VCP 511: Diagnostic Laboratory Section

Blood urea nitrogen

METHOD OF ESTIMATION (UREASE - GLDH METHOD)

• Serum or plasma is choice of specimen; anticoagulant containing any nitrogen must be avoided. The anticoagulant choice is EDTA.
• Urea can be estimated by any of the following methods
  • Chromatographic technique.
  • Absorbance spectrophotometry.
  • Dipstick Method
• In absorbance spectrophotometry urea is estimated by Urease-GLDH method

Principle

• Enzymatic determination of urea is done according to the following reactions:

Reagent composition

• UREA U.V (S.L) R1
  • Tris buffer, (pH 7.60) - 100 mmol/L
  • ADP - 0.7 mmol/L
  • a -Ketoglutarate - 9.0 mmol/L
  • Urease  > 6500 U/L
  • GLDH  > 1100 U/L
• UREA U.V (S.L) R2
  • NADH - 0.25 mmol/L
  • 2-Oxoglutorate - 5 mmol/L

Laboratory procedure

	Standard	Sample
Working reagent	1000 µL	1000 µL
Standard	10  µL	-
Sample	-	10 µL
Mix and read the optical density (T1) 30 second after the sample or standard addition. Take second reading (T2) exactly 60 sec. after the first reading.

Calculation

Urea standard concentration is 50 mg/dl