The document outlines various methods for estimating blood urea nitrogen (BUN) and creatinine levels, highlighting key techniques such as the diacetylmonoxime method and the Berthelot reaction for urea estimation. It also describes the Jaffe reaction for creatinine, detailing both manual and automated approaches for measurement. Each method is characterized by specific reactions, conditions, and detection wavelengths that are critical for accurate assessment.

1. METHODS FOR DETECTION OF BLOOD
UREA NITROGEN & CREATININE
Karthik G Kamath K
Associate Professor
Department of Biochemistry

2. DETERMINATION OF UREA (BUN)
Urea is reported as urea / BUN as mg/dl
Mol.wt of urea = 60, mol.wt of urea nitrogen = 28
Urea = 2.14 x urea nitrogen

3. METHODS FOR UREA (BUN) ESTIMATION
Diacetylmonoxime (DAM) method
Berthelot reaction method
Rate of reaction method

4. ESTIMATION OF UREA USING
DIACETYLMONOXIME METHOD
Urea reacts with DAM in hot acidic medium
In the presence of thiosemicarbazide and ferric ions
To form pink colored compound
Measured photometrically on green filter / 520 nm

5. BERTHELOT REACTION METHOD
Urease splits urea into ammonia and CO2
Ammonia reacts with phenol in presence of hypochlorite and form indophenol
Indophenol with alkali gives a blue-colored complex
Intensity is read at 546nm / green filter
Color is stable for 12 hrs

6. RATE OF REACTION METHOD
(UREASE GLDH METHOD)
Urease hydrolyses urea into ammonia and CO2
NADH is oxidized to NAD+
in the presence of ammonia, α-ketoglutarate, and
glutamate dehydrogenase (GLDH)
The rate of decrease in OD is measured every 30 sec at 340 nm
Which is directly proportional to urea concentration

7. RATE OF REACTION METHOD
(UREASE GLDH METHOD)
Prewarm working solution (GLDH, -
α ketoglutarate, urease, & NADH + diluent) to
room temp
Add 1ml of working reagent into a cuvette
Add 0.01ml serum, mix and note the change in OD/min (▲A ‘T’) after delay time for
3 mins
Add 1 ml of working reagent + 0.01 ml of urea-nitrogen standard and mix well
note the change in OD/min (▲Std) after delay time for 3 mins
▲AT x 20/▲Std

8. METHODS FOR CREATININE ESTIMATION
Based on Jaffe’s method – manual & automated method

9. ESTIMATION OF CREATININE USING JAFFE’S
REACTION
Creatinine reacts with alkaline picrate to form red orange colored product
Measured at 520 nm / green filter
Automated Method
Carried out at 30°C
First reading at 20 sec (non creatinine chromogens reacts with alkaline picrate fast)
Creatinine reacts with alkaline picrate 20-80sec. Second reading at 80sec
Time interval eliminates protein interference
Same procedure for standard
OD of T (80s – 20s) x conc. of std/ OD of S (80s -20s)