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Clinical Chemistry 44: 1964-1973, 1998;
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(Clinical Chemistry. 1998;44:1964-1973.)
© 1998 American Association for Clinical Chemistry, Inc.

General Clinical Chemistry

Luminometric single step urea assay using ATP-hydrolyzing urease

Birgitta Näslunda, Lars Ståhle, Arne Lundin1, Björn Anderstam, Peter Arner, and Jonas Bergström

1 BioThema AB, Strandvägen 36, S-130 54 Dalarö, Sweden.
a Address correspondence to this author at: Department of Pharma Marketing, Roche AB, Liljeholmsstranden 5, Box 47327, S-100 74 Stockholm, Sweden. Fax 46-87440681; e-mail birgitta.naslund{at}roche.com.


   Abstract
Top
 Abstract
Introduction
 principle of the assay
 Materials and Methods
 Results
 Discussion
 References
 
An automatic enzyme kinetic luminometric method for determination of small quantities of urea in biological fluids and in microdialysates is presented. The method is based on the ATP-hydrolyzing urease reaction [urea amidohydrolase (ATP-hydrolyzing); EC 3.5.1.45], monitored by a luciferin-luciferase ATP reaction. The assay range is 100 pmol to 50 nmol with a detection limit of 5 µmol/L in the sample, compared with detection limits of 0.1 mmol/L in earlier spectrophotometric methods. To reduce the non-urea-dependent ATPase activity (vblank) and to increase the urea-dependent activity, 1,2-propanediol was included. Assay conditions were optimized by multivariate analysis. Recoveries of urea added to blood dialysate and plasma were 96–103%. No analytical interference of common metabolites, drugs, or other additives was observed. The total CVs (6 days and six concentrations, 1.2–21.8 mmol/L) were 3.6–8.5%. The results obtained with the present assay were highly correlated for dialysate (r = 0.979) and for plasma (r = 0.978) with those obtained by a spectrophotometric kit method with slopes of 1.02–1.03 and intercepts of 0.08–0.23 mmol/L.


   Introduction
Top
 Abstract
 Introduction
principle of the assay
 Materials and Methods
 Results
 Discussion
 References
 
The major nitrogenous end product of protein metabolism in mammals is urea, which is almost exclusively excreted by the kidneys. When renal function is reduced, urea accumulates in the body fluids. The blood concentration of urea is followed in patients with renal insufficiency for evaluation of the protein intake, renal function, and the effect of blood dialysis treatment. Urea seems to be freely distributed in the body water of humans (1) . Microdialysis techniques offer the advantage of continuously monitoring metabolite concentrations in the body fluid, for example, in blood and adipose tissue (2) . A small dialysis probe is implanted and perfused continuously with a microinfusion pump, and microdialysate samples are frequently collected. However, in microdialysis the recovery of endogenous metabolites may not be complete. The unbound extracellular tissue concentration must then be estimated by special techniques (3)(4) . In such situations it may be useful to determine urea in the microdialysate as a quality control of changes in the recovery of the microdialysis probe.

Several chemical or enzymatic methods based on hydrolysis of urea by urease (urea amidohydrolase) for the determination of urea in human blood and serum have been developed (5)(6)(7)(8) and references in 8. However, the detection limits for the common enzymatic ultraviolet or colorimetric methods used are reported to be 0.1 mmol/L in the sample, corresponding to 7.5–20 nmol of urea in the assay (8) . These detection limits are at or above the concentrations that are common in, for example, microdialysate samples. These methods are therefore not sensitive enough for clinical and biochemical studies when the concentration is low and the sample volume available is small. Higher sensitivity with the measuring range of 0.01–0.3 mmol/L was obtained with an amperometric method including enzyme probes (9) . A lower detection limit, 0.5 nmol, for a chemiluminometric method involving the three immobilized enzymes urease, glutamate dehydrogenase, and glutamate oxidase and based on hydrogen peroxide analysis has been reported (10) .

We previously have published luminometric methods for studies of fat metabolism (11)(12)(13)(14) and carbohydrate metabolism (15) . In the present investigation, we developed an assay for urea based on ATP-hydrolyzing urease activity monitored by the firefly luciferase reaction. The method is suitable for both plasma and microdialysates and is to be used when the sample volume is small, for example, in pediatrics, in microdialysis, and when several compounds should be determined in the same sample.


   principle of the assay
Top
 Abstract
 Introduction
 principle of the assay
Materials and Methods
 Results
 Discussion
 References
 
ATP-hydrolyzing urease was used in the present method. This enzyme, present in yeast, catalyzes two discrete and sequential reactions (16)(17)(18) , the biotin-dependent urea carboxylase activity (reaction a) and the allophanate hydrolase activity (reaction b). These reactions yield an irreversible degradation of urea and ATP in the presence of carbonate and the production of ammonia, ADP, and inorganic phosphate. The rate of ATP hydrolysis is measured in a luminometric luciferin-luciferase reaction and is proportional to the amount of urea present. The rate of ATP hydrolysis follows first-order kinetics with respect to ATP. By measuring the light intensity at two time points, the rate of ATP hydrolysis could be determined.


Net reaction:


   Materials and Methods
Top
 Abstract
 Introduction
 principle of the assay
 Materials and Methods
Results
 Discussion
 References
 
apparatus
Luminometric technique.
A 1251 luminometer with a temperature-controlled sample carousel for 25 cuvettes and 3 dispensers from BioOrbit Oy was used. Data were transferred and automatically calculated on a Macintosh computer using a Microsoft Excel program obtained from BioThema AB.

reagents
Chemicals.
Urease [urea amidohydrolase (ATP-hydrolyzing); EC 3.5.1.45; synonyms: urease (ATP-hydrolyzing), urea amidolyase, ATP–urea amidolyase, and UALase; CAS reg. no. 72561-06-9] from Candida sp. was obtained from Sigma Chemical Co. 1,2-Propanediol (propylene glycol), diethylene glycol, and 1,2-butanediol were purchased from Fluka Chemie AG. The following chemicals were obtained from Merck: Trizma base, EDTA, magnesium acetate, potassium hydrogen carbonate, 1,2-ethanediol, 1-propanol, 2-propanol, 1,3-propanediol, 1,3-butanediol, 2,3-butanediol, and 1,4-butanediol. Glycerol was obtained from Sigma. Potassium acetate was obtained from BDH. Urea of biopure grade was obtained from Apoteksbolaget AB. The ATP Monitoring Kit was obtained from BioThema AB. All chemicals were of analytical grade. The water used was of reagent grade.

Chemicals and solutions used in the interference experiments.
Sodium hydrogen carbonate, creatine, glucose, oxalic acid, sodium fluoride, pyruvate, epinephrine bitartrate, and the amino acids except phenylalanine were obtained from Sigma. Phenylalanine was obtained from Apoteksbolaget AB. L-lactate (1 mol/L) was purchased from Boehringer Mannheim. Uric acid, sodium citrate, hemoglobin, ascorbic acid, and acetone were obtained from Merck. Vamin®, an amino acid solution used for infusion (100 mmol/L), was obtained from KabiVitrum. Insulin (Actrapid; 105 IU/L) was purchased from Novo Nordisk A/S. Inutest, containing inulin (polyfructosan-s; 250 g/L), was obtained from Laevosan-Gesellschaft. Aminohippurate, 200 g/L, was purchased from Merck Sharp & Dohme International. Mebumal (pentobarbital; 60 g/L) was obtained from NordVacc. Heparin (5 x 106 IU/L) was purchased from Lövens Läkemedel. Stock solutions were prepared or diluted to 10-fold above the final concentration in the sample with physiological NaCl obtained from Kabi Pharmacia and neutralized with sodium hydroxide, if necessary.

procedures
Buffers and stock solutions.
The following buffers were prepared: 200 mmol/L Tris acetate–4 mmol/L EDTA buffer, pH 7.75, and dilution buffer containing 100 mmol/L Tris acetate–2 mmol/L EDTA, pH 7.75 (this buffer is now obtained with the ATP Monitoring Kit); they were stored at room temperature. Potassium acetate, 2 mol/L, and magnesium acetate, 0.5 mol/L, were prepared in water and were stored at 4 °C. Potassium hydrogen carbonate, 0.5 mol/L in water, was prepared daily and was kept at room temperature. The urease enzyme was dissolved to 20 kU/L in 20 mmol/L Tris acetate buffer, pH 7.75, containing 250 g/L glycerol, and was stored at -20 °C (17) .

Reagents for the luminometric assay.
The stock analysis buffer was prepared 1 day before use (for 150 assays) by mixing 200 mmol/L Tris acetate–4 mmol/L EDTA buffer, pH 7.75 (67.5 mL), water (27 mL), 1,2-propanediol (27.5 mL), and 2 mol/L potassium acetate (3.3 mL). This solution was stable for at least 1 week at room temperature. The analysis buffer (for 90 assays) was prepared for each day by mixing stock solution (75 mL), 0.5 mol/L magnesium acetate (1.08 mL), and 0.5 mol/L potassium hydrogen carbonate (1.08 mL). The same buffer was used for the calibrators as for the samples. The ATP Monitoring Reagent was dissolved by addition of 5 mL of water to one bottle of freeze-dried reagent (containing firefly luciferase, luciferin, bovine serum albumin, and inorganic pyrophosphate). The urease reagent was prepared by a 10-fold dilution of ATP-hydrolyzing urease (100 µL of enzyme solution and 900 µL of analysis buffer).

Calibrators.
The urea (Apoteksbolaget AB, biograde) calibrator stock solution (100 mmol/L) was prepared in water and was stored in 0.5-mL portions at -20 °C. Calibrators (13 points) from 10 µmol/L to 40 mmol/L were prepared by dilution (1:1) in dilution buffer (see above).

The ATP standard from the ATP Monitoring Kit was dissolved by addition of 10 mL of water with a cannula (and a second cannula for the outlet of air) to the bottle (can be stored in portions of 2 mL at -20 °C).

Luminometric assay.
The 25 samples in the sample carousel were automatically analyzed in 65 min. Analysis buffer (860 µL) was added to cuvettes. Samples (20 µL) were then added in the following order: dilution buffer (blank), the 13 urea calibrators in increasing concentrations, and the samples (diluted to a urea concentration <2.5 mmol/L). The luminometric procedure included preincubation for 10 min at 25 °C, addition of 100 µL of ATP Monitoring Reagent through the first dispenser, and then addition of 10 µL of ATP calibrator through the second dispenser. Finally, 10 µL of the urease reagent was added through the third dispenser. The kinetic decrease in light emission was determined at the fixed time points of 40 and 80 s after the urease reagent addition. The rate was calculated as (ln light emission at 40 s - ln light emission at 80 s)/(80 s - 40 s) x 60 s and is expressed as percentage/min (the rate constant of the decay of light in %/min). The final concentrations in the cuvette containing 1 mL of solution including the components of the ATP Monitoring Reagent were as follows: 0.2–800 µmol/L urea, 16 mmol/L Mg2+, 50 mmol/L K+, 6 mmol/L HCO3-, 0.1 µmol/L ATP, 2.5 mol/L 1,2-propanediol, 20 U/L ATP-hydrolyzing urease, 2 mmol/L EDTA, firefly luciferase, 100 mg/L D-luciferin, 4 mg/L L-luciferin, 1 g/L bovine serum albumin, 2 µmol/L inorganic pyrophosphate, and 90 mmol/L Tris acetate, pH 7.75.

specimens
Samples included blood dialysate obtained during dialysis of patients with kidney disease, plasma obtained from human heparin-treated blood, and samples from adipose tissue microdialysate. Samples were stored at -20 °C until analysis. The samples were diluted 10-fold with dilution buffer.

optimization of the urea assay
Effect of alcohols.
Alcohols affected the non-urea-dependent activity of the ATP-hydrolyzing urease (vblank, i.e., the velocity of ATP hydrolysis in the blank). Therefore, a screening of one to four carbon alcohols was performed. The final concentrations of the alcohols in the assay were 0.15 and 1.5 mol/L. From this test the compounds that exhibited the highest response factors, i.e., the highest ratio between the velocity of ATP hydrolysis in the presence (2 µmol/L) and absence of urea, were chosen. In a second experiment, urea calibration curves at seven diol concentrations between 0.5 and 4 mol/L for 1,2-propanediol, 1,3-propanediol, and 1,3-butanediol were performed. From this experiment 1,2-propanediol was chosen.

Experiments with multivariate data analysis.
Further optimization of the assay was performed by a combination of statistical design methodology and multivariate data analysis by means of the partial least-squares analysis method (see review in 19) and by response surface methodology (20) . The sequence of experiments started with two fractional factorial designs to localize the region of the optimum and a final experiment, which was essentially a central composite design, to characterize the response surface in detail (Table 1 ). In experiment 1, the effects of K+, Na+, pH, MgAc2, and diol on the Vmax/vblank ratio were studied using a 25–2 fractional factorial design (Table 1 ). In the second experiment, the effects of Cl- vs Ac-, Na+ vs K+, Mg2+, and diol on Vmax/vblank were studied by means of a 24–1 design (Table 1 ). These data were analyzed by partial least-squares analysis as shown by Ståhle et al. (21) for HPLC assays. In the final experiment, the effects of K+, Mg2+, and diol on Vmax and vblank were studied separately by means of the central composite design (Table 1 ). The choice of Vmax/vblank as the outcome variable in the first two experiments was made as a quick way to reach the region of the optimum. For finer adjustment and choice of final conditions, we separately estimated response surfaces for Vmax and vblank in the last experiment. In this analysis, all previous data were also included.


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  		Table 1. Experimental design for the multivariate analysis.

analytical performance
Analytical recovery.
Samples (20 µL) from dialysate and plasma diluted 5-fold and 10-fold were mixed with 20 µL of 2.5 mmol/L urea calibrator and were analyzed. The control samples were mixed with the corresponding volume of dilution buffer.

Precision study.
The total, within-run, and between-day variations were determined over a 2-week period (during 6 days, analyses of six samples, each repeated four times, were analyzed). Five samples containing between 1.76 and 21.8 mmol/L urea from blood dialysate and one microdialysate sample from adipose tissue containing 1.16 mmol/L urea were diluted 10-fold and were kept frozen in aliquots (100 µL) at -20 °C until analysis. Separate 40 mmol/L urea calibrators were prepared, diluted to 0.1 mmol/L, and stored under the same conditions as the samples.

Comparison of methods.
Comparisons were performed with samples obtained from dialysate and from heparinized blood, which were analyzed both by the present method and using a urea kit. The latter method included urease and glutamate dehydrogenase and is based on NADH measurement (7) . A spectrophotometer, Multistat III plus, and a microcentrifugal analyzer from Instrumentation Laboratories were used. A Seronorm sample (batch no. 184, Nycomed Pharma AS) was run with this method and compared with the present (10-fold diluted) method.

Interference studies.
The effects of different potential interfering agents on the assay were investigated (22) using a dialysate sample. The factor stock solutions (50 µL) were diluted 10-fold with the dialysate sample (450 µL), producing a urea concentration of 1.2 mmol/L. These samples (not diluted further) were analyzed and compared with samples to which the corresponding volume of physiological NaCl had been added.

statistical methods and calculations
Multivariate analysis of the optimization studies was performed as described above. ANOVA (variance components analysis) correlation, determination of CVs, and Student's t-test were performed according to Snedecor and Cochran (23) . The linear regression analysis was performed according to Draper and Smith (24) after calculating logarithms of light emission and concentration values. The concentrations of urea in samples were automatically calculated (see above) using an extended linearization plot described previously for glycerol (14) .


   Results
Top
 Abstract
 Introduction
 principle of the assay
 Materials and Methods
 Results
Discussion
 References
 
The activity of ATP-hydrolyzing urease was potassium-dependent, was stimulated by magnesium and carbonate, and was completely inhibited by avidin, in agreement with an earlier report (16) . However, we recognized that the non-urea-dependent ATPase activity was substantial. This is shown in Fig. 1 as the nonoptimized assay system. We concluded that this enzyme activity was not caused by contaminating urea in the enzyme preparation because extensive washing or pretreatment of the enzyme with jack bean urease did not abolish the non-urea-dependent ATPase activity.



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  		Figure 1. The effect of 1,2-propanediol on the ATP-hydrolyzing urease activity and on the vblank (shown in the inset).

The urea concentration in the cuvette is given. The concentration of diol was 2 mol/L ({bigtriangleup}), 2.5 mol/L ({square}), 3 mol/L ({blacktriangleup}), or 3.5 mol/L ({diamondsuit}). ({blacksquare}), nonoptimized system without diol.

effect of alcohols
We discovered that alcohols interacted with the enzyme activity of ATP-hydrolyzing urease. The effect was on either the non-urea or the urea-dependent enzyme activity or on both activities. Therefore, different alcohols were screened at the concentrations of 0.15 and 1.5 mol/L (Table 2 ). All the alcohols studied except 1,2-ethanediol and diethylene glycol reduced the non-urea-dependent ATPase activity. The butanediols were most effective and decreased the background by 47–65%, followed by the propanediols with a reduction by 28–42%. The greatest response to urea was obtained by ethanol and diethylene glycol. This was followed by 1,2-ethanediol and the propanediols, which increased the response by >25%. Further studies with 1,2-propanediol, 1,3-propanediol, and 1,3-butanediol were performed. Calibration curves were determined for the different diols, using seven concentrations between 0.5 and 4 mol/L in the final assay medium. Data from these studies revealed that both Vmax and Km for urea were increased, whereas the vblank was reduced for the three respective diols (data not shown). The aim was to obtain an assay system with a low background (vblank) and with a high potency to respond to urea i.e., a high v/vblank ratio. The Vmax/vblank ratio was the highest for 1,2-propanediol, which therefore was chosen for further optimization of the assay.


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  		Table 2. Screening of different alcohols and the effect on the blank and on the response to urea in the luminometric urea assay.

assay optimization
Because several factors could influence the urease activity, it was necessary to use a multivariate design and analysis for the final optimization of the assay. The results from this study showed that pH, Na+, and HCO3- concentrations did not interact with the determination of urea in the range investigated. The K+, Mg2+, and diol concentrations all influenced the Vmax/vblank ratio; there was also a tendency to a better performance with Ac- replacing Cl-. In the final experiment, we could identify a region in the Mg2+-K+-diol plane where Vmax attained a maximum (Mg2+ = 24.5 %/min, K+ = 58.9 %/min, and diol = 2.11%/min). No minimum could be identified for vblank; it continued to decrease with increasing concentrations of diol and magnesium. In the analysis of Vmax, both potassium and diol contributed significantly to the response surface (P <0.0001), whereas Mg2+ was not quite significant (P = 0.052). For vblank, diol and Mg2+ were significant, as was the interaction between these two factors (P <0.0001). Thus, the choice of final conditions had to be balanced between the influences on Vmax and vblank, respectively. We show the response surfaces in the K+-diol plane and in the Mg2+-diol plane for Vmax and vblank separately in Fig. 2 .



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  		Figure 2. Results obtained from the multivariate data analysis where the Vmax and vblank, respectively, are a function of diol, K+, and Mg2+.

The Vmax response surfaces are shown in the K+-diol plane (a) and in the Mg2+-diol plane (b). The vblank response surfaces are shown in the K+-diol plane (c) and in the Mg2+-diol plane (d).

The presence of potassium, magnesium, and diol inhibited the light emission. Therefore, we also had to consider the light emission when choosing the conditions. A compromise between the results from the multivariate analysis and the light emission was made to define the best assay conditions. The light emission in the final assay system was 10% in the presence of potassium, propanediol, and with an increase of magnesium concentration from 10 to 16 mmol/L, as compared with the usual ATP monitoring assay. However, this particular assay has the advantage of being independent of the absolute light emission. Therefore, the reduction of light did not reduce the sensitivity of detection.

Before optimization, the Vmax was 8.1%/min, the Km was 1.9 µmol/L, and the blank was 10%/min. After optimization, the Vmax was was 78.8%/min, the Km was 19.6 µmol/L, and the blank was 3.39%/min. Consequently, optimization produced a 29-fold increase of the maximal response to urea (Vmax/vblank ratio), leading to a similar increase of the measuring range for urea. After optimization, the following results (mean ± SD) were obtained for eight independent urea calibration curves (5 µmol/L to 10 mmol/L in the sample) determined on separate days: The slope obtained from logarithmic values was 1.016 ± 0.065; the intercept was 0.004 ± 0.007; the coefficient of correlation was 0.999; the Km was 19.6 ± 0.68 µmol/L; the Vmax was 78.8 ± 2.63%/min; and the vblank was 3.39 ± 0.22%/min. The detection limits of urea in the sample, defined as 3 SD of the reagent blank based on 10 blank points obtained with four different concentrations of 1,2-propanediol in an optimized enzyme assay system, were as follows: 5 µmol/L urea for 2 and 2.5 mol/L diol, 10 µmol/L urea for 3 mol/L diol, and 50 µmol/L urea for 3.5 mol/L diol. The velocity vs substrate curves for 1,2-propanediol concentrations between 2 and 3.5 mol/L are shown in Fig. 1Up . A curve from a nonoptimized system without diol is also shown in Fig. 1Up .

final assay conditions
The concentrations of the assay mixture, including the components of the ATP Monitoring Reagent, were are follows: 0.2–800 µmol/L urea, 16 mmol/L Mg2+, 50 mmol/L K+, 6 mmol/L HCO3-, 0.1 µmol/L ATP, 2.5 mol/L 1,2-propanediol, 20 U/L ATP-hydrolyzing urease, 2 mmol/L EDTA, firefly luciferase, 100 mg/L D-luciferin, 4 mg/L L-luciferin, 1 g/L bovine serum albumin, 2 µmol/L inorganic pyrophosphate, and 90 mmol/L Tris acetate, pH 7.75.

calibration curve
Data on velocity (v) and substrate (S) were plotted according to Lundin et al. (14) . For the determination of vblank (at the S concentration of zero) a plot of the linear regression of vobserved vs the Sadded in the linear part of the calibration curve in the assay (0–3.125 µmol/L) was used. The vblank was then subtracted from the v of the individual calibrator points, and the Vmax (intercept) and Km (slope) were calculated from an Eadie-Hofstee plot (v vs v/S). Finally, the Vmax and Km values were used to plot v/Vmax vs S/(S+Km). This is a way to linearize kinetic substrate assays, based on the Michaelis-Menten equation. The curve is a straight line when both axes are linear as well as when both axes are logarithmic. This was confirmed for a urea concentration up to 40 mmol/L, i.e., far above the Km value. However, the maximum recommended concentration in the sample is 2.5 mmol/L because at higher concentrations a small error in the reaction rate determined will produce a large error in the concentration calculated. A typical calibration curve using a logarithmic plot is shown in Fig. 3 . The assay using 2.5 mol/L 1,2-propanediol was linear up to 40 mmol/L. Thus, with a detection limit of 5 µmol/L, the measuring range of the assay is between 100 pmol and 50 nmol in the sample, corresponding to 5 µmol/L to 40 mmol/L.



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  		Figure 3. The urea calibration curve (10 µmol/L to 40 mmol/L in the sample), using the logarithmic version of the Lundin plot (14).

analytical recovery
The recovery of urea added to dialysate and to plasma was 96–103%. No correlation between the dilution factor (5- or 10-fold) and recovery of urea from the samples at the two dilutions tested was observed.

precision
The precision of the assay was determined by assessing six analyte concentrations between 1.16 and 21.8 mmol/L. The results (Table 3 ) show an acceptable performance and no relation between CV and sample mean. This indicates that the log-transform, made to obtain homoscedasticity, was appropriate to allow for the variance component analysis. The geometric means were virtually identical with the arithmetic means. The total CV for all samples was 6.7%, the within-run CV was 4.7%, and the between-day CV was 4.7%.


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  		Table 3. Precision of the luminometric urea assay.

comparison of methods
The results obtained by the present luminometric assay and by a conventional automatic spectrophotometric kit method were compared. The lowest concentration in the sample routinely used for the Multistat spectrophotometric method was 1 mmol/L, corresponding to 2 nmol in the sample. Below this concentration the Multistat method did not give reproducible results. According to the description sheet for this method, the measuring range was 0.94–15 mmol/L. In the comparison experiments, the concentration range of urea was 0.65–29.6 mmol/L for 39 dialysate samples and 2.9–22.4 mmol/L for 51 plasma samples (Fig. 4 ). The funnel shape in this plot supported the observation that the CV is constant. The coefficients of correlation between the methods were 0.979 for dialysate and 0.978 for plasma samples. The Seronorm sample contained 6.17 mmol/L with the present method and 6.24 mmol/L with the designated comparison method.



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  		Figure 4. Correlations between the present method and the spectrophotometric kit method for dialysate ({blacksquare}) and plasma ({square}) samples.

interference study
The influence of different factors that might interfere with the assay was investigated. No interference of common physiological compounds, drugs, or additives used in routine clinical chemistry was observed in the assay (Table 4 ). The concentrations of the compounds used were at least one order of magnitude higher than the expected concentration in the sample.


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  		Table 4. Compounds that did not interfere with the luminometric urea assay.


   Discussion
Top
 Abstract
 Introduction
 principle of the assay
 Materials and Methods
 Results
 Discussion
References
 
In this investigation we developed a simple, sensitive, and automatic method for the determination of small amounts of urea. The method is specifically designed for determination of low concentrations of the metabolite in biological fluids and in microdialysates when conventional techniques fail because of small sample volumes.

The present assay is based on an ATP-hydrolyzing urease, urea amidolyase (16)(17)(18) . The enzyme also exhibited ATPase activity in the absence of urea. This activity was abolished by avidin, which confirmed that this was a biotin-containing enzyme (16) . We established that the enzyme activity observed in the blank was not because of urea in the enzyme preparation. The activity of the enzyme in the absence of urea and also the observation of a relatively limited response to added urea would have produced a urea assay with a too narrow substrate range.

Previously, we used 500 mL/L 1,2-propanediol to preserve the activity of synthetases and oxidases in the freezer (11)(15) . It was found that the non-urea-dependent ATP-hydrolyzing urease activity was absent after being kept in 500 mL/L 1,2-propanediol. However, the urea-dependent ATPase activity was increased. Therefore, we investigated the effect of other alcohols. We found that several alcohols changed the enzyme activity. Some alcohols reduced the ATPase activity in the absence of urea. Certain alcohols stimulated enzyme activity. This suppression of blank ATPase activity by alcohols has not been reported previously. However, it has been shown that the kinetics of enzymes can be changed by polyhydric alcohols (25) and that aspartase is activated by glycerol, ethylene glycol (1,2-ethanediol), and propylene glycol (1,2-propanediol) (26) . Moreover, other biotin-dependent enzymes can be activated by 100 mL/L ethanol (27) and by cryoprotectants (28) . In this assay, we included 1,2-propanediol to reduce the background activity and to increase the response to urea. Some of the other alcohols we tested had similar effects on the enzyme. 1,2-Propanediol was chosen because it exhibited the highest Vmax/vblank ratio. In addition to an increased Vmax and reduction of the vblank, the Km was increased by the diol. This diol is usually not present in human tissues. All the alcohols tested at 150 mmol/L had an effect on the assay. However, several of the alcohols are not naturally present in the tissues, and furthermore, such high concentrations are unlikely to occur. For comparison, the glycerol concentration is in the micromolar range.

The incubation system also included the components ATP, magnesium, potassium, and carbonate in addition to 1,2-propanediol. The effects of these components on the reaction were suspected to be dependent on each other. This would make it laborious to optimize one component at a time. Instead the multivariate method (19) , allowing variation of several factors simultaneously, was utilized. The aim of the optimization was to obtain the most active, stable, and reliable enzyme system exhibiting a low background activity and high Vmax and Km. This was made possible by the use of combined experimental design and multivariate analysis. The choices of optimization methods and criteria are discussed in detail by Ståhle et al. (21) .

We chose 2.5 mol/L 1,2-propanediol for the assay because the response to urea was high and the detection limit was low at that concentration. Concentrations of 1,2-propanediol >2.5 mol/L increased the detection limit and therefore reduced the linear range. Compared with a system without it, diol increased the Km 10-fold, which increased the linear substrate range of the assay. When an extended linear plot was used, a dynamic range for the assay even above Km was obtained (14) . However, it is advisable to dilute the samples to a maximum of 2.5 mmol/L urea because a small error in the determination of the activity would produce a large error in urea estimation at higher concentrations.

The small amount of urea needed for this luminometric assay allows a diminutive sample volume. Compared with an earlier published spectrophotometric method (8) , the present method is at least 75-fold more sensitive and is also simpler than an earlier luminometric method (10) . The present automatic luminometric assay is based on a single enzyme step, which is coupled to a luminometric ATP monitoring reaction. No interference with the assay was observed by any of the studied compounds except for alcohols at nonphysiologically high concentrations. However, it is recommended to check the system for every new compound that might be present in the sample.

In summary, we have developed a sensitive, automatic, and simple luminometric assay for urea. The sensitivity of the method allows determination of urea using small sample volumes.


   Acknowledgments
 
We acknowledge Monica Eriksson and Eva Sjölin for excellent technical assistance. This investigation was supported by grants from MFR (project nos. 1002, 01034, and 09069) and Hospal International, Lyon, France.


   Footnotes
 
Clinical Research Centre and Divisions of 1 Medicine, 2 Clinical Pharmacology, 3 Renal Medicine, and 4 Baxter Novum, Karolinska Institute, Huddinge University Hospital, S-141 86 Huddinge, Sweden.


   References
Top
 Abstract
 Introduction
 principle of the assay
 Materials and Methods
 Results
 Discussion
 References
 

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