxidase. That nascent oxygen reacts with 4-aminoantipyrine, and in the presence of phenol, this reaction produces quinoneimine, which is a colored compound that can be analyzed using colorimetric analysis. The intensity of the color produced correlates directly to the concentration of glucose in the sample. The colorimetric analysis is performed at 505 nm and compared to the standard, which is treated similarly.

Glucose + 02+ H2O >>>>>>>>>>>>>>>>>>>> Gluconic acid + H2O2

Glucose Oxidase (GOD)

H2O2 >>>>>>>>>>>>>>>>>>>> H2O + [O]

Peroxidase (POD)

[O] + 4-amino-antipyrine + phenol >>>>>> Quinoneimine + H2O