Answer to Question #124985 in Biochemistry for K Ganesh

Question

In a procedure for testing blood glucose, a drop of blood is placed on a paper strip impregnated with the enzyme glucose oxidase and all the reagents necessary for the reaction.
The H2O2 produced causes a color change on the paper that indicates how much glucose is present. Since glucose oxidase is specific for the beta anomer of glucose and doesn’t act on alpha anomer, in spite of this specificity, the reaction is used in clinical assay for measuring total blood glucose which consists of a mixture of beta D glucose and alpha D glucose. Explain how this assay

Answer 1

Glucose is the most important carbohydrate fuel in the body. In the fed state, the majority of circulating glucose comes from the diet. In the fasting state, gluconeogenesis and glycogenolysis maintain glucose concentrations. Very little glucose is found in the diet as glucose. Most is found in more complex carbohydrates that are broken down to monosaccharides though the digestive process. About half of the total carbohydrates in the diet are in the form of polysaccharides and the remainder as simpler sugars.

The concentration of glucose in the blood is maintained at a relatively stable concentration from 80 to 120 mg/dl. The strong reducing properties of glucose makes it relatively easy to measure and thus the clinical estimation of circulating glucose was one of the earliest tests available to the clinician.

Glucose oxidase and reagents to measure the generation of hydrogen peroxide can be bonded to filter paper and the system used to measure glucose concentrations in a drop of capillary blood. This has resulted in the most important change in diabetes management since the introduction of insulin.

During measurement of glucose, a drop of whole capillary blood is placed on the reagent bonded to the paper strip. Instead of using a known volume of blood, an excess of blood is exposed to a fixed quantity of glucose oxidase for a finite period of time to estimate concentration. After the specified time, usually 1 minute, the excess blood is removed by washing or wiping and the color is allowed to develop. The concentration is then estimated by comparing to a color chart, or by using a portable reflectance meter specific to the reagent strip, to measure the developed color. 

The principle behind this process is that the aldehyde group of glucose can undergo condensation with aromatic compounds to yield a colored product. In the most commonly used condensation reaction, o-toluidine reacts with glucose to form a glucosamine that has an intense green color. The color is then measured spectrophotometrically to estimate the glucose concentration. The reaction is rapid, and the intense color allows a high degree of sensitivity. Other aldoses can cross react, but only mannose and galactose give a highly colored product. These sugars are not found in great concentrations in the blood and their cross reactivity is ordinarily not significant. o-Toluidirie has the drawback of being highly corrosive and toxic. For this reason, this method is rapidly being phased out of the clinical laboratory.

The enzyme glucose oxidase reacts with glucose, water, and oxygen to form gluconic acid and hydrogen peroxide. The hydrogen peroxide is then used to oxidize a chromogen or the consumption of oxygen measured to estimate the amount of glucose present. Glucose oxidase is specific for β-d-glucose, so cross reaction with other sugars is not a problem. In aqueous solution, approximately $66$ percent of d-glucose is in the β state and $34$ percent exists as α- d-glucose.