Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
1998-09-17
2004-02-24
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
Reexamination Certificate
active
06696266
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to an assaying composition for high sensitive assay of a small amouunt of ammonia and/or ammonium ions using NAD synthetase. More particularly the present invention relates to a method for assaying ammonia and/or ammonium ions contained in a liquid sample which comprises subjecting the sample to a reaction in the presence of an NAD synthetase, deamidated NAD, ATP, Mg
2+
ions and/or Mn
2+
ions, a dehydrogenase capable of forming ammonia molecules from an amine substrate and an oxidized NAD, and the amine substrate, and then determining the amounts of the components consumed or generated by the reaction. The present invention further relates to an assaying composition for ammonia and/or ammonium ions comprising containing NAD synthetase, deamidated NAD, ATP, Mg
2+
ions and/or Mn
2+
ions, a dehydrogenase capable of forming ammonia molecules from an amine substrate and oxidized NAD, and the amine substrate.
BACKGROUND OF THE INVENTION
Ammonia and/or ammonium ions are generated during protein metabolism in vivo and are used for biosynthesis of other nitrogen compounds in the liver. Excess ammonia and/or ammonium ions are excreted in urine after synthesis of urea. If the liver is injured or fails, abnormal ammonia metabolism will occur and high level of ammonia and/or ammonium ions in blood will be observed. Consequently, assaying ammonia and/or ammonium ions in blood is an indicator of the liver failure. Prior known assay methods of ammonia and/or ammonium ions are not sufficiently sensitive and thus cause erroneous measurements.
Creatinine is important in the diagnosis of renal functions, however prior known assay have low sensitivity which results in incomplete diagnosis.
Hitherto known assay methods of ammonia and/or ammonium ions using NAD synthetase (EC 6. 3. 1. 5 and EC 6. 3. 5. 1) are: a method assaying directly a reduced NAD using oxidoreductase and its substrates from oxidized NAD generated by enzymatic action of NAD synthetase; a method for measuring a small amount of ammonia by colored formazan with diaphorase and tetrazolium salt in a reaction mixture; and a method for measuring generated colored hydrogen peroxide by combination of oxidase system therewith. The reaction systems are illustrated as follows. (Japanese Patent Unexamined Publication No. 59-198995, U.S. Pat. No. 4,767,712, ibid. U.S. Pat. No. 5,206,146, Japan Pat. Unexam. Publ. No.63-185378 and U.S. Pat. No. 4,921.786). In these reaction systems, neither P1 (oxidized product of S1) nor P3 (oxidized product of S3) reaction product generate the cycling reaction.
wherein:
E1: dehydrogenase which catalyzes a reaction with consuming oxidized NAD and substrate S1, while generating reduced NAD and P1.
E2: active substance which catalyzes a reaction with consuming reduced NAD and S2, while generating oxidized NAD and P2.
S1: reduced substrate of E1.
S2: oxidized substrate of E2.
P1: oxidized substrate of S1.
P2: reduced substrate of S2.
wherein:
E3: dehydrogenase which catalyzes a reaction with consuming coenzyme oxidized NAD and substrate S3, while generating reduced NAD and P3.
E4: active substance which catalyzes a reaction with consuming coenzyme reduced NAD and substrate S4, while generating oxidized NAD and P4.
E5: oxidase which catalyzes a reaction with oxidizing P4 by enzymatic action while generating hydrogen peroxide and S4.
S3: reduced substrate of E3.
S4: oxidized substrate of E4.
P3: oxidized substrate of S3.
P4: oxidized substrate of S4.
In assaying a small amount of ammonia or ammonium generated from decomposed creatinine by an action of creatinine deiminase in the assay of creatinine in a specimen, direct assay of reduced NAD has a problem due to lower sensitivity, and the method with assaying formazan is highly sensitive but has an adsorption problem of pigment to an equipment. Further, oxidase system is easily affected by reduced substance such as ascorbic acid and has turned out to be expensive due to the use of a number of enzymes.
Accurately assaying ammonium and/or ammonium ions present in minute amounts has been difficult until now. Consequently, there is a need for an inexpensive and high sensitive assay method and an assaying composition.
SUMMARY OF THE INVENTION
We have tried to solve the above problems in assaying a small amount of ammonia and/or ammonium ions by using NAD synthetase, and made efforts to find a highly sensitive assaying method. Namely, ammonia and/or ammonium ions and a deamidated-NAD are reacted by an action of NAD synthetase in the presence of ATP, Mg
2+
ions and/or Mn
2+
ions, under the combination of a dehydrogenase capable of forming ammonia molecules from an amine substrate and an oxdized NAD,such as L-amino acids dehydrogenase and D-amino acids dehydrogenase, with the amine substrate, to form AMP and oxidized NAD. The thus formed oxidized-NAD is reacted with the amine substrates by an action of a dehydrogenase capable of forming ammonia molecules from an amine substrate and an oxidized NAD, to construct ammonia cycling and form the oxidized substrate and ammonia and/or ammonium ions. The thus accumulated reduced NAD is directly or continuously measured, or the oxidized substrate formed from the substrate accompanied by the formation of reduced NAD is measured continuously, to successfully measure ammonia and/or ammonium ions with high sensitivity. The present invention was completed by finding the above method.
An object of the present invention is to provide a method for assaying ammonia and/or ammonium ions contained in a liquid sample which comprises subjecting the sample to a reaction in the presence of an NAD synthetase, deamidated-NAD, ATP, Mg
2+
ions and/or Mn
2+
ions, a dehydrogenase capable of forming ammonia molecules from an amine substrate and an oxidized NAD, and the amine substrate,and then determining the amounts of the components consumed or generated by the reaction.
Another object of the present invention is to provide an assaying composition for ammonia and/or ammonium ions comprising containing NAD synthetase, deamidated NAD, ATP, Mg
2+
ions and/or Mn
2+
ions, a dehydrogenase capable of forming ammonia molecules from an amine substrate and an oxidized NAD, and amine substrate.
The present invention is explained in details as follows.
A reaction system of the present invention is summarized as follows.
wherein:
E6: dehydrogenase which catalyzes a reaction generating reduced NAD, and P6 and ammonia molecules from substrate S6, and coenzyme oxidized NAD.
S6: reduced substrate of E6.
P6: oxidized product of S6.
Liquid samples or specimens of the present invention can be the samples containing ammonia (including ammonium ions), further the samples containing ammonia liberated or generated from the other enzyme reactions, which can be liberated or generated ammonia, or liberated or generated from acid or alkaline hydrolysis system, for example specimens such as blood, serum and urine. Examples of the above enzyme reaction systems can be any enzyme systems generating ammonia. Examples of enzyme reaction systems, in which their enzyme actions or substrates involved in these reactions are measured, are illustrated as follows.
(1) creatinine deiminase (EC 3. 5. 4. 21)
creatinine+H
2
O→N-methylhidantoin+NH
4
+
(2) guanine deaminase (EC 3. 5. 4. 3)
guanine+H
2
O→xanthine+NH
4
+
(3) adenosine deaminase (EC 3. 5. 4. 4)
adenosine+H
2
O→inosine+NH
4
+
Examples of a dehydrogenase capable of forming ammonia molecules from an amine substrate and an oxidized NAD, and the amine substrate are preferably L-amino acids dehydrogenase and its substrates. Any L-amino acids dehydrogenase, which uses coenzyme oxidized NAD formed by enzymatic reaction of NAD synthetase and substrate L-amino acids, and forms 2-oxo acids and ammonia from L-amino acids, can be used without limitation. Examples of enzymes and substrates will be illustrated hereinbelow and are enzymes and substrates such as amine substrate in
Etoh Takashi
Takahashi Mamoru
Asahi Kasei Pharma Corporation
Gitomer Ralph
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