A u t o m a t e d
Micromeasurement
Urease
a n d the Berthelot
of Urea
Using
Reaction
RONALD J. CRENO, B.S., MT(ASCP), ROBERT E. WENK,
M.D.,
AND PATRICIA BOHLIG, MT(ASCP)
Division of Clinical Pathology, State University of New York, Upstate Medical Center, Syracuse, New Y
13210, and Pathology Department, United States Public Health Service Hospital,
Boston (Brighton), Massachusetts 02135
ABSTRACT
Creno, Ronald J., Wenk, Robert E., and Bohlig, Patricia: Automated micromeasurement of urea using urease and the Berthelot reaction. Amer. J. Clin.
Path. 54: 828-832, 1970. T h e method described is based on Kaplan's modification of the Chaney and Marbach procedure: urea is hydrolyzed by urease to
form ammonia, which is reacted with alkaline hypochlorite and phenol
(Berthelot reaction). The procedure is rapid, precise, sensitive, specific, and
simple. Dialysis is unnecessary and only 0.05 ml. of sample is required. An
unmodified AutoAnalyzer is used; the manifold is compatible with a micromethod for glucose using o-toluidine.
AUTOMATED METHODS which utilize urease
and alkaline phenol for the determination
of urea offer rapid, precise, specific, and
sensitive alternatives to manual or other
automated procedures. 12 ' 13
The method presented in this paper offers these advantages in addition to requiring only 0.05 ml. of specimen, eliminating
the dialysis procedure, and permitting a
greater number of samples to be processed
per hour with less carryover. The reagents
are the same as those in "stat" manual
analysis, and the AutoAnalyzer * modules
are compatible with a recently described
micromethod for glucose.11
Materials and Methods
Reagents
Phenol-nitroprusside
reagent. Prepare
quantity sufficient to make one liter. T o
500 ml. of distilled demineralized water in
Received March 6, 1970; accepted for publication April 6, 1970.
Reprint requests should be sent to: Robert E.
Wenk, M.D., Lenox Hill Hospital, 100 East 77th
St., New York, N. Y. 10021.
• Technicon Instrument Corporation, Ardsley,
New York.
a 1-liter volumetric flask, add 50 Gm. of
phenol (Mallinckrodt, Reagent Grade) and
0.25 Gm. sodium nitroprusside (Fisher Scientific Co., cat. no. 83004). Dilute to volume with distilled demineralized water.
Store in an amber glass bottle at 4 to 10 C.
This reagent is stable for two months.
Alkaline hypochlorite reagent. Prepare
quantity sufficient to make one liter. T o a
1-liter volumetric flask containing 600 ml.
of distilled demineralized water, add 25
Gm. of sodium hydroxide pellets (Mallinckrodt Reagent Grade). Allow to cool
and add 40 ml. of 5.25% (w/v) sodium hypochlorite solution (Chlorox, T h e Chlorox
Company). Dilute to volume and add 0.50
ml. Brij-35 wetting agent (0.3%, w/v; Technicon, cat. no. 80947). Store in an amber
glass bottle at 4 to 10 C. This reagent is
stable for three months.
EDTA 1.0% (w/v), pH 6.50. Dissolve
10 Gm. of disodium ethylenediaminetetraacetic acid in 500 ml. of distilled demineralized water. Adjust pH to 6.30 with 1 N
sodium hydroxide. Add 400 ml. of water
and readjust the p H to 6.50. Dilute to one
liter total volume.
828
December 1970
AUTOMATED ENZYMATIC MICROMEASUREMENT OF UREA
o
o
829
o\
of
SAMPLE
'°o0o°7
MODULE
, 0 8 1 DISTILLED D E M O R A L I Z E D
H;0
. 0 6 5 WASTE FROM COLORIMETER
.05 1 DISTILLED DEMINERALIZED
H20
. 0 2 0 UREASE ENZYME
. 0 1 0 SAMPLE
. 0 4 5 AIR
. 1 1 0 DUMMY TUBE
10.
. 0 5 6 REAGENT
. 0 8 1 SAMPLE
I
WASH
. 0 5 6 REAGENT E
SAMPLE SPEED 4 0 SAMPLES/HR. (II MINUTES)
SAMPLER CAM RATIO 1/2 (SAMPLE/WASH RATIO.)
WAVELENGTH 5 4 0 m / i . (NO.3 APERTURE
ON REF-
2N H2S04
ERENCE PHOTOCELL.)
4. 8 MILLIMETER FLOW CELL.
5. USE MICRO SAMPLING TUBE.
*
DEBUBBLER
EVEN NUMBERS ON TOP OF BRIDGE.
COLORIMETER
Fie. 1. Automated micro urea nitrogen manifold. Berthelot Methodology. Single mixing coil =
SMC; pulse suppressor = PS.
Slock urease solution in 50% glycerol (80
units per ml.; Sigma Chemical Co., cat. no.
108B6370). Store at 4 to 10 C. This reagent is stable for four months.
Working urease. Dilute stock urease
with 1% EDTA reagent by adding 1.0 ml.
of stock urease to 99.0 ml. EDTA. Store at
4 to IOC. when not in use in urea nitrogen
analysis. This reagent is stable for 12 hours
at room temperature.
Urea Nitrogen Working Standards. 10,
30, 50, 70, 100, 150 mg. urea nitrogen per
100 ml. (Harleco, cat. no. 64338-C).
Equipment
AutoAnalyzer, Technicon Corporation,
Ardsley, New York, with five modules (Fig.
1). No modifications are necessary.
Calibration
A standard curve is obtained before each
batch of patient specimens. The curve
should be linear to 150 mg. urea nitrogen
per 100 ml.
Procedure
Refer to flow diagram (Fig. 1).
1. The recorder is set at 0% transmission while pumping water. Reagents are
then pumped through the manifold for
10 min.
2. A reagent baseline is set at 98% transmission. No "noise" should be noted during the baseline recording.
3. Standards, controls, and patient specimens are aligned and sampled in the
usual way.
The analysis requires 0.05 ml. of sample,
which is diluted with distilled demineralized water and mixed with urease. The
enzyme hydrolyzes urea, forming ammonia
at 55 C. The ammonia formed reacts with
phenol-nitroprusside reagent followed by
alkaline hypochlorite solution. The mixture is reheated at 55 C. to permit color
development of indophenol. The color intensity is measured and recorded at 540 rm.
830
A.J.C.P.—Vol.
CRENO ET AL.
urease-Berthelot method. Student's t test
for differences between means of paired
samples was applied. Results indicated a
significant difference between the methods
(#. < 0.001).
One hundred patient specimens, 34 in
the abnormal elevated range, 33 in the
normal range, and 33 in the abnormally
decreased range, were selected from the
parent population by the use of a random
number table. The urea nitrogen concentrations obtained with the urease-Berthe-
150
1
L
20
i |
j_
1
l_
50
30
IC
1
\
I v
1v T ^
54
Table 1. Random Analysis of Serum Pools for Urea
Nitrogen by the Automated Berthelot Method
t
1 1ft
?,o«) IC
V / ^
V \1 \1 vi V
Fie. 2. Standard curve (40 samples/hour) followed
by five 10 mg. per 100 ml. standards.
Experimental Studies
Carryover. The small carryover of the
system is illustrated in Figure 2. A standard
of 150 mg. urea nitrogen per 100 ml. followed by a 10 mg. per 100 ml. standard
shows approximately 2% carryover.
Precision. Assayed serum from two pools,
one normal (containing 15 mg. per 100
ml.) and one abnormal (containing 55 mg.
per 100 ml.), were analyzed daily for 30
days from cups spaced randomly among
other specimens. The results are shown in
Table 1.
Recoveries. Four serum pools containing
16.6, 17.0, 26.0, and 34.0 mg. urea nitrogen
per 100 ml., respectively, were each split
into four parts. T o the four parts, four
aqueous standards of 10, 40, 80, and 150
mg. urea nitrogen per 100 ml. were added.
Analysis of these specimens produced the
results shown in Table 2.
Comparison of methods. We measured
the urea nitrogen concentrations in more
than 1,000 specimens of serum and plasma
referred from the clinical services of the
State University Hospital at Syracuse, New
York, by both the standard AutoAnalyzer
diacetyl monoxime procedure6-10 and the
Urea Nltrogen
Specimen
Normal
Pool
(mg./lOOml.)
Abnormal
Pool
(mg./lOOml.)
1
2
3
4
5
15
17
16
14
15
54
54
56
50
55
6
7
8
9
10
15
15
15
15
14
54
52
55
55
53
11
12
13
14
IS
16
14
15
15
15
57
54
55
52
53
16
17
18
19
20
15
15
16
16
16
56
56
58
58
58
21
22
23
24
25
15
15
15
15
15
57
56
57
56
56
26
27
28
29
30
16
16
15
15
15
57
57
55
55
54
Mean
S.D.
C.V.
15.2000
±0.6643
±4.3700
55.1666
±1.9312
±3.5000
December 1970
AUTOMATED ENZYMATIC MICROMEASUREMENT OF UREA
831
Table 2. Recover)' of Added Urea Nitrogen (U.N.) from Plasma by the Automated
Berthelot Method*
Urea
Assay Value
of
Nitrogen
Expected
Actual
Per Cent
Added
U.N. Value
U.N. Value!
Specimen
U.N. Poolf
Recovery
22
10
21
1
34.0
95
2
26.0
10
18
18
100
3
17.0
10
14
14
100
4
16.6
10
13.3
14
105
1
2
3
4
34.0
26.0
17.0
16.6
40
40
40
40
37
33
29
28.3
36
34
29
29
97
103
100
102
1
2
3
4
34.0
26.0
17.0
16.6
80
80
80
80
57
53
49
48.3
54
54
50
48
95
100
102
99
1
2
3
4
34.0
26.0
17.0
16.6
150
150
150
150
92
88
84
83.3
91
88
84
84
99
100
100
101
M EAN
99.9
* Values of urea nitrogen given in mg. per 100 ml.
t Patient pooled plasma.
t Average of three replicate recoveries.
lot and diacetyl nionoxime methods were
compared. A regression line was fitted and
showed a positive slope of 0.82. The correlation coefficient was r = 0.992.
Specificity. Some false elevation was produced by amines (Table 3).2>4 Uric acid
inhibits the color reaction but only in
concentrations exceeding the physiologic
range. Bilirubin does not interfere with
the method.
Discussion
Previously described urease-Berthelot
AutoAnalyzer procedures have excellent
sensitivity, precision, and specificity. However, they required larger samples and dialysis, and are slower because the carryover
necessitates that wash cups be inserted between samples. 12 ' 1S
The present method eliminates significant carryover and maintains linearity
throughout the physiologic range of urea
nitrogen levels in serum. There is high
sensitivity in the low range so that the addition of urea to produce peaks large
enough to read (as in the standard diacetyl
nionoxime method) is not necessary.1
The specificity of the procedure has been
reviewed by Kaplan. 5 Under the conditions
of alkaline pH, using nonlipemic sera, the
automated method offers the same degree
of specificity as the manual procedure. 3 In
addition, the closed system eliminates error
produced by ammonia contamination of
the atmosphere—the only common source
of potentially serious error. 5 - 7
Plasma or serum may be used without
dialysis or protein precipitation; the turbidity caused by protein precipitation has
been prevented by dilution. 5 Sera with visible hemolysis produce slightly higher values (10 to 15%) than nonhemolyzed sera.
Others have noted the same result, although the effect is reversed in specimens
from patients with severe uremia. It is generally thought that hemolysis produces an
elevation of only about 1 mg. per 100 ml.
of urea in the normal range of 9 to 19 mg.
per 100 ml.
Day-to-day variability is acceptable and
832
CRENO ET AL.
A.J.C.P.— Vol. 54
Tabic 3. The Effect of Bilirubin and Some Amine-containing Substances
upon Urea Nitrogen Determinations
Interferant
(100 mg./lOO ml.
aqueous standards)
Urea Nitrogen Equivalent
(mg./lOO ml.)
Creatinine
Alanine
Phenylalanine
Glucosamine
Uric acid
Glutamine
p-aminophenol
Citrulline
Bilirubin
2
5
1
0
Inhibits reaction*
11
13
1
4t
Mixed Standards
(l:l(v/v) urea
nitrogen
lOOmg./lOOml.
plus interferant
100 mg./lOO ml.)
Interference as
Apparent Urea
Nitrogen
(mg./lOO ml.)
50
52
50
52
10
56
6
52
50
0
2
0
2
-40
6
6
2
0
* At physiologic concentrations of uric acid (both normal and abnormal) this substance did not inhibit the
Berthelot reaction. Aqueous standards of 4, 7, 15, and 30 mg./lOO ml.9were tested,
f The bilirubin standard was a 20 mg./lOO ml. aqueous standard.
Laboratory, Technicon Instruments Corporation.
2. Boleter, W. T., Bushman, C. J., and Tidwell,
P. W.: Spectrophotometry determination of
ammonia as indophenol. Anal. Chem. 33: 592594, 1961.
3. Chaney, A. L., and Marbach, E. P.: Modified
reagents for determination of urea and ammonia. Clin. Chem. 8: 130-132, 1962.
4. Fenton, J. C. B.: The estimation of plasma
ammonia by ion exchange. Clin. Chim. Acta
7: 173, 175, 1962.
5. Kaplan, A.: Urea nitrogen and urinary ammonia. Standard Methods in Clinical Chemistry. 5: 245-256, 1965.
6. Marsh, W. H., Fingerhut, B., and Kirsch, E.:
Determination of urea nitrogen with the diacetyl method and an automatic dialyzing apparatus. Amer. J. Clin. Path. 28: 681-688,
1957.
7. Martinek, R. G.: Clinical estimation of urea
nitrogen in biologic fluids by a modified
Berthelot reaction. Amer. J. Med. Techn. 26:
1964.
8. Searcy, R. L., Gough, G. S., Korotzer, J. L., and
Bergguist, L. M.: Evaluation of a new technique for estimation of urea nitrogen in
serum. Amer. J. Med. Techn. 23: 255-262,
1961.
9. Shinowara, G. Y.: Spectrophotometric studies
on blood serum and plasma. Amer. J. Clin.
Path. 24: 696-710, 1954.
10. Skeggs, L. T.: An automatic method for color.
metric analysis of urea nitrogen. Amer. J.
Clin. Path. 28: 311-322, 1957.
11. Wenk, R. E., Creno, R., Loock, V., and Henry,
J. B.: Automated micro-measurement of glucose by means of o-toluidine. Clin. Chem. 15:
1162-1170, 1969.
Acknowledgment. Albert J. Schneider, M.D., and
John Bernard Henry, M.D., gave advice in this 12. Wilcox, A. A., and Sterling, R. E.: The use of
the Berthelot reaction in the automated analstudy and in the preparation of the manuscript.
ysis of serum urea (abstract). Clin. Chem. 8:
427, 1962.
References
13. Wilson, B. W.: Automated estimation of urea
using urease and alkaline phenol. Clin. Chem.
1. AutoAnalyzer Method File, N methodology,
12: 360-368, 1966.
urea nitrogen. Ardsley, New York, Technicon
is comparable with the precision of the
standard diacetyl monoxime method. The
present method yields coefficients of variation of 4.4% in the normal range and 3.5%
in the elevated range. Recovery studies indicate excellent accuracy in the normal
and elevated ranges, averaging 99.9%.
Comparison of the diacetyl monoxime
procedure and the urease-Berthelot method
shows good correlation. It appears that the
measured values of the present method average about 3 mg. higher than those produced by the diacetyl monoxime method,
at a significance level of p < 0.001. About
8% of the serum urea nitrogen determinations in the borderline abnormal range of
22 to 24 mg. per 100 ml. are 1 to 3 mg.
per 100 ml. higher by the Berthelot procedure than with the diacetyl monoxime
method.
Two other features are noteworthy. The
absence of a dialyzer module and the small
sample size render the method suitable for
use with an automated micromeasurement
of glucose by means of o-toluidine. 11