Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Patent
1996-07-18
1998-02-17
Gitomer, Raph
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
435962, C12Q 132
Patent
active
057190364
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND
1. Field of the Invention
The present invention relates to a method for quantitatively determining potassium ions which is useful for clinical tests.
2. Description of the Background Art
Japanese Unexamined Patent Publication/PCT No. 1-503596 discloses that a glycerol dehydrogenase may be used in a quantitative determination method for potassium ions. However, when ammonium ions are present in a sample, they interfere with a glycerol dehydrogenase reaction and thus an accurate determination cannot be achieved.
As a method for eliminating ammonium ions in a sample, there is known a Chemical Analysis II (Nitrogen-Containing Components), 2nd edition, Tokyo Kagaku Dojin Co., Ltd. (1979)!.
It is known that a glutamine synthetase produces glutamine in the presence of adenosine triphosphate (ATP) using glutamic acid and ammonium ions as Ltd. (1982)!. However, it has never been reported that the glutamine synthetase is used for the elimination of ammonium ions in a sample.
NADH or NADPH used in the method of eliminating ammonium ions in a sample by pretreating the sample with a glutamate dehydrogenase give an influence upon a glycerol dehydrogenase reaction, and raise the background. These facts constitute obstacles to the determination of potassium ions in the sample. Therefore, this method can not be applied to a method for quantitatively determining potassium ions in a sample with a glycerol dehydrogenase.
SUMMARY OF THE INVENTION
It is the object of the present invention to provide a method for quantitatively determining potassium ions using a glycerol dehydrogenase. Even though ammonium ion or hydroxylamine is contained in a sample, the method can quantitatively determine potassium ions of an extremely low concentrations.
The method of the invention for quantitative determination of potassium ions is characterized in that a sample is pretreated with a glutamine synthetase in the presence of glutamic acid and ATP prior to a method for quantitatively determining potassium ions in a sample using a glycerol dehydrogenase. This is illustrated schematically below. ##STR1##
According to the invention, it is possible to quantitatively determine potassium ions of extremely low concentrations even in a sample containing ammonium ions or hydroxylamine.
In the present invention, any sample may be used; for example, body fluids such as blood and urine.
Now, preferred embodiments of the method of the invention for quantitative determination of potassium ions will be described below.
An aqueous medium is added to a sample, if necessary, then, 1-10 mM ATP, 1-10 mM glutamic acid, 2-50 mM magnesium and 1-100 KU/liter, preferably 1-50 KU/liter of a glutamine synthetase are added to the sample. The resultant solution is incubated at 20.degree.-40.degree. C. for 3-5 minutes to thereby carry out a pretreatment. Then, preferably in the presence of a chelating agent, 0.2-50 mM coenzyme, 0.05-2 KU/liter of a glycerol dehydrogenase and 2-500 mM substrate for the glycerol dehydrogenase are added and reacted at 8.degree.-50.degree. C. for 1-5 minutes. By measuring the activity of the glycerol dehydrogenase, it is possible to quantitatively determine the corresponding potassium ions. When at least one of the coenzyme used for the reaction, the glycerol dehydrogenase and the substrate for the glycerol dehydrogenase can be added after the pretreatment reaction, two other substance(s) can be added prior to the pretreatment reaction.
As an aqueous medium, any buffer may be used as long as it does not contain ammonium ions nor potassium ions. For example, glycine buffer, Tris buffer, Good's buffer, Veronal buffer, barbital buffer and 20 mM or below borate buffer may be enumerated. Preferably, these buffers have a concentration of 20-1000 mM and a pH value of 7-9.5.
The glutamine synthetase used in the invention may be any glutamine synthetase as long as it belong to the Enzyme No. 6.3.1.2. For example, natural enzymes derived from animal organisms, plant seeds, microorganisms, etc. or enzymes obtained by modifying such natural
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Kayahara Norihiko
Tadano Toshio
Umemoto Masao
Gitomer Raph
Kyowa Medex Co., Ltd.
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