Principle of Measurement

1. Urease–GLDH Method (Enzymatic UV)

(Most widely used, highly specific)

How it works:

1. Urease enzyme hydrolyzes urea → ammonia + carbon dioxide.

2. Ammonia reacts with α-ketoglutarate in the presence of GLDH (glutamate dehydrogenase).

3. NADH is oxidized to NAD⁺, causing a decrease in absorbance at 340 nm.

4. The rate of absorbance decrease is proportional to urea concentration.

Advantages:

• Very specific

• Minimal interference

• Ideal for automated chemistry analyzers

2. Berthelot (Indophenol) Colorimetric Method

(Common in semi-auto analyzers)

How it works:

1. Urease converts urea to ammonia.

2. Ammonia reacts with phenol and hypochlorite → blue indophenol dye.

3. Absorbance measured at 580–600 nm.

Advantages:

• Stable color formation

• Suitable for manual and semi-automatic systems

3. Diacetyl Monoxime (DAM) Method (Older method)

Forms a yellow complex; used mainly in research.

Reagent Components

Urease–GLDH Reagents May Contain:

• Urease enzyme

• GLDH enzyme

• NADH

• α-ketoglutarate

• Buffer solution

• Surfactants and stabilizers

• Preservatives

Berthelot Reagents May Contain:

• Phenol

• Sodium nitroprusside

• Hypochlorite

• Buffer solution

Packaging Formats

• Two-reagent systems (R1 buffer + R2 enzyme)

• Single-reagent kits (less common)

• Analyzer-specific liquid cartridges

Typical volumes: 25 ml, 50 ml, 100 ml, 250 ml.