Urease generation

Biopsy (rapid) urease HP urease generates ammonia, which causes a color change Test of choice at endoscopy >90% sensitive and specific easily performed rapid results (usually within 24 hours) tests for active HP infection antibiotics, bismuth, and PPIs may cause false-negative results test may yield false-negatives in active ulcer bleeding available as gel tests, paper tests, and tablets... 

In order to make a useful biosensor, enzyme has to be properly attached to the transducer with maintained enzyme activity. This process is known as enzyme immobilization. The choice of immobilization method depends on many factors such as the nature of the enzyme, the type of transducer used, the physiochemical properties of analyte, and the operating conditions [73]. The major requirement out of all these is its maximum activity in immobilized microenvironment. Enzyme-based electrodes provide a tool to combine selectivity of enzyme toward particular analyte and the analytical power of electrochemical devices. The amperometric transducers are highly compatible when enzymes such as urease, generating electro-oxidizable ions, are used [74]. The effective fabrication of enzyme biosensor based on how well the enzyme bounds to the transducer surface and remains there during use. The enzyme molecules dispersed in solutions will have a freedom of their movement randomly. Enzyme immobilization is a technique that prohibits this freedom of movement of enzyme molecules. There are four basic methods of immobilizing enzymes on support materials [75] and they are physical adsorption, entrapment, covalent bonding, and cross-linking, as shown in the Fig. 36. 

Recently, the old alkaline phenol method has been revived, and is being widely used in clinical laboratories, without protein preclpltatlon(27). In this procedure, the serum is added to an alkaline phenol reagent, and the ammonia generated from urea is determined either after the action of urease or after strong alkaline treatment of the serum. The objection to this procedure is first, that all urease is rich in ammonia, and second, the color produced with alkaline phenol is not specific for ammonia. It will react with other compounds, especially for those that liberate ammonia. By this procedure one obtains a useful number from the point of view of determining whether the patient has nitrogen retention, but a value which is somewhere between a urea and an N.P.N. determination. 

The above discussion does not mean that the use of urease and subsequently the use of an ammonia electrode is not practicable for a urea determination. Unfortunately, the commercial company that produced the urea analyzer, chose a conductivity procedure, which happens to be unsuitable for the laboratory of Neonatology. Had they chosen the ammonia electrode, which happens to be a relatively good electrode, and is especially specific, since only ammonia and not potassium can pass an air space, then the instrument could have been made highly specific for urea. In this case an ammonia determination would be done initially and then subtracted by the computer, from the amount which has been generated subsequently. In any case, with present technology, sensitivity is not adequate to use less than approximately 15fil of serum. 

Enzyme sensors are based primarily on the immobilization of an enzyme onto an electrode, either a metallic electrode used in amperometry (e.g., detection of the enzyme-catalyzed oxidation of glucose) or an ISE employed in potentiometry (e.g., detection of the enzyme-catalyzed liberation of hydronium or ammonium ions). The first potentiometric enzyme electrode, which appeared in 1969 due to Guilbault and Montalvo [140], was a probe for urea with immobilized urease on a glass electrode. Hill and co-workers [141] described in 1986 the second-generation biosensor using ferrocene as a mediator. This device was later marketed as the glucose pen . The development of enzyme-based sensors for the detection of glucose in blood represents a major area of biosensor research. 

Tor [7] developed a new method for the preparation of thin, uniform, self-mounted enzyme membrane, directly coating the surface of glass pH electrodes. The enzyme was dissolved in a solution containing synthetic prepolymers. The electrode was dipped in the solution, dried, and drained carefully. The backbone polymer was then cross-linked under controlled conditions to generate a thin enzyme membrane. The method was demonstrated and characterized by the determination of acetylcholine by an acetylcholine esterase electrode, urea by a urease electrode, and penicillin G by a penicillinase electrode. Linear response in a wide range of substrate concentrations and high storage and operational stability were recorded for all the enzymes tested. 

Figure 15.2 Reaction mechanism of urease. Ni 1 binds urea and acts as a Lewis acid to polarise the carbonyl group, making its carbon more electrophilic, while Ni 2 facilitates deprotonation of a bound water molecule to generate a nucleophilic hydroxyl species. (From Ragsdale, 1998. Copyright 1998, with permission from Elsevier.)...

The electrode is an ammonium ion-selective electrode surrounded by a gel impregnated with the enzyme urease [Fig. 6.13 (34)]. The generated ammo-... 

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). 

Fig. 8 Synthesis of amino acids by a multienzyme system consisting of leucine dehydrogenase (LeuDH) catalyzing the reductive amination of the corresponding keto acid, L-lactate dehydrogenase (l-LDH), and lactate for the regeneration of NADH and urease for the in situ generation of ammonia. The coenzyme NAD+ was covalently bond to dextran, enzymes and dextran-coupled NAD+ were...

M Polarization of the carbonyl bond by coordination activates it for nucleophilic attack. This situation is assumed to occur by the bridging hydroxide, which appears to be most suitably arranged when the picolinamide-0 is located in a bridging position, although its transient shift to a terminal site cannot be ruled out. The failure of 32 to hydrolyze picolinamide confirms that generation of a nucleophilic hydroxide is required for the reaction to proceed (82). In subsequent work, 33 was then found to serve as a valuable functional model for the urease active site (see Section III.B.4). 

Numerous dinickel complexes have been reported as models for urease. As depicted in Scheme 5, a dinickel complex [Ni2( 4-0H)( j,-H20)(bdptz)(H20)2][0Ts]3 is capable of hydrolyzing a bound amide substrate by intramolecular nucleophilic attack of a coordinated hydroxide ion, either from a bridging position or from a transient terminally bound form. This amide hydrolysis mimics the hydrolysis of urea by urease in that a hydroxide nucleophile is generated by the dinickel center, and the coordination of the substrate to the dinickel center as well as a nickel-bound hydroxide ion serving as the nucleophile are crucial to hydrolysis. Protonation of the amine group by an acidic residue in the active-site results in loss of ammonia. 

Urea undergoes hydrolysis in the presence of acids, bases, or the enzyme urease (isolable from jack beans generated by many bacteria, such as Micrococcus ureqey... 

Enzymatic methods for the measurement of urea are based on preliminary hydrolysis of urea with urease (urea amidohydrolase, EC 3.5.1.5 main source jack bean meal) to generate ammonium ion, which is then quantified. This approach has been used in end-point, kinetic, conductimet-ric, and dry chemistry systems. ... 

By co-immobilization of a proton-consuming enzyme. This was successfully done with urease, where in situ formed ammonia neutralized the generated protons in an extraordinarily effective manner [99]. 

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