JP2980811B2 - Determination method of ammonium ion - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for quantifying ammonium ions which is useful as a reagent for a clinical test.

[0002]

2. Description of the Related Art As a method for quantifying ammonium ions in a sample using an enzyme reaction, oxoglutarate and ammonium ions as substrates and reduced nicotinamide adenine dinucleotide (NAD) as coenzymes are known.
H) using a glutamate dehydrogenase reaction [Clinical Chemical Analysis II, pp. 57-59, 1979,
A method using a glutamine synthetase reaction using glutamate and ammonium ions as substrates in the presence of Tokyo Chemical Dojin] and adenosine triphosphate (ATP) is known [enzyme, Dickson Webb, p. 396,
1970, published by Hakusuisha].

It is known that glycerol dehydrogenase is activated by ammonium ions. [Enzyme, Dixon Webb, p. 396, 1970]
It is not known that it is used for the determination of ammonium ion.

[0004]

The method of quantifying ammonium ions in a sample using an enzymatic reaction using ammonium ions as a substrate has a low measurement sensitivity. Development is desired.

[0005]

SUMMARY OF THE INVENTION The present invention comprises reacting glycerol dehydrogenase with a substrate and a coenzyme of glycerol dehydrogenase in the presence of a sample containing ammonium ions, and measuring the amount of change in the substrate or coenzyme before and after the reaction. And a method for quantifying ammonium ions.

The principle of the present invention is based on the fact that the enzyme reaction is activated by ammonium ions, and the degree of activation is proportional to the concentration of ammonium ions.

The present invention is applicable to a case where a biological sample such as serum (136 to 147 mM of sodium ion and 3.5 to 5.0 mM of potassium ion) containing a large amount of contaminant ions such as sodium ion and potassium ion is used as a sample. Since the glycerol dehydrogenase reaction is not affected by these contaminant ions, a trace amount of ammonium ion as an activator of the enzyme can be determined.

The method of the present invention can be applied to any sample, for example, to a sample derived from a living body such as blood, serum, and urine.

The glycerol dehydrogenase used in the present invention is an enzyme belonging to the enzyme number [EC1.1.1.6.], Which is a natural enzyme such as a microorganism-derived enzyme, or modified by genetic manipulation, protein engineering or the like. Things can be used.

As a substrate for glycerol dehydrogenase, when a dehydrogenation reaction of the enzyme is used, a substrate used for dehydrogenation of glycerol dehydrogenase, for example, glycerol, 1,2-propanediol, 2,3-butanediol, preferably Use glycerol. Also,
When a reverse reaction (reverse reaction) of the dehydrogenation reaction of the enzyme is used, dihydroxyacetone or the like can be used.

As a coenzyme, nicotinamide adenine dinucleotide (NAD) is used. When a reverse reaction of glycerol dehydrogenase is used, reduced nicotinamide adenine dinucleotide (NADH) is used.

[0012] The method of measuring the amount of change in the substrate or coenzyme before and after the glycerol dehydrogenase reaction includes:
Substrate for glycerol dehydrogenase activity before and after the reaction,
Any method may be used as long as it measures the change in the product or coenzyme.In the case of a dehydrogenation reaction, for example, a method in which the change in a substrate such as glycerol is measured using glycerol oxidase, etc. Represents a method of measuring the amount of decrease in NAD or the amount of increase in NADH by spectrophotometry or fluorescence spectrometry. And the method of measuring the decrease in NADH or the increase in NAD by spectrophotometry or fluorometry.

Hereinafter, preferred embodiments of the present invention will be described. If necessary, add an aqueous medium to the sample, and then use 0.1% when using glycerol dehydrogenase dehydrogenation.
-100 mM, preferably 0.5-5 mM NAD and 10-500 mM, preferably 20-250 mM glycerol, 0.2--2 mM when the reverse reaction of the enzyme is used.
M NADH and 0.2 to 5 mM, preferably 0.5 to
After addition of 2 mM dihydroxyacetone, 0.05 to 2 KU / l of glycerol dehydrogenase is added, preferably in the presence of a chelating agent, at 8 to 50 ° C., preferably 15 to 40 ° C., for 1 to 10 minutes, preferably Is 1-5
After reacting for minutes, the corresponding ammonium ion can be quantified by measuring the increase or decrease of NAD or NADH.

As the aqueous medium, ammonium ion,
Any buffer may be used as long as it does not contain potassium ions or a large amount of borate ions, and examples thereof include Tris buffer, glycine buffer, veronal buffer, and barbital buffer. The concentration of these buffers is between 20 and
1000 mM, preferably 50-500 mM, p
H has a pH of 7 to 10, preferably 8 to 9.5.

The chelating agents used in the present invention include ethylenediaminetetraacetic acid (EDTA), 2-hydroxyethylethylenediaminetriacetic acid (HEDTA), 1,2-diaminocyclohexanetetraacetic acid, and nitrilotriacetic acid (NT).
A), diethylenetriaminepentaacetic acid (DTPA), glycol ether diamine ^{-N, N, N,, N} , - tetraacetic acid (GEDTA), ethylenediamine diacetic acid (EDD
A), dihydroxyethylglycine (DHEG), ethylenediaminedipropionate hydrochloride (EDDP), 1,
2-bis (o-aminophenoxy) ethane-N, N, N
^{,, N,} - tetraacetic acid tetra potassium salt (BAPT
A), hydroxyethyliminodiacetic acid (HIDA) and the like, which can be used at a concentration of 0 to 50 mM.

In the present invention, polyethylene glycol, alkyl phenyl ether,
A surfactant such as polyoxyethylene polyoxypropylene condensate and the like, albumin, magnesium chloride, calcium chloride and the like may be added.

[0017]

【Example】

Example 1 (1) Preparation of Ammonium Ion Standard Solution Ammonium chloride (manufactured by Wako Pure Chemical Industries, Ltd.) was diluted with distilled water, and ammonium ion concentrations of 0.1, 0.25 and 0.1.
5, standard solution for 0.75 mM ammonium ion calibration curve was prepared.

(2) Determination of ammonium ion A standard solution for ammonium ion calibration curve 0.050 was placed in a test tube.
glycerol (Boehringer Mannheim,
2.0 ml of 250 mM Tris-HCl buffer (pH 9, 25 ° C.) containing 20 mg / ml of high purity product and 1.5 mg / ml of NAD (Boehringer Mannheim) was added,
Preincubation was performed at 37 ° C. Next, 37
Aqueous solution (referred to as enzyme solution) containing 0.25 U / ml or 0.5 U / ml of glycerol dehydrogenase (manufactured by Oriental Yeast Co., Ltd.) incubated at ° C.
After adding 0 ml and stirring, the change in absorbance at 340 nm (2 to 3 minutes after the start of the reaction; hereinafter referred to as OD value) was measured with a spectrophotometer (UV3400, manufactured by Hitachi, Ltd.). The obtained calibration curve is shown in FIG.

According to FIG. 1, the calibration curve of ammonium ion showed linearity in the concentration range of 50 to 1000 μmol / l when the enzyme amount was 0.25 U / ml and 0.5 U / ml.

Example 2 In Example 1, sodium ion (106 m
M, 148 mM or 191 mM) was measured in the same manner, but the OD value of the glycerol dehydrogenase activity obtained was the same as the OD value obtained in Example 1.

Example 3 In Example 1, 150 mM of sodium ion was added to the sample.
The measurement was performed in the same manner except that 3.9 mM or 7.8 mM of potassium ion was added, and the OD value of the glycerol dehydrogenase activity obtained was the same as the OD value obtained in Example 1. .

From the above, it was found from Examples 2 and 3 that the measurement of ammonium ions was not affected by the contaminant ions.

[0023]

According to the present invention, there is provided a quantitative method capable of measuring a low concentration of ammonium ion even in the presence of contaminating ions.

[Brief description of the drawings]

Fig. 1 Calibration curve of ammonium ion

[Explanation of symbols]

 ─ ○ ─ ・ ・ ・ ・ ・ ・ ・ ・ ・ Enzyme 0.5 U / ml used ─ ● ─ ・ ・ ・ ・ ・ ・ ・ Enzyme 0.25 U / ml used

Claims (6)

(57) [Claims] 1. A method for reacting a glycerol dehydrogenase with a glycerol dehydrogenase substrate and a coenzyme in the presence of a sample containing an ammonium ion, and measuring a change in the substrate or coenzyme before and after the reaction. Quantitation method. 2. The method according to claim 1, wherein the coenzyme is nicotinamide adenine dinucleate.
Reotide or reduced nicotinamide adenine dinucleotide
The method according to claim 1, wherein the quantification is otide. 3. Substrate of glycerol dehydrogenase
Is glycerol, 1,2-propanediol, 2,3-butane
From the group consisting of tandiol and dihydroxyacetone
The method according to claim 2, which is selected. 4. Glycerol dehydrogenase and glycerol
Roll, 1,2-propanediol and 2,3-butanegio
Glycerol dehydrogen selected from the group consisting of
Substrate of ninase and nicotinamide adenine dinucleotide
Sulfide in the presence of a sample containing ammonium ions
The amount of substrate change before and after the reaction
Reduction or reduction of tinamide adenine dinucleotide
To increase the amount of the original nicotinamide adenine dinucleotide
Determination of ammonium ion characterized by measurement
Law. 5. Glycerol dehydrogenase, dihydrogen
Roxyacetone and reduced nicotinamide adenine
Nucleotides in the presence of samples containing ammonium ions
The reaction is performed under
Seton change or reduced nicotinamide adenine
Nucleotide reduction or nicotinamide adenine
Measuring the amount of increase in dinucleotides.
Determination method of ammonium ion. 6. Glycerol dehydrogenase, glycerol
Roll and nicotinamide adenine dinucleotide?
Quantitative reagent for ammonium ion.