Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Patent
1996-01-23
1999-10-05
Gitomer, Ralph
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
435 14, 435 15, C12Q 140
Patent
active
059622482
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a quantitative determination method for chloride ions in an organism which is applicable to clinical tests.
2. Description of the Prior Art
There is known a quantitative determination method for chloride ions comprising reacting an .alpha.-amylase with a chloride ion-containing sample and a substrate, such as p-nitrophenylmaltoside or starch, by utilizing the phenomenon that an .alpha.-amylase deactivated by a (1974)!. There is known another quantitative determination method for chloride ions wherein 2-chloro-4-nitrophenyl-.beta.-D-maltoheptaoside is used as a substrate in order to simplify the process for measuring the .alpha.-amylase activity in the above quantitative determination method
As a method for measuring the activity of an .alpha.-amylase, there is known a method comprising producing maltose by using an oligosaccharide as a substrate, converting the resultant maltose into glucose and quantitatively determining the glucose obtained to thereby measure the 705 (1979)!.
Since glucose coexisting in a sample influences a quantitative determination method for a substance in a sample, a method is disclosed in Japanese Unexamined Patent Publication No. 5-76397 wherein glucose in a sample is eliminated by using hexokinase or the like.
Out of the quantitative determination methods for chloride ions, the method using a synthetic substrate such as 2-chloro-4-nitrophenyl-.beta.-D-maltoheptaoside for measuring the activity of an .alpha.-amylase must use the two-point calibration method because linearity in calibration curves is hard to obtain. The known method by using starch as a substrate for an .alpha.-amylase reaction and measuring the reaction product, i.e. a reducing sugar such as glucose and maltose, cannot accurately determine the amount of chloride ions when a blood sample is used, because glucose and maltose are present in blood. Therefore, if a method of newly using a maltooligosaccharide as a useful substrate for an .alpha.-amylase reaction is employed, it will be impossible to achieve an accurate determination because of the interference of glucose and the like present in blood.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a quantitative determination method for chloride ions which can accurately determine the amount of chloride ions even when blood is used as a sample.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a calibration curve using maltopentaose as a substrate.
FIG. 2 is a calibration curve using maltotetraose as a substrate.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The method of the invention for quantitative determination of chloride ions relates to a method of determining, in an aqueous medium, chloride ions in a sample by using an .alpha.-amylase which has been deactivated by a chelating agent. The method of the present invention is characterized by adding to a sample in advance adenosine triphosphate and an enzyme having glucokinase activity to thereby eliminate glucose in the sample, deactivating the enzyme having glucokinase activity and determining the amount of glucose produced by a reaction of .alpha.-amylase activated by the chloride ions using an oligosaccharide as a substrate for the reaction.
The method of the invention has been achieved based on the following finding that, in the above method of determining in an aqueous medium chloride ions in a sample by using an .alpha.-amylase which has been deactivated by a chelating agent, the sample is pretreated, the oligosaccharide is used as substrate and the .alpha.-amylase activity activated by the chloride ion is determined by measuring the amount of glucose as a final product, the amount of chloride ions can be accurately determined. In particular, the method for the pretreatment of a sample has been established based on the following finding that a more accurate determination of chloride ions can be achieved by adding, to a sample, not only an enzyme having glucokinase activity for elimination of gl
REFERENCES:
patent: 4427771 (1984-01-01), Misaki et al.
patent: 5384247 (1995-01-01), Berry et al.
patent: 5719036 (1998-02-01), Tadano et al.
Levitzki A., The Allosteric Activation of Mammalian Amylase by Chloride, Eur J Biochem 41 171-180, 1974.
Ono T., A New Enzymatic Assay of Chloride in Serum, Clin Chem 34(3) 552-553, 1988.
Kayahara Norihiko
Tadano Toshio
Umemoto Jun
Gitomer Ralph
Kyowa Medex Co., Ltd.
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