EXPERIMENT 14
MEASUREMENT OF UREA NITROGEN IN SERUM
(DIACETYL MONOXIME METHOD)
AIM AND REQUEST
1. Comprehend the method for measurement of urea nitrogen in serum and its
clinical value.
2. Review the bio-synthesis of urea and its significance.
PRINCIPLE
Urea reacts with diacetyl monoxime under strong acidic conditions catalyzed by
ferric ions, and condense to a red product with the existing of thiosemicarbazide. The
intense red colour formed is directly measured to calculate the urea content in serum
or plasma with the omitting of removing plasma protein. The reaction formulas are
described as bellow.
N
H3C
O
H3C
N OH
CH3
+
+
H2N
NH
3+
H , Fe
H2N
O
O
H3C
N
H2O
(diacetyl monoxime)
H3C
H3C
N
N
OH
N
red colour compound
Thiosemicarbazide intensify the reaction and stabilize the red product to light.
PROCEDURE
Prepare three 13 x 100 mm tubes and labell them. Add reagents as described
in the table.
1
Reagents (mL)
Serum
Working urea
standard solution
Colour reagent
control
0
0
standard
0
0.1
6.1
6.0
sample
0.1
0
6.0
Mix all tubes well. Keep them in a boiling waterbath for 10 minutes. Remove
from waterbath and cool the tubes for 5 minutes. Set the spectrophotometer to zero with
control at 520nm and measure the absorbance (A) of the other tubes. Measurement
should be taken within 30 minutes since the red color will fade after 2 hours.
CALCULATION
Aspecimen
Urea concentration (mmol/L) = ────── ×5.0
Astandard
REFERENCE RANGE
2.85 mmol/L ～ 8.2 mmol/L
CLINICAL INTERPRETATION
Elevated serum urea levels may be due to renal failure, acute upper
gastroinEXPERIMENTinal hemorrhage, sustained hyperpyrexia, etc.
Decreased serum urea levels could be due to pregnancy, intravenous infusion, low
antidiuretic hormone secretion, low protein intake, severe liver diseases, inborn
errors of urea cycle and SIADH (Syndrome of inappropriate ADH secretion).
REAGENTS
1. Colour reagent
Solution Ⅰ: Add slowly 50 ml of Conc. H2S04 (Analar grade, AR) and 50 mL
orthophosphoric acid (H3PO4) to 800 ml distilled water and mix. Then add 0.05 mL
100g/L ferric chloride hexahydrate (FeCl3·6H2O) and make the volume up to 1000
mL in a volumetric flask.
Solution Ⅱ: Thiosemicarbazide 0.4 g and diacetyl monoxime 2.0 g dissolved
in distilled water and make up to 100 mL.
Mix solution Ⅰand solution Ⅱ (1:1) and store in a brown bottle at 40C.
2. Stock urea standard solution
Weigh 3.0 g of analytical-grade dry urea and dissolve it with distilled water, make
the volume up to 1000 ml in a volumetric flask..
3. Working urea standard solution
2
Dilute 10 ml of stock urea standard to 100 ml with distilled water. The concentration
of this standard solution is 5.0 mmol/L.
METHOD EVALUATION
1.There are several methods for urea test, the method described here and urease
method, however, are more commonly used. Urease method is a good method with
high specificity and high precision, but it is difficult to popularize because of its
complicated procedure and time-consuming. This method is a relatively simple and
precise method for clinical application.
2. Although urea test and non-protein nitrogen (NPN) test have almost the same
clinical significance, NPN test has been gradually substituted by urea test in clinic
since the later is more simple.
3