Urea, enzymatic determination

For the diagnosis of kidney function, creatinine determinations in serum are increasing in popularity over urea measurements, since the creatinine level is not affected by a high protein diet or by metabolic rate, as is the case for urea. Enzymatic methods use either creatinine amido-hydrolase (EC 3.5.2.10) or creatinine iminohydrolase (EC 3.5.4.21) ... 

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. 14.8 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.

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... 

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. 

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. 

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. 

Glucose [50-99-7] urea [57-13-6] (qv), and cholesterol [57-88-5] (see Steroids) are the substrates most frequentiy measured, although there are many more substrates or metaboUtes that are determined in clinical laboratories using enzymes. Co-enzymes such as adenosine triphosphate [56-65-5] (ATP) and nicotinamide adenine dinucleotide [53-84-9] in its oxidized (NAD" ) or reduced (NADH) [58-68-4] form can be considered substrates. Enzymatic analysis is covered in detail elsewhere (9). 

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. 

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. 

Total protein, albumin, urea (standard methods) and middle molecules (MM) were determined in citrated plasma [6]. The trypsin-like activity (TLA) of plasma was measured using the chromogenic peptide substrate (Z-glycyl-glycyl-L-arginine-4-nitroanilide) [7]. Evaluation of anti-enzymatic potential in plasma was based on concentrations of the main protease inhibitors -proteinase inhibitor (ttj-PI) and aj-macroglobulin (a -M). Student s t-test was used for statistical analysis. 

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. 

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. 

Urea is most commonly assayed by combined urease methods, in which the urea is first converted to two ammonium ions. The ammonium generated is then measured by either enzymatic or chemical methods. Urea nitrogen values determined by this method (mg/ml) are converted to urea values by the use of appropriate factors (2.14 for urea in mg/ml, 0.357 for urea in mmol/L) (Emeigh Hart and Kinter 2005). 

Enzymatic activity, however, is not merely associated with covalent structures, but chiefly with tertiary structure which is still more difficult to determine. The crucial role of tertiary structure is proved by the fact that denaturation brings about inactivation. Even with proteins which may be reversibly denatured, such as chymotrypsin and trypsin, activity is lost as long as denaturation persists. Ribonuclease appeared for a while to be an exception, since it was still active in 8 M urea. But it was shown later that phosphate ions, at a concentration as low as 0.003 M, and polyphosphates induced in urea-denatured ribonuclease spectral changes usually associated with refolding (164). It could then be assumed that ribonucleic acid, the actual substrate, was also able to refold the denatured form and prevent inactivation in this way. In other words, even in ribonuclease, the active center is probably not built by adjacent residues in a tail or a ring, but by some residues correctly located in space by the superimposed... 

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