Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Patent
1997-10-16
2000-12-19
Prats, Francisco
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
435 18, 435 25, 424617, 424618, 424630, 424646, 2525193, 25251932, 2525195, 2525203, 2525212, 436 79, 436 80, 436 81, 436 84, 536 2624, 536 2625, C12Q 132, A61K 3324, H01B 112, C07H 19207
Patent
active
061626155
DESCRIPTION:
BRIEF SUMMARY
The invention concerns stabilized aqueous solutions of a coenzyme for hydrogen-transferring enzymes as well as their use for determining a corresponding analyte (substrate) in a reduced form or the enzyme activity of a corresponding dehydrogenase. The stabilized solution contains a heavy metal salt and optionally a completing agent as a further component and hence enables the determination in an alkaline environment preferably at a pH value of 8.5 to 10.0.
The determination of enzyme activities (or substrate concentrations) in particular in blood serum or blood plasma plays an important role in clinical chemical diagnostics. Test procedures are often used for this which are based on the reduction of nicotinamide adenine dinucleotide ("NAD") or nicotinamide adenine dinucleotide phosphate ("NADP") and the photometric detection of the change of absorbance behaviour in the ultraviolet wavelength range (.lambda.=334, 340 or 365 nm) which occurs in this process. When. choosing suitable test conditions this change is linearly proportional to the enzyme activity (or substrate concentration) to be determined.
The method described in Eur. J. Chin. Chem. Chin. Biochem. 31, 897 (1994) and Eur. J. Chin. Chem. Chin. Biochem. 32, 639 (1994) is nowadays generally recommended to determine the enzyme activity of for example lactate dehydrogenase (LDH, E.C.1.1.1.27). The test principle is that lactate is oxidized to pyruvate with the simultaneous reduction of a coenzyme such as NAD or NADP to NADH or NADPH. Such a conversion, which in this case is for example catalysed by LDH, takes place in an alkaline medium (pH 9.4) i.e. under conditions under which it is known that NAD or NADP is unstable. This instability is manifested by a relatively rapid increase in absorbance (the so-called reagent blank) in the relevant wavelength range for the measurement so that the reagent combination already becomes useless after a short period (3 months) even with refrigerated storage (2.degree. to 8.degree. C.). This is a particular problem for the production of ready-to-use liquid reagents with long-term stability which are intended to enable the user to carry out analyses in the daily routine as simply and reliably as possible.
A process for stabilizing aqueous coenzymes using chelating agents and azides is known from JP 84/82398. However, a disadvantage of this process is that it is necessary to add azide which on the one hand has been classified as cancerogenic and in addition has an inhibitory effect on many enzymes. Moreover this stabilization process has clear limits at higher pH values and temperatures.
The object of the present invention is therefore to provide an improved stable liquid reagent containing a coenzyme for hydrogen-transferring enzymes which is suitable for the determination of dehydrogenase activity or of corresponding substrates.
The invention is achieved by an aqueous solution which contains a heavy metal salt at a concentration of approximately 1 to 60 mM preferably at a concentration of 2 to 10 mM. The pH value of corresponding working solutions is in the alkaline range preferably above pH 8.5, particularly preferably between pH 8.5 and 10.0. Working solutions are understood according to the invention to mean that the stabilized coenzyme solution is mixed with a solution which is essentially composed of a substance buffering in the alkaline range. It has proven to be advantageous that the ratio of stabilizer to buffer solution is ca. 1:5. Water-soluble salts of acids with heavy metal ions such as e.g. Cu.sup.2+ and/or Co.sup.2+ and also other heavy metals such as for example iron, manganese, zinc, nickel, silver, magnesium, calcium or palladium have proven to be suitable as the heavy metal salt. The acid residues of common acids known to a person skilled in the art come into consideration as the counterion i.e. the so-called acid anion such as for example sulfate, phosphate, the acid residues of carboxylic acids and halogenides. Copper and cobalt sulfate and/or the corresponding chlorides have proven to be particul
REFERENCES:
patent: 3331752 (1967-07-01), Struck et al.
patent: 4162194 (1979-07-01), Pierre et al.
patent: 5036000 (1991-07-01), Palmer et al.
Green et al, Can. J. Biochem. 57:995-999 (1979).
Prats Francisco
Roche Diagnostics GmbH
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