Clinical Chemistry 43: 1932-1936, 1997;
(Clinical Chemistry. 1997;43:1932-1936.)
© 1997 American Association for Clinical Chemistry, Inc.
Kinetic assay of serum and urine for urea with use of urease and leucine dehydrogenase
Yoshitaka Morishita1,a,
Kiyoshi Nakane1,
Toshiaki Fukatsu1,
Nobuo Nakashima1,
Katsumi Tsuji2,
Yoshihiro Soya2,
Keizo Yoneda2,
Shigeki Asano2 and
Yoshihisa Kawamura2
1
Department of Clinical Laboratory, Nagoya University Hospital, 65 Tsurumai-cho, Showa-ku, Nagoya, 466, Japan.
2
Tsuruga Institute of Biotechnology, Toyobo Co.,
Ltd.,10–24 Toyo-cho, Tsuruga, 914, Japan.
a Author for correspondence. Fax 81-52-744-2611.
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Abstract
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We describe a new kinetic assay for determining urea in serum or urine
with use of urease (EC 3.5.1.5) and leucine dehydrogenase (EC 1.4.1.9).
The latter enzyme is suitable for the kinetic assay of
NH4+ because itsKmvalue for NH4+ at pH 8.75
is large (~500 mmol/L). Interference from endogenous
NH4+ in serum or urine is obviated by
subtraction of the assayed endogenous NH4+value in a sample blank. For serum, within-assay CVs (n = 10) were
0.39–0.58%; day-to-day CVs (n = 10) were 1.56–2.30%. In urine,
within-assay CVs (n = 10) were 0.86–1.15%. Analytical recovery
of urea (0.893–71.4 mmol/L) added to patients' sera (urea 6.14
mmol/L) was 99.2–105.2%. The calibration curve for serum was linear
through zero for urea concentrations up to 142.9 mmol/L and for urine
up to 714.3 mmol/L. No influences of added ammonium ion, bilirubin,
hemoglobin, ascorbic acid, or Intralipid were observed. The regression
equations for this method (y) and conventional methods
(x = Determiner-LUN for serum assays, Serotec UUR-R
for urine) were: y = 1.016x - 0.12
mmol/L (r = 0.999,
Sy|x = 0.34 mmol/L, n = 100)
for sera, and y = 1.070x -
12.6 mmol/L (r = 0.998,
Sy|x = 7.41 mmol/L, n = 100)
for urine.
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Introduction
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Urea nitrogen (UN)1
in serum or urine has been
measured by many methods (1)(2) that are based
on enzymatic reaction with urease (EC 3.5.1.5) and glutamate
dehydrogenase (EC 1.4.1.4). Endogenous NH4+ in
serum and urine is a potential interference, because
NH4+ produced from urea by urease is determined
in the reaction system. The NH4+ in serum and
urine has been eliminated with the glutamate dehydrogenase and
2-oxoglutaric acid before addition of urease. But in some urine that
has a large amount of endogenous NH4+, the
elimination of the NH4+ may not be complete,
even in samples diluted 10–20-fold.
Other approaches to avoiding interference from
NH4+ have been described. To eliminate
much of the NH4+ in urine, a recycling system
(NADH
NAD+) with isocitrate dehydrogenase (EC 1.1.1.42)
has been used (3). Alternatively, ADP (produced from ATP
by urease (4)) can be determined instead of
NH4+. However, the cost of urea determination
in these methods is very high; moreover, the former method was linear
only to urea concentrations of ~10 g/L, and the latter method was
carried out at 376 nm, a wavelength unavailable on many automated
analyzers.
We investigated a new enzymatic kinetic assay of serum or urine UN by
using leucine dehydrogenase (LED; EC 1.4.1.9) and urease
(5)(6). Interference from endogenous
NH4+ in serum and urine is avoided by
subtracting the endogenous NH4+ value assayed
in a sample blank.
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Assay Principle
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Endogenous NH4+ in serum or urine is
allowed to react with 2-ketoisohexanoic acid, NADH, and LED. The
reaction rate at which NADH is oxidized to NAD+ depends on
the amount of the endogenous NH4+ measured
(I).
Next, urease is added to the reaction system, and the
oxidation rate of NADH to NAD+ by both the
NH4+ produced from urea and the endogenous
NH4+ is measured (II). UN in the
sample is calculated from the differences of oxidation rate between
I and II.
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Materials and Methods
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Apparatus.
This proposed method and the conventional
(comparison) methods were performed with the Hitachi Model 7150
automated analyzer.
Reagents.
Urease (111 kU/g) and LED (45.1 kU/g;
Km(NH4+) = 500
mmol/L (5)) were purchased from Toyobo. Analytical-grade
ammonium chloride, L-ascorbic acid, urea, and NADH were
from Wako Pure Chemical Industries. 2-Ketoisohexanoic acid sodium salt
was from Nacalai Tesque (Kyoto, Japan);
N,N-bis(2-hydroxyethyl)glycine (Bicine) from
Dojindo Labs (Kumamoto, Japan); bilirubin from Sigma Chemical Co., and
Intralipid 10% from KabiVitrum AB.
Reagent 1 (R1) for the new method contained 2-keto-isohexanoic acid
3.0 mmol/L, ß-NADH 0.3 mmol/L, and LED 1.5 kU/L in 100 mmol/L Bicine
buffer (pH 8.75). Reagent 2 (R2) contained urease, 70 kU/L, in R1.
Calibrators were physiological saline, with and without 17.86 mmol/L
UN.
Assay procedure.
UN in serum or urine was measured with
the Rate-Analysis system B mode of the Hitachi 7150 automated analyzer,
as shown Fig. 1
. Serum (15 µL) or urine (3 µL) and R1 (300 µL) were
mixed, and the reaction rate was measured at measurement points 6–24,
from 1 min to 5 min after the mixing (test I). Next, R2 (100
µL) was added and the reaction rate was measured at points 30–50
(from 6 to 10 min) at 340 nm (test II). The lag time, to
allow the urease reaction to proceed sufficiently, was set at 1 min
(measurement points 25–30). A linear calibration curve based on the
absorbance of the 0 (physiological saline) and 17.86 mmol/L UN
calibrators was used to estimate the UN concentrations of the samples.
Comparison method.
The enzymatic method eliminated
endogenous NH4+ in serum. The Determiner-LUN
reagent kit from Kyowa Medex (Tokyo, Japan) was used for serum UN
determination; another enzymatic method eliminated endogenous
NH4+ in urine, the Serotec (Sapporo, Japan)
UUR-R reagent kit for urine UN determination.
Samples.
L-Consera N (Nissui Pharmaceutical Co., Tokyo,
Japan) was used as control serum. Patients' serum and urine samples
were from patients in Nagoya University hospital.
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Results
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optimization studies
Optimization studies of this proposed method were carried out with
the calibrator (UN 17.86 mmol/L), a patient's serum (UN 18.29 mmol/L),
and L-consera N control serum (UN 5.82 mmol/L).
Effects of pH.
The effect of pH on the serum UN
determination was examined in 100 mmol/L Bicine buffer at various pH
values (8.00, 8.25, 8.50, 8.75, 9.00, and 9.25). With increasing pH
values, the reaction rate (A/min) for assay of the
calibrator increased, but the UN values of the patient's serum and
L-consera N remained virtually unchanged (Fig. 2
a). We chose to use Bicine buffer, pH 8.75.
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Figure 2. Effects of (a) pH, (b)
2-ketoisohexanoic acid, (c) LED activity, and (d)
urease activity.
The reaction rate (A/min; oxidation rate from NADH to
NAD+) of UN calibrator ( , 17.86 mmol/L UN), patient's
serum ( , 18.29 mmol/L UN), and L-consera N (•, 5.82 mmol/L UN) was
measured under various conditions.
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Effects of 2-ketoisohexanoic acid.
We examined the
effects of concentrations of 2-ketoisohexanoic acid on the serum UN
determination. With increasing concentrations of 2-keto-isohexanoic
acid (0.3, 0.5, 1.0, 3.0, 5.0, and 10.0 mmol/L), the reaction rate
(A/min) of the calibrator increased, reaching almost the
maximum at 5.0–10.0 mmol/L 2-keto-isohexanoic acid, whereas the UN
values of the patient's serum and L-consera N were almost unchanged
(Fig. 2b
). We chose 10 mmol/L as the 2-ketoisohexanoic acid
concentration in R1 and R2 to obtain the maximum reaction rate.
Effects of LED activity.
With increasing LED activity
(0.1, 0.2, 0.5, 1.0, 2.0, and 4.0 kU/L), the rate of reaction
(A/min) of the calibrator increased proportionally, whereas
UN values of the patient's serum and L-consera N did not indicate much
change (Fig. 2c
). We added 1.5 kU/L LED to R1 and R2.
Effect of urease activity.
With increasing urease
activity (5.0, 10.0, 20.0, 50.0, 70.0, and 100.0 kU/L), the reaction
rate of the calibrator (A/min) increased, with the maximum
occurring at 70–100 kU/L, whereas the UN values of the patient's
serum and L-consera N did not show any marked change at urease of 50
kU/L or greater, as shown in Fig. 2d
. We thus added 70 kU/L urease to
R2.
Time course.
Typical time courses in this proposed
kinetic assay of serum UN are shown for the calibrator, the patient's
serum, and L-consera N in Fig. 3
. The first reaction is the kinetic assay of the endogenous
NH4+ (points 6–24); the second reaction is the
kinetic assay of both endogenous NH4+ and
NH4+ produced from urea by urease (points
30–50). Within 1 min (from points 25 to 30), the urease reaction is
ended.
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Figure 3. Time course of reaction.
Calibrator (, 17.86 mmol/L UN); patient's serum (, 18.29 mmol/L
UN); L-consera N (•, 5.82 mmol/L UN).
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assay evaluation
Precision.
The serum UN assay samples were calibrator,
L-consera N, and a patient's serum, which contained 17.91, 5.83, and
18.17 mmol/L UN, respectively. For the urine UN assay, we used a
calibrator and two patients' urines, with respective UN contents of
17.99, 93.68, and 575.49 mmol/L. Within-assay CVs were measured with
n = 10, and day-to-day CVs were determined from assays performed
on 10 days (n = 10). As shown in Table 1
, serum UN within-assay CVs ranged from 0.39% to 0.58%;
day-to-day CVs were 1.56–2.30%; in urine UN determinations,
within-assay CVs (n = 10) were 0.86–1.15%.
Detection limit.
We examined the detection limit of this
UN determination by assaying physiological saline (NaCl 9 g/L, the 0 UN
calibrator) 10 times. The result (mean ± SD) was 0.012 ±
0.018 mmol/L. The detection limit, mean for physiological saline + 3.0
SD, was 0.066 mmol/L.
Analytical recovery.
For UN additions of 0.89, 1.79,
2.68, 3.57, 7.14, 10.71, 17.86, 26.79, 35.71, 53.57, and 71.43 mmol/L
to patients' serum (UN 6.14 mmol/L), from 99.2% to 105.2% was
recovered (mean 102.2%).
Linearity of calibration curve.
We examined the
linearity of the calibration curve in duplicate with the UN
calibrators. The calibration curve was straight up to at least 142.9
mmol/L for serum UN determinations and up to 714.3 mmol/L in urine UN
determinations (Fig. 4
).
Interferences.
Various substances were examined for
their potential effects on this UN determination. One volume of
examined substance was mixed with nine volumes of patient's serum (UN
16.29 mmol/L), L-consera N (UN 5.82 mmol/L), or a patient's urine (UN
324.1 mmol/L).
Ammonium chloride was added to the patient's serum and L-consera N at
concentrations of 0.056–0.56 mmol/L, and to the patient's urine at
55.6–166.7 mmol/L. Assay of UN was virtually unaffected (Fig. 5
). Bilirubin, hemoglobin, ascorbic acid, and Intralipid were
added to the patient's serum and L-consera N in the same way and
assayed. We found no interference with serum UN determination from
bilirubin up to 0.342 mmol/L, hemoglobin 0.059 mmol/L, ascorbic acid
1.14 mmol/L, and 50-fold-diluted (0.2%) Intralipid.
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Figure 5. Interference of added NH4+ (as
various concentrations of ammonium chloride) to samples of patient's
serum (, 16.29 mmol/L UN), L-consera N (•, 5.82 mmol/L UN), and
patient's urine ( , 324.1 mmol/L UN).
The concentration of NH4+ shown indicates the
concentration in the sample.
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Correlation.
Possible correlation between this proposed
method (y) and the comparison methods (x) was
examined (Fig. 6
). The correlation between values obtained with our method and
the Determiner LUN kit for 100 patients' sera was: y =
1.016x - 0.12 mmol/L (r = 0.999,
Sy|x = 0.34 mmol/L); comparison
with the Serotec UUR-R kit for 100 patients' urines gave:
y = 1.070x - 12.6 mmol/L
(r = 0.998, Sy|x =
7.41 mmol/L).
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Figure 6. Correlation between this proposed method and conventional
methods: comparison with (a) Determiner LUN kit for 100
patients' sera, and (b) Serotec UUR-R kit for 100
patients' urines.
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Discussion
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After detailed optimization studies, we investigated the new
enzymatic kinetic assay of serum or urine UN determination by LED and
urease with the Hitachi 7150 automated analyzer, which automatically
calculated the difference between the endogenous
NH4+ reaction (test I) and the
reaction of both the endogenous NH4+ and the
NH4+ produced from urea by urease (test
II). Characteristic features of this method are:
1) Endogenous NH4+ in serum or urine does
not affect the UN determination, so that urine, even when containing a
high concentration of endogenous NH4+, need not
be diluted before assaying.
2) This method yields linear results for large concentrations of UN—up
to 142.9 mmol/L in serum and up to 714.3 mmol/L in urine—because the
large Km value of LED for
NH4+, ~500 mmol/L, is suitable for the
kinetic assay of NH4+.
3) No influences of added ammonium ion, bilirubin, hemoglobin, ascorbic
acid, or Intralipid were observed.
4) Good precision, reasonable analytical recovery without interference,
and good correlation with conventional methods were observed.
The procedure takes 10 min and should be applicable to other discrete
automated analyzers. The reagents could be prepared as liquid reagents.
We therefore conclude this proposed method may be useful in routine
clinical diagnosis.
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Footnotes
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1 Nonstandard abbreviations: UN, urea nitrogen; LED,
leucine dehydrogenase; and Bicine,
N,N-bis(2-hydroxyethyl)glycine. 
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Why ""Urea Nitrogen"" When Urea is Measured?
Clin. Chem.,
April 1, 1998;
44(4):
894 - 895.
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Abstract
Introduction
