Enzymatic determination of urea

Urea in aqueous solutions is broken down quantitatively into ammonium in the neutral range (pH at approximately 7) by the enzyme urease . The reaction can be represented by the following equation  

NH + is then subsequently determined analytically. The determination limit and accuracy are not dependent on the enzymatic transformation but on the analytical detectability of the ammonium formed. 

It is advisable to carry out the process of determination photometrically, employing Berthelot s colour reaction. This reaction is based on the formation of the indole dye indophenol blue. Firstly, hypochlorite is used to convert NH4 into chloramine under alkaline conditions. 

Chloramine reacts with phenolate in the presence of sodium nitroprusside as catalyst and in several reaction steps forms the dye indophenol blue, which is yellow in the non-dissociated and blue in the dissociated state. This reaction can be represented by the following empirical formula  

2 mg/ml urease + 50 mmol/1 phosphate buffer, pH = 6,5 Phenol solution  

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Fig. 14.8 Single-channel FIA system for enzymatic determination of urea based on potentiometric (pH) measurements. (Reproduced from [26] with permission of the American Chemical Society).

Figure 4.6. Flow-injection manifold employed for the enzymatic determination of urea by potentiometric measurement of pH. The sample S (30 jjlL) is injected into a carrier stream of 1.0 mM TRIS buffer in 0.14 M NaCl (pH 7.70), containing dissolved urease, and then passed to the reaction coil a, placed in a themostated water jacket, where the enzymatic degradation of the injected urea takes place. The sample zone is then led to a capillary glass flowthrough electrode (pH) and finally via the reservoir, housing the reference electrode (REF), to waste, W.

J. Rflii5ka, E. H. Hansen, A. K. Ghose, and H. A. Mottola, Enzymatic Determination of Urea in Serum Based on pH Measurement with tfie Flow Injection Method. Anal. Chem., 51 (1979) 199. 

T. D. Yerian, G. D. Christian, and J. Rflii ka, Enzymatic Determination of Urea in Water and Serum by Optosensing Flow Injection Analysis. Analyst, 111 (1986) 865. 

The use of enzymatic techniques in the field of water analysis is described in Section 4.2, taking as an example the enzymatic determination of urea, e.g. in swimming pool water. 

The sequence of operations in enzymatic determination of urea is summarized in outline in the following pipetting chart. 

A new kinetic enzymatic method for the routine determination of urea in semm has been evaluated. This method is based upon an enzymatic reaction and formation of a coloured complex. The method is based on a modified Berthelot reaction. The reaction was monitored specRophotomebically at 700 nm (t = 25 0.1 °C). The optimal pH value, chosen for the investigation of complex, is 7.8. 

For monitoring catalytic (enzymatic) products, various techniques, such as spectrophotometry [32], potentiometry [33,34], coulometry [35,36] and amperometry [37,38], have been proposed. An advantage of these sensors is their high selectivity. However, time and thermal instability of the enzyme, the need of a substrate use and indirect determination of urea (logarithmic dependence of a signal upon concentration while measuring pH) cause difficulties in the use and storage of sensors. 

All enzymatic methods for the determination of urea are based on urea hydrolysis by urease (EC 3.5.1.5) ... 

Blood glucose and blood urea nitrogen (BUN) are probably the two most frequently performed clinical tests. In the procedures described in Table 24.2, the total of all reducing sugars is measured, and so results tend to be high. But these methods have been adopted as standard ones for many years. The enzymatic determination of glucose (Chapter 22) is an established method, and dedicated enzymatic glucose analyzers are now widely used. 

Fig. 14.7 System for the enzymatic potentiometric determination of urea including a dialyser and an immobilized-enzyme reactor. (Reproduced from (27] with permission of Elsevier).

In Section 4.1 the potentiometric determination of urea via enzymatic degradation by urease according to the reaction ... 

In Fig. 4.36 is shown the readouts obtained in this system when injecting a series of ammonium chloride standards where, in order to increase the sensitivity, the sample zone was stopped in the flow cell (in this case for 16 s). To the right in the same figure is shown the recorder outputs for the determination of urea, enzymatically degraded to ammonia by means of urease. In the latter case, the chasing zone (b) consisted of... 

A second application of the stopped-flow technique is for kinetic measurements. In this application, the flow is Slopped with the reaction mixture in the flow cell where the changes in the concentration of reactants or products can be monitored as a function of time. The stopped-flow technique has been used for the enzymatic determination of glucose, urea, galactose, and many other substances of interest in clinical chemistry. 

Most biosensors described in the literature for the determination of urea are potentiometric based on NH4 or HCOj" sensitive electrodes [181, 182]. Osaka and co-workers constructed a highly sensitive and rapid flow injection system for urea analysis with a composite film of electropolymerised inactive PPy and a polyion complex [183]. Pandey and co-workers fabricated a urea biosensor based on immobilised urease on the tip of an ammonia gas electrode (diameter 10 pm) made from a PPy film coated onto a platinum wire [170]. The enzymatic response was achieved in the wide range of 0.001-0.05 M with a stability of more than 32 days. Cho and co-workers [184] developed a procedure for urea determination by crosslinking urease onto PANI-Nafion composite electrodes, which could sense the ammonium ions efficiently. Such a urea biosensor has a detection limit of about 0.5 pM and a response time of 40 seconds. 

The next group of multimembrane systems comprises membranes sensitized biologically using immobilized enzymes or microorganisms. Species that are directly sensed by an ISE are produced in the enzymatic reaction of the analyte. Examples of such sensors are those used for determination of urea in milk, based on immobilized urease and measurement of a pH change. An example of the application of bacteria strains is the use of immobilized recombinant Escherichia coli coupled with a pH electrode. Such electrodes have been used for determination of cephalosporins. When this bacterial strain is coupled with a CO2 gas sensor, glutamic acid determination can be carried out. 

There is also a solid-state version of the Severinghaus electrode which utilizes a Pd/PdO internal pH electrode [102] and Cl2-electrode based on an Ag/AgCl internal electrode [103]. Enzymatic Severinghaus electrodes include, for example, electrodes for the determination of urea [100] and lysin [104]. 

The most important degradative method for the determination of urea in the natural water samples is based on its conversion to carbon dioxide and ammonia by hydrolysis obtained with a nickel metalloenzyme (urease). In the manual procedure outlined by McCarthy [89] for the analysis in seawater, the enzymatic hydrolysis of urea was carried out at 50°C for 20 min, in the range of pH from 6.4 to 8.0, using a solution of crude lyophilized jack beam urease. After the samples were cooled at room temperature, NH4 concentration was determined by manual colorimetric method after cooling the samples at room temperature. The ambient concentration of NH4 and the analytical blank (NH4 contained in the reagents and in the urease solution) have to be subtracted for any sample to obtain the concentration of urea. In this reference study, the precision (RSD) was 1% at the concentration of urea equal to 1 pmol N A manual indirect methodology was also described by Katz and Rechnitz [209] and the method was revised in other following studies [9,53,197,198]. It persists with minor modifications in recent works on the field and in culture experiments [71,199-202] and for determination of isotope ratio in urea by elemental... 

In conclusion, the utilization of the enzymatic hydrolysis for the determination of urea in natural waters should be discouraged in high-resolution environmental studies, in particular when high salinity low-urea samples have to be analyzed. Its application in the monitoring of eutrophic aquatic habitats would require an accurate determination of the blanks, as well as salinity and internal standard corrections for each sample. 

A non-linear regression analysis is employed using die Solver in Microsoft Excel spreadsheet to determine die values of and in die following examples. Example 1-5 (Chapter 1) involves the enzymatic reaction in the conversion of urea to ammonia and carbon dioxide and Example 11-1 deals with the interconversion of D-glyceraldehyde 3-Phosphate and dihydroxyacetone phosphate. The Solver (EXAMPLEll-l.xls and EXAMPLEll-3.xls) uses the Michaehs-Menten (MM) formula to compute v i- The residual sums of squares between Vg(,j, and v j is then calculated. Using guessed values of and the Solver uses a search optimization technique to determine MM parameters. The values of and in Example 11-1 are ... 

H.C. Tsai and R.A. Doong, Simultaneous determination of pH, urea, acetylcholine and heavy metals using array-based enzymatic optical biosensor. Biosens. Bioelectron. 20, 1796-1804 (2005). 

Urea in kidney dialysate can be determined by immobilizing urease (via silylation or with glutaraldehyde as binder) on commercially available acid-base cellulose pads the process has to be modified slightly in order not to alter the dye contained in the pads [57]. The stopped-flow technique assures the required sensitivity for the enzymatic reaction, which takes 30-60 s. Synchronization of the peristaltic pumps PI and P2 in the valveless impulse-response flow injection manifold depicted in Fig. 5.19.B by means of a timer enables kinetic measurements [62]. Following a comprehensive study of the effect of hydrodynamic and (bio)chemical variables, the sensor was optimized for monitoring urea in real biological samples. A similar system was used for the determination of penicillin by penicillinase-catalysed hydrolysis. The enzyme was immobilized on acid-base cellulose strips via bovine serum albumin similarly as in enzyme electrodes [63], even though the above-described procedure would have been equally effective. 

Note that instruments 1-4 were photometric devices with less then 0.01 absorbance accuracy evaluated against reference neutral filters at 546 nm and A = 1.000, traceable to INM. The bandwidth provided by the interference filters equipping the absorption photometers was within the range of 4-10 nm. Instrument 5 was a 10 nm bandwidth photometric device with less than 1.0% absorbance linearity, evaluated at 405 nm and 500 nm, against liquid absorbance RMs type 16.02 and 16.03 [5], Enzymatic colorimetric methods for determination of glucose and urea were used. The o-cresoftalein colorimetric method was used for calcium determination. 

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