Which method is used as the reference method for enzymatic glucose measurement because it does not use hexokinase?

• A. Hexokinase method
• B. Glucose oxidase method
• C. Glucose-6-phosphate dehydrogenase method
• D. Diaphorase method

Answer: (C)

The main point is understanding what makes a glucose assay a reference method: it must be highly specific for glucose and provide a highly accurate, traceable result with minimal interference. The standard reference enzymatic approach achieves this by using two enzymes in sequence. First, hexokinase converts glucose to glucose-6-phosphate using ATP, effectively trapping glucose in a form that is specific to glucose and reducing interference from other substances present in blood. Then glucose-6-phosphate is oxidized by glucose-6-phosphate dehydrogenase, generating NADPH from NADP+. The amount of NADPH produced is proportional to the glucose concentration and is measured spectrophotometrically, providing a precise and reliable result.

If a method is said to avoid hexokinase altogether, it would have to rely on a different enzymatic reaction, such as glucose oxidase, which converts glucose to gluconic acid and hydrogen peroxide. While glucose oxidase methods are common in routine testing, they are not considered the reference because they can be more susceptible to interference from sample oxygen levels and other factors, leading to less consistent results in some situations. The glucose-6-phosphate dehydrogenase approach, in fact, would still require glucose to be converted to glucose-6-phosphate at the start, which typically involves hexokinase, so it does not truly provide a hexokinase-free reference.

In short, the recognized reference method for enzymatic glucose measurement is the hexokinase-based method, chosen for its superior specificity and reliability.