Determination of Urea in Blood and Urine

with Diacetyl Monoxime-Glucuronolactone

Reagent

Tsufomu Momose, Yosuke Ohkura, and Jun Tomita

A simple method for determination of urea in blood and urine with diacetyl monoxime-
glucuronolactone reagent is presented. The stability of the color produced permits
analysis of a large number of samples at one time. Deproteinization of blood is accom-
plished with sodium tungstate and alum.

N UMEROUS STUDIES have been reported in the literature on the photo-

metric determination of urea in biological fluids with diacetyl monoxime
as the color-developing agent. The reaction is photosensitive, does not
follow Beer’s law, and requires special precautions for reproducible re-
sults. In many proposed procedures, oxidants were added in the reaction
mixture to destroy hydroxylamine which might be generated during the
reaction by the strong acidity, and decrease the produced color. The
oxidants employed have included persulfuric acid (5, 8, 12, 17), arsenic
acid (1,2,6,10,19,20), perchioric acid (ii), chloramine T (9), and ferric
ammonium sulfate (18). Substances other than oxidants have also been
added to the reaction mixture: phenylanthranilic acid (21) and phena-
zone (3) to intensify the color and, more recently, thiosemicarbazide (4)
to minimize the photosensitivity of the reaction.
n-Glucuronolactone or n-glucuronic amide was found, in the authors’
laboratory, to produce an intense and photostable color when added to
the reaction mixture. A simple, accul-ate, and reproducible method for
determination of urea ill blood and urine was then developed utilizing
this reaction.

From the Departmnemst of Analytical (‘lmemsiistry, Faculty of Pim:mrmmm:mceutieal Sciences, Kyuslmu

University, Fukuoka, Japan.
The authors express their gratitude to Dr. Junji Nagai for the supply of biological samples,
and to the Research Institute of Cliugai Seiyaku Kai,mmslsiki Knisha, Nihoimbashi Tionelso, Chuo-Kui,
Tokyo, for time supply of glucurommolaetone.
Received for publication May 19, 1964; accepted for publication July 10, 1964.

113
114 MOMOSE ET AL. Clinical Chemistry

Experimental
Reagents
1. Sodium tungskute solution Dissolve 10.0 gm. Na0\V04 . 2110 in
water and dilute to 100 ml.
2. Alum solution 1)issolve 9.6 gm. KA1(S04)0. 121120 in water and
dilute to 100 ml.
3. Diacetyl monoxime-giucuronolactone reagent Dissolve succes-
sively 5.0 gm. diacetyl monoxime, 5.0 gm. n-glucuronolactone, and 25 ml.
glacial acetic acid in water and dilute to 500 ml. Store in a brown bottle.
This solution is stable at room temperature for at least 3 months.
4. P/u ospluoric acid solution 1)ilute 600 ml. phosphoric acid (85-
90%) with water to 1 L.

Procedure for Blood or Serum

Dilute 0.1 ml. of blood or serum with 2.9 ml. of water in a test tube, add
0.5 ml. sodium tungstate solution, and mix. Add 0.5 ml. of alum solution
to the mixture and mix well. Transfer to a centrifuge tube, cover with a
piece of aluminum foil, and centrifuge. Pipet 1.0 ml. of the clear super-
natant solution into a test tube, add successively 1.0 ml. of diacetyl mo-
lloxime-glucuronolactone reagent and 5.0 ml. of phosphoric acid solution,
and mix well. At the same time, prepare a reagent blank by addiiig the
color developing reagents to 1.0 ml. of water. Cover each tube with a
piece of aluminum foil.
Pack the test tubes in a heating basket (13), heat them in a boiling
water 1)ath for 40 mm., cool in running water, and shake each tube. I1eas-
ure the absorbance of the sample at 475 m against the reagent blank,
and read the value of urea nitrogen on the calibration curve, prepared as
described below.

Procedure for Urine

Dilute 1.0 ml. of urine with water to a filIal volume of 1 L. Treat 1.0 ml.
of this dilution in the same way as the deproteinized blood solution, and
measure the absorbance against a reagent blank.

Calibration Curve

Dissolve 429 mg. of dried urea in water and fllake up to I L. Dilute 100
ml. of this solution with water to make 1 L. The resulting solution con-
tains 2 mg. urea nitrogen per 100 ml. IJsing this solution, prepare dilu-
tions corresponding to 0.1, 0.2, 0.3, 0.5, 1 .0, and 1.5 mg. urea nitrogen per
100 nIl.
Pipet three 1.0-nIl. aliquots of each solution into test tubes, aiicl three
1.0-ml. aliquots of water for blanks. Add the color developing reagents
Vol. Il, No. 2, 1965 UREA DETERMINATION 115

to all tubes all(l develo1) color iii the sanl(’ way as for the deproteinized
1)100(1 solution. looi the 3 reageiit blanks aimd nieasure the absorbances of
the standar(ls against this i)ool(’(l blank.
A typical calibration curve is shown in Fig. 1. For blood, the urea nitro-

Urea nitrogen concentration

01 02 0.3 QS 1.0 t.c #{248}w4./ IOOMI.

0_s

I-

o6

I,

C)

0.2

Urea nitrogen value

Fig. 1. Calibration curve.

gen value, calculated in mg./100 ml.; is obtained by multiplying the mg./

100 ml. of diluted urea solution by 40. For urine, tile urea nitrogen value
(gm./100 nIl.) is the same as tile number of mg./l00 ml. of the dilute solu-
tiOll.

Absorption curves in this study were measured by a Shimazu auto-

matic recording spectrophotometer.* Absorbances were measured by a
Hitachi spectrophotometert with a cuvet of 10 mm. optical path length.

Results and Discussion

Diacetyl monoxirne gives an intense orange color with urea when
heated in the presence of glucuronolactone in moderately concentrated
Model SV-50A, Shimazu.
I Model EPU-2, Hitachi.
116 MOMOSE fT AL. Clinical Chemistry

phosphoric acid. Tile color is stable in daylight and has an absorption

maximum at 475 mt. In the absence of glucuronolactorie, however, the
developed color is photosensitive and has a maximum at 478 mj.. (ilu-
curonolactone, therefore, may play an important role ill the color forma-
tion. The absorption curves of the developed color and of a reagent
blank are shown in Fig. 2.
The concentration of diacetyl monoxime affected the color intensity,
and the described concentration, 1%, was selected to give maximum in-
tensity with a lower blank. The 1% concentration of glucuronolactone
was selected as the optimum for stabilization of color developed iii a wide
range of urea concentrations (0.2-2.0 mg./100 ml.).

08

0.6

1.

04

0.2

0
400 450 4’15 500

Wave length

Fig. 2. Absorption curves: Curve 1, urea standard (2 aug. urea nitrogen per 100 ml.) vs.
reagent blank; curve 2, reagent blank vs. water blank.
Vol. II, No. 2, 1965 UREA DETERMINATION 117

A moderately high phosphoric acid concentration gave the most in-

tense and stable color development. Other strong acids of various COIl-
centrations tested showed 110 advantage over phosphoric. Those studied
were sulfuric, hydrochloric, nitric, and perchloric. With the proposed
method, the color intensity increased rapidly with tile heating time for
the first 30 mm. and reached its maximum in 40.
The stability of this color is compared ill Fig. 3 with those developed
by diacetyl monoxime-thiosemicarbazide reagent (4) and diacetyl mo-
noxime-arsenic acid reagent (10). Urea nitrogen solution, 1 mg./100 ml.,
was developed and exposed to daylight. The first intensity of absorbance,
in each case, was taken arbitrarily as 100%. The proposed method is
practically unaffected by the daylight in a laboratory, permitting analy-
sis of a large number of samples at the same time without protection from
ordinary daylight. Direct sunlight, however, must be avoided.
Blood may be deproteinized by trichloroacetic acid or by a mixture of
‘I’
100

$0

60

40
4,
-4
-I
-4
,0
a
43
cc

20

0 20 50 $0

Time of Exposure am.

Fig. 3. Conuparison of color stability: Curve 1, present nsetliod; curve 2, diaectyl monoximmme-
timiosemicarbazide method; curve 3, diacetyl momuoxime-arsenic acid method.
118 MOMOSE fT AL. Clinical Chemistry

Table 1. REC OVERY OF ADDED UREA

Recome red

anij.te Iii it mt Added Quantity r/r

BLOOD (NO./100 ML.)

10 19.2 99
1 9.3 20 29.3 100
40 50.3 103
10 16.4 97
2 6.7 20 26.8 101
40 47.2 101
10 26.3 101
3 16.2 20 36.5 102
40 57.5 103
10 22.7 102
4 12.5 20 32.5 100
40 52.3 100
10 26.5 98
5 16.7 20 36.5 99
40 56.8 100

SERUM (No./100 ML.)

10 34.5 101
1 24.4 20 44.3 100
40 64.8 101
10 41.5 98
2 31.7 20 51.5 99
40 71.3 99
10 48.0 102
3 37.8 20 58.0 101
40 78.0 101

URINE (GM./100 ML.)

0.25 1.11 100

1 0.86 0.50 1.36 100
1.00 1.87 101
0.25 0.50 96
2 0.26 0.50 0.76 100
1.00 1.26 100
0.25 0.86 96
3 0.62 0.50 1.11 98
1.00 1.63 101
0.25 0.93 100
4 0.68 0.50 1.20 104
1.00 1.70 102
0.25 1.04 104
5 0.78 0.50 1.28 100
1.00 1.76 98
0.25 0.59 96
6 0.35 0.50 0.85 100
1.00 1.34 99
Vol. II, No. 2, 1965 UREA DETERMINATION 119

sodium tungstate and sulfuric acid (7), but the deproteinizing agent em-
ployed in the method presented is more convenient to prepare, store, and
use. This deproteinizing agent was first used in tile determination of
blood sugar (14), and later in the determinations of acetone and aceto-
acetic acid in blood (15) and creatinille and creatine in serum (16). With

Table 2. PARALLEL TESTS WITH DIACF’[’YL MONOXIME-ARSENIC ACID METHOD

Met Ii ad

Sample Proposed I)iacetyl mono.ri,ne-aroenic acid

BLOOD (Mo./100 ML.)

1 12.0 12.2
2 9.8 9.3
3 14.7 14.0
4 6.8 6.4
5 8.8 8.8
6 21.3 20.5
7 26.5 25.8
8 10.3 10.5
9 14.7 14.3
10 12.3 12.7
11 14.3 14.4
12 21.2 21.5

SERUM (MG./100 ML.)

1 17.5 17.3
2 20.0 20.3
3 11.0 11.5
4 8.7 9.0
5 11.0 11.0
6 25.2 25.3
7 13.6 13.2
8 32.5 31.5
9 l1.9 11.8

URINE (GM./100 ML.)

1 0.56 0.54
2 0.55 0.54
3 0.49 0.49
4 0.87 0.87
5 0.58 0.59
6 0.95 0.95
7 0.92 0.93
8 0.80 0.82
9 0.57 0.56
10 0.73 0.74

*Normal clinical ehemnistry sertmm, contaimuing 11.7 mg. urea nitrogemi per 100 mnl. (Hyland
Laboratories).
120 MOMOSE fT AL. Clinical Chemistry

Table 3. PRECISION OF PROPOSED METHOD FOR UREA NITROGEN

Coefficient of
variation
Sample Mean vatue Standard deviation (%)

Blood (mg./100 ml.) 11.2 0.33 3.0

21.6 0.30 1.4
Serum (ing./100 ml.) 14.5 0.37 2.6
Urimie (gni./100 ml.) 0.21 0.006 2.9
0.66 0.008 1.2
1.19 0.009 0.7

urine, the sample is so diluted that deproteinization is usually unneces-

sary.
Recovery tests were performed by adding known amounts of urea to
blood and urine samples. The results are shown in Table 1. An average
recovery of about 100% was obtained.
The results of parallel tests with a diacetyl monoxime-arsenic acid
method (4) on blood and urine samples are shown in Table 2. In the latter
method, the color development and subsequent procedure were per-
formed under cautious protection from light. Results reported were the
mean values of 3 determinations. There was excellent agreement be-
tween the 2 methods.
Substances otiler than ureid compounds which might occur in blood or
urine did not influence the color development, even in a concentration of
200 mg./100 ml. Those studied were creatine, creatinme, uric acid, am-
monia, histamine, choline, chondroitine, glutathiolle, nicotinic acid, lactic
acid, 3-hydroxybutyric acid, 2-ketoglutaric acid, oxaloacetic acid, ascor-
bic acid, inositol, glucose, and 17 different amino acids. Some ureid com-
pounds such as alloxan, allontoin and citrulline, and tryptophane did pro-
duce a weak color with the present reagent, hut no practical interference
was observed when their concentration was reduced to 8 mg./100 ml.
Some preservatives or anticoagulants which might he added to blood
had no influence on the urea nitrogen value. Those tested were sodium
citrate, sodium oxalate, sodium fluoride, and EDTA. Thymol and cresol,
however, caused a slight decrease in the color and hence should not be
used as preservatives.
The precision of the method was examined by performing 48 separate
analyses on samples of blood, serum, and urine. The results are sum-
marized in Table 3.
References
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plasma, or imrimIe. Seandinar. J. Cliii. 4 Lab. Invest. 11, 122 (1959).
2. Caraway, W. T., and Fammger, H., Improved ultramicroprocedimre for determination of siren
nitrogen in serimmu. Am. J. Clin. Path. 26, 1475 (1956).
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3. Ceriotti, G., and Spumidrio, L., A spectrophotonmetrie mmiethod for deter1IiiIimtio1i of urea.
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