A Simple Method for the Determination of Blood

Urea Nitrogen, with Special Reference to

Automatic Colorimetric Analysis

Helmut J. Richter and Yvette S. Lapointe

RECOMMENDATIONS for automatic colorimetric analyses for the deter-

mination of blood urea nitrogen (B.IJ.N.) are based upon the work of
L. T. Skeggs (1) and W. H. Marsh, (2) a modification of the car-
bamido-diacetyl reaction. B.U.N. determinations are routinely per-
formed automatically in this laboratory; however, for emergency
specimens, when the instrument is set up for another test, for night
staff work, and also in case of instrument repair, we had to develop a
“manual” method for the determination that would correspond as
closely as possible to the automatic procedure and if possible not
require a separate set of reagents. We also wished to leave our pro-
cedure for the preparation of protein-free filtrates (modified Folin-
Wu) unchanged in respect to other tests. As an initial step in devel-
oping this procedure, we measured the amounts of reagents the
automatic procedure required for one cycle, and performed separate
nitrogen determinations on the solution from the discard side of the
dialyzer and the dialyzed solution with a set of urea standards, 10 to
150 mg./100 ml. urea nitrogen. We found that on the average 20%
of the urea in the sample is being dialyzed. With this data on hand
we set up our manual B.U.N. method as follows.
REAGENTS
Diacetyl Monoxime Solution. Diacetyl monoxime,t 10 Gm., is dis-
Prom the Dept. of Biochemistry, Ottawa Civic Hospital, Ottawa, Ontario, Canada.
The authors wish to acknowledge the interest of Dr. M. 0. Klotz, Chief Pathologist, under
whose direction this work was made possible.
Received for publication April 4, 1959.
Auto-Analyzer, Technicon Instruments Corp., Chauncey, N. Y.
tEastman Organic Chemicals.

617
618 RICHTER & I.APOINTE Clinical Chemistry

solved in a two liter volumetric flask in approximately 1000 ml. dis-

tilled water. Sodium chloride, 300 Gm., is added with more distilled
water. The solution is made up to volume, filtered, and stored in an
amber glass bottle.
Sulfuric Arsenic Pent oxide Solution. In a large Erlenmeyer flask
are placed 1100 ml. distilled water and 226 Gm. arsenic pentoxide.
With constant stirring, 1770 Gm. concentrated sulfuric acid is intro-
duced at a rate that will provide for maximum development of heat
without causing violent boiling. The mixture is stirred until all the
arsenic pentoxide is dissolved. The solution is allowed to cool to room
temperature, and filtered through a fritted glass funnel.

PROCEDURE
1. Protein-free filtrates:
Unknown Blank
14 ml. distilled water 16 ml. distilled water
2 ml. oxalated blood 2 ml. 10% sodium tungstate sol.
2 ml. 10% sodium tungstate sol. 2 ml. 2/3 N sulfuric acid sol.
2 ml. 2/3 N sulfuric acid sol.
2. In 60 ml. pyrex glass bottles with ground glass stoppers are
placed 2 ml. of the protein-free ifitrate from the Unknown, and 2 ml.
from the Blank ifitrate for the reagent Blank bottle.
3. To each bottle is added 8.5 ml. of distilled water and the solution
is well mixed.
4. With constant rotating of the bottle, 8.5 ml. of diacetyl mo-
noxime solution is added. The contents are thoroughly mixed.
5. Again with constant rotating of the bottles, 8.5 ml. of arsenic
sulfuric acid solution is added and thoroughly mixed.
6. The stoppered bottles are placed in a constant temperature bath
of 100#{176}
for exactly 20 minutes.
7. The bottles are then allowed to gradually cool in ambient air
for at least 15 minutes, and then further cooled in a 25#{176} water bath
for at least another 15 minutes. The bottles are kept away from
direct light during the heating and the cooling periods.
8. The absorbance of the yellow solution is read on a spectro-
photometer, against the reagent Blank at 475 m. (We use a Cole-
man Universal spectrophotometer, 13 mm. square cells).
9. The results are read in milligrams per 100 milliliters B.U.N.
from a graph which has been obtained by running a series of stand-
ards containing 10 to 60 mg./100 ml. urea nitrogen through the above
Vol. 5, No. 6, 1959 DETERMINATION OF BLOOD UREA NITROGEN 619

procedure. If the color is too intense, for high values, the yellow
solution can be diluted with distilled water and the result be corre-
spondingly corrected with a dilution factor.
It is important that the solution in the 60 ml. bottle is well mixed
after the addition of each reagent, and that the temperature of the
heating bath be constant and the time of heating strictly adhered to.
Using test tubes or centrifuge tubes instead of Pyrex bottles is not
advisable because thorough mixing is then difficult and equal heating
along the length of the tube is not guaranteed. If a brownish-red
turbidity appears in the bottles during or after heating, the yellow.
colored substance is decomposed and the spectrophotometric values
are invalid. This turbidity appears when the hot bottles are being
cooled down too quickly, and also occasionally in cases of exceeding-
ly high concentrations of B.TJ.N. In the latter case the analysis is
repeated with less filtrate, adding distilled water* to make up to the
original volume, and the dilution factor is noted. The yellow color
is stable for one hour if kept away from direct light.

RESULTS
Table 1 shows the results of 30 consecutive determinations of
B.U.N. done automatically on plasma and by the manual method on

*using Blank filtrate to make up to the original volume of 2 ml. instead of distilled water

does not affect the final result.

Table 1. B.1J.N. DETERMINATIONS (COMPARISON OF THE AUTOMATIC AND MANUAL MErwoD)

Automatic ‘ManuaZ” method Automalic ‘ManuaZ” method

B.U.N. B.U3. B.t7.N. B.uJ,r.
Specimen No. (mg./100 ml.) (mg./100 ml.) Specimen No. (m g./IOO ml.) (mgJlOO ml.)

1 18 18 16 25 25
2 30 31 17 15 18
3 20 19 18 31 35
4 8 7 19 64 63
5 45 43 20 23 24
6 12 12 21 18 19
7 31 30 29 27 25
8 29 28 23 19 18
9 74 76 24 15 13
10 10 9 25 22 24
11 10 10 26 13 12
12 21 18.5 27 35 37.5
13 14 14 28 10 8.5
14 51 53 Hyland standard 12 11.5
15 50 54 Lab-Trot standard 13.5 14
620 RICHTER & LAPOINTE Clinical Chemistry

Table 2. Raoovzav TESTS or Uana ADDED TO BlooD SPECIMENS (MANUAL METHOD)

Urea nitrogen Urea nitrogen Total urea

in blood specimen added nitrogen found
Specimen no. (nig./100 rat.) (mg./100 ml.) (ssg./100 ml.) Recovery %

1 7 100 108 100.9

2 9 50 58.5 99.0
3 10 25 36 102.8
4 18 20 36.5 96.1
5 14 40 54.5 100.9
6 11 5 16 100
7 10.5 10 21 102.4
8 11 60 70 98.6
9 8 4.8 13 101.6
10 8 7 15 100
11 8 12 20.7 103.5
12 8 46.5 53.5 98.2

whole blood. Table 2 shows recovery tests of urea added to the blood
specimens, determined by the “manual” method, expressed as B.U.N.

SUMMARY
The accepted automatic procedure for the determination of blood
urea nitrogen has been developed into a “Manual” method, useful to
users and nonusers of the instrument. This procedure is also very
suitable for the determination of urea nitrogen in small specimens.
REFERENCES
1. Skeggs, L. T., Am. J. Clin. Pathol. 28, 311 (1957).
2. Marsh, W. H., Fingerhut, B., and Kirsch, E., Ass. J. GUn. PaUzol. 28, 681 (1957).