Process and reagent for the fully enzymatic determination of ure

Details Process and reagent for the fully enzymatic determination of ure Process and reagent for the fully enzymatic determination of ure

1986-01-10

1988-02-23

Rosen, Sam

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving urea or urease

435 15, C12Q 158

Patent

active

047270250

DESCRIPTION:

BRIEF SUMMARY
DESCRIPTION

The invention concerns a process and reagent for the enzymatic determination of urea, especially in body fluids, such as serum, plasma and urine.
Within the scope of analytical chemistry, the quantitative determination of urea plays an especially important part in clinical-chemical diagnosis. The urea concentration in body fluids, such as serum or urine, gives the clinical physician important indications regarding the kidney function, furthermore in the case of dialysis patients regarding the degree of action of extra- and intracorporeal blood washing.
In comparison with purely chemical or partly enzymatic photometric determination processes (e.g. the methods used especially frequently with the use of Fearon's reagent or of the urease/Berthelot reaction (for the description see e.g. R. J. Henry, D. C. Cannon, J. W. Winkelmann (eds.), Clinical Chemistry: Principles and Techniques, 2nd edition, Harper and Row, Hagerstown, Md., U.S.A. (1974), pages 503-526), fully enzymatic analysis methods achieve more and more importance. Their important advantages, in comparison with the above-mentioned processes, are the high specificity, their nm-disturbance, e.g. towards medicament influences, temperatures and pH variations, the avoidance of the use of corrosive, strongly acidic or alkaline or toxic reagents, their universal usability not only for the manual carrying out of the test but also on the most varied automatic analysers, as well as the possibility to be able to evaluate the measurement results, without the concurrent use of standards, with the help of precisely defined conversion factors, whereby the conditions of the Lambert-Beer Law are, in principle, ensured over the whole extinction range usable on the photometer.
Hitherto, two different processes have been known for the fully enzymatic urea determination.
In one of these processes, urea is split hydrolytically with urease E.C. 3.5.1.5 into two molecules of ammonia and one molecule of CO.sub.2 ; from the resultant ammonia, there is formed glutamate in the presence of .alpha.-ketoglutarate, NADH and glutamate dehydrogenase E.C. 1.4.1.3. The decrease of the NADH concentration in the reaction mixture, which can be measured photometrically at 334, 340 or 365 nm, is proportional to the amount of urea used (H. U. Bergmeyer (ed.), Methoden der enzymatischen Analyse, 3rd edition, Vol. II, Verlag Chemie, Weinheim (1974), p. 1842). According to the second process (U.S. Pat. No. 3,655,516), urea is hydrolysed in an ATP-dependent reaction by means of urea amidohydrolase E.C. 6.3.4.6, whereby one molecule of ADP is formed per mole of reacted urea. This is converted back into ATP with phosphoenol pyruvate and pyruvate kinase E.C. 2.7.1.40 and the liberated pyruvate is reduced to lactate in the presence of NADH and lactate dehydrogenase E.C. 1.1.1.27. As in the first-mentioned process, the measurement parameter is here also the decrease of the NADH concentration in the reaction mixture.
Of the two processes, up to today, only the first has found wide use in routine analysis.
In spite of the marked advantage which the fully enzymatic determination of urea has provided in comparison with the purely chemical or partly enzymatic methods of analysis, nevertheless both the abovementioned enzymatic processes display several substantially identical disadvantages: On the one hand, NADH is relatively unstable in aqueous solution, even in the neutral pH range, so that the storage stability of the reagent solutions ready for use is only ensured for a comparatively short period of time, i.e. about 1 day at room temperature or 3 days in the case of storage at 2.degree. to 8.degree. C. Furthermore, test batches according to the sample/reagent blank measurement process sometimes give rise to falsely increased urea values if it is necessary to work with sample materials which are turbid or absorb strongly in the range of 334 to 365 or if comparatively high concentrations are present of ammonia or pyruvate which are always present in traces in serum or urine. Thus, for exact

REFERENCES:
patent: 4465770 (1984-08-01), Modrovich
Trijbels et al.--Chem. Abst., vol. 66, (1967), p. 43898d.
Vander Drift et al.--Chem. Abst., vol. 75, (1971), p. 84644x.

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