Quantitative Determination of Glucose in Biochemistry

Glucose Overview

• Glucose is a major source of energy for most cells.
• Insulin facilitates glucose entry into cells.
• Diabetes (hyperglycemia) results from an inability of insulin to be produced or secreted.

Types of Diabetes

• Insulin-dependent diabetes mellitus (IDDM)
• Non-Insulin-dependent diabetes mellitus (NIDDM)
• Gestational diabetes
• Diabetes insipidus

Glucose Oxidase (GOD) Principle

• GOD catalyzes the oxidation of glucose to gluconic acid.
• Hydrogen peroxide (H2O2) is detected by a chromogenic oxygen acceptor, phenol, 4-aminophenazone (4-AP) in the presence of peroxidase (POD).
• The intensity of the color formed is proportional to glucose concentration in the sample.

Assay Conditions

• Wavelength: 546nm (490-550)
• Cuvette: 1cm light path
• Temperature: 37ºC or 15-25ºC
• Adjust the instrument to zero with reagent blank

Procedure

• Pipette into a cuvette:
  • Blank: 1.0 ml R2
  • Standard: 1.0 ml R2, 10 µl standard
  • Sample: 1.0 ml R2, 10 µl sample
• Mix and incubate for 10 min at 37ºC or 30 min at room temperature (15-25ºC)
• Read the absorbance (A) of the samples and standard, against the blank

Calculation

• Concentration of glucose (mg/dl) = A Sample / A Standard * 100 (standard conc.)
• 1 mg/dl = 0.0555 mmol/L

Expected Value

• Plasma, serum: 60-110 mg/dl (~ 3.33-6.1 mmol/L)

Clinical Significance

• Causes of hyperglycemia:
  • Pancreatitis
  • Diabetes mellitus
  • Obesity
  • Acromegaly
  • Hyperthyroidism
  • Cushing's syndrome
  • Administration of certain drugs
• Causes of hypoglycemia:
  • Insulinemia
  • Addison's disease
  • Galactosemia
  • Alcohol ingestion
  • Severe liver disease

Dental Treatment

• Maximum acceptable levels of blood glucose for removal of teeth in diabetics:
  • 180 mg/dl (before meal)
  • 234 mg/dl (2 h after a meal)
• High blood glucose levels reduce nitric oxide secretion, leading to poor circulation and slow-healing sockets.