Quantitative Analysis - Enzymatic Method

Glucose Oxidase

• Saifer Gernstenfield

• Coupled enzyme reaction (Trinder’s Reaction) – Colorimetric

• Measures B-D-Glucose

  • Indirect Measurements of glucose, based with hydrogen peroxide na na proproduce.

  • Kun damot na proproduce na hydrogen peroxide, damo gihapon it glucose level.

• For urine and whole blood glucose rapid reagent strip testing

• Also for automated methods for plasma and serum

• Interference by: Uric Acid, Bilirubin, Ascorbic Acid

• Increased Uric Acid, Ascorbic Acid, Bilirubin, Tetracycline, Hemoglobin and Glutathione 🡪 Falsely Low Glucose Result

ENZYMES:

• Glucose oxidase

• Peroxidase

Glucose oxidase - oxygen consumption

• Polarographic glucose oxidase method

• O2 consumption is determined by O2 electrode

• Less common than hexokinase method. Commonly used for glucose meter testing

• Accurate and precise method, virtually no interferences

ENZYME:

• Glucose Oxidase

Hexokinase Method

• Most specific and reference method

• Based on formation of NADH followed by increase in absorbance at 340nm (directly proportional to glucose concentration)

• Falsely Low: Gross hemolysis (inhibits peroxidase), extremely increased Bilirubin

• Not affected by Ascorbic Acid and Uric Acid

• Specific glucose method which employs G6PD as a second coupling step requiring Magnesium

ENZYMES

• Hexokinase

• G6PD

For example

How do it measure glucose?

• This is your tube, it contains serum, glucose molecules, tapos mayda kita light source, once this light passes through the tube, it ig meameasure iton intensity han light na naka pass through han tube this detector.

• This detector will measure the glucose level.

• Wavelenthg selector will just select the specific wavelength of glucose.

Specimen Handling

• Standard clinical specimen: VENOUS PLASMA GLUCOSE

• For daily monitoring: CAPILLARY BLOOD

• Separate plasma from cell WITHIN 1 HOUR

Random Blood Sugar

• Requested during insulin shock and hyperglycemic ketonic coma

• Normal: <140 mg/dL

Fasting Blood Sugar

• Fasting blood glucose should be obtained in the morning after an approximately 8 – 12 hours fast (not longer than 16 hours)

• Reference Values: 70 – 100 mg/dl

Point of care testing

• For daily monitoring, specimen is capillary blood

• Strips impregnated with glucose oxidase and peroxidase

• Principle: Color change read by Reflectance Photometry

2hr post prandial blood sugar

• Glucose load of 75g (adults) or 1.75g/kg (children) of glucose is administered and a specimen is drawn 2 hours later.

• Normal value: <140 mg/dL

• Diabetic: >180 mg/dL

glycosylated hemoglobin

• Index for long term plasma glucose control 2 – 3 months period, indicating compliance and efficacy of Diabetes Mellitus therapy