JPH0671437B2 - Reagent for quantifying magnesium ion - Google Patents

Description

TECHNICAL FIELD The present invention relates to a magnesium ion quantification reagent for quantifying magnesium ion in a reagent.

(Prior Art) At present, the determination of magnesium ion is roughly divided into two. One is quantification by instrumental analysis method typified by atomic absorption method, and the other is quantification by chemical method such as xyrelidyl blue method. The former method gives relatively accurate results, but requires specially expensive equipment,
The operation is also complicated. Further, in the latter quantification, there is a problem in that the effect of coexisting substances is unavoidable and only a result including a large error factor is obtained because of lack of specificity for magnesium ion.

In order to solve this, various reagents for quantifying magnesium ions have been proposed in recent years that utilize an enzymatic reaction. This is a reagent utilizing the fact that a kinase enzyme such as hexokinase expresses its activity using a complex of magnesium and adenosine 5'-triphosphate as a substrate. Specific examples include reagents that utilize a coupling reaction with hexokinase (hereinafter abbreviated as HK) glucose-6-phosphate dehydrogenase (hereinafter abbreviated as G6PDH) [Methods of Enzymatics, Analysis Third Edition 592- 597
(1985). ].

In addition, glucokinase, which is an enzyme similar to HK (hereinafter Glc
Reagents utilizing the conjugation reaction between G6PDH and G6PDH have also been proposed [see the 25th Annual Meeting of the Japan Society for Clinical Chemistry, p. 75 (1985) or JP-A-62-151200]. ].

Further, glucerose kinase (hereinafter abbreviated as GlyK), glycerol 3-phosphate oxidase (hereinafter abbreviated as GOD) and peroxidase (hereinafter abbreviated as POD)
Reagents using three enzyme reaction systems have been proposed [Clinica Chemistry, 32, No. 4, 629, (1986).
reference. ].

On the other hand, although it is not a reagent for quantifying magnesium ions,
A composition containing a chelating agent has also been proposed (Japanese Patent Publication Sho
60-27516, JP-B-60-36760, JP-B-61-17467, JP-A-57
-102185, JP-A-62-104598. ).

However, the chelating agent in these compositions is merely added for the purpose of stabilizing the composition.

(Problems to be Solved by the Invention) In the above-mentioned reagents for quantifying magnesium ion, magnesium ion is converted into adenosine 5'-triphosphate (hereinafter referred to as "adenosine 5'-triphosphate").
It is quantified as a complex with ATP).

However, in addition to magnesium, ATP forms a complex with divalent metal ions such as manganese, calcium, and zinc, and these can also serve as substrates for enzymes, so these metal ions coexist in the sample. Then, there is a risk that an error may occur in the determination of magnesium ion.

(Means for Solving Problems) The inventors of the present invention have conducted extensive studies to solve such problems, and as a result, if divalent metal ions other than magnesium are masked with a chelating agent, precise magnesium is obtained. The inventors have found that it is possible to quantify the ion concentration and completed the present invention.

That is, the gist of the present invention is a reagent for quantifying magnesium ion which is quantified as a complex with magnesium 5'-triphosphate, and which contains a chelating agent.

The basic measurement principle of the present invention is not to directly quantify magnesium ions, but to utilize an enzymatic reaction in which a complex is once formed with ATP and the complex is used as a substrate. The following three examples can be given as such specific examples.

(In the formula, ATP is adenosine 5'-triphosphate, ADP is adenosine 5'-diphosphate, NADP ⁺ is oxidized nicotinamide adenine dinucleotide phosphate, HK is hekikinase, and G6PDH is glucose-6-phosphate dehydration. Represents an elementary enzyme.) (In the formula, GlcK represents glucokinase.) (In the formula, GlyK is glycerol kinase, GOD is glycerol-3-phosphate oxidase, POD is peroxidase, 4AA is 4-aminoantipyrene, and HDCBS is 2-nitrodoxy-3.5.
-Represents dichlorobensulfonic acid. ) The measurement is to measure the increase in absorption of NADPH produced by at 340 nm and the increase of absorption of red product at 510 nm. Regarding the concentration of each component of these reagents, first, for a system using HK and GlcK, HK or GlcK was 0.1 to 50u / ml, G6PDH was 0.1 to 50u / ml, and ATP was 0.
It is preferable that the content is 01 to 20 mM, NADP is 0.1 to 20 mM, and glucose is 1 to 100 mM. Especially HK or GlcK is 0.1-10u / ml,
G6PDH 0.2-10u / ml, ATP 0.05-10mM, NADP 0.2-10m
M and glucose are preferably 1 to 50 mM.

In the system using GlyK, GlyK is 0.01 to 0.5u / m.
l, GOD 1-50u / ml, POD 0.1-50u / ml, ATP 0.5-5mM, 4
AA 0.1 to 5 mM, HDCBS 0.05 to 50 mM, glycerose 0.1 to
It is preferably 10 mM. Especially GlyK 0.01 ~ 0.1u / ml,
GOD 1-20u / ml, POD 0.1-10u / ml, ATP 0.1-5mM, 4AA
0.1 to 3 mM, HDCBS 0.05 to 10 mM, glycerol 0.2 to 5 m
It is preferably M.

The source of HK and GlcK used in the present invention is not limited, and various sources such as those derived from microorganisms and those derived from animals can be used.
Yeasts are preferable for HK, and microorganisms such as Bacillus are preferable for GlcK. The source of GlyK is not limited, and those derived from microorganisms and animals and plants can be used. Among them, those derived from microorganisms such as Bacillus and Candida are preferable. The source of other coupling enzymes is not limited, and those derived from microorganisms and animals can be used.

Further, in addition to the above, stabilizers, excipients, preservatives, activators and the like can be used without any trouble.

As the chelating agent used in the present invention, any chelating agent having a chelate stability constant represented by a logarithmic value with magnesium and other divalent metal ions having a difference of 2 or more may be used. Examples include ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA), 1,2-bis (O-aminophenoxy) ethane-N, N, N ', N'-tetraacetic acid (hereinafter B
APTA), trans-1,2-cyclohexanediamine-N, N, N ', N'-tetraacetic acid (hereinafter abbreviated as CyDTA), dihydroxyethylglycine (hereinafter abbreviated as DHEG), ethylenediamine-N, N'-dipropionic acid (hereinafter abbreviated as EDDP), glycol ether diamine-
Examples thereof include N, N, N ′, N′-tetraacetic acid (hereinafter abbreviated as GEDTA), hydroxyethyliminoniacetic acid (hereinafter abbreviated as HIDA), and nitrilotriacetic acid (hereinafter abbreviated as NTA).
The amount used is preferably about 0.001 to 10 mM.

To measure magnesium ions using the reagent of the present invention, for example, the reagent is kept at the measurement temperature (any of 25 ° C., 30 ° C., and 37 ° C.) for about 3 minutes, and then the cell chamber is kept warm for spectrophotometry Put in a meter, add the sample, mix, and measure the increase in absorbance. The reaction time after adding the sample can be arbitrarily selected from 1 to 30 minutes, but in order to adapt to the measurement conditions of the automatic analyzer, etc.
About 5 minutes is preferable.

(Examples) The present invention will be specifically described below with reference to Examples.

Example 1, Comparative Example 1 ATP1mM, NADP0.5mM, Glucose 20mM, GEDTA1mM, HK (from Baker's yeast, purchased from Oriental Yeast Co., Ltd.) 0.6u / ml, G6PDH (from Baker's yeast, Oriental Yeast Co., Ltd.) (Purchased from K.K.) 0.5u / ml of reagent for quantitative determination of magnesium was prepared.

Using this reagent, a manganese ion addition test was conducted to investigate the effect of manganese ion.

The measurement was carried out by adding 0.01 ml of Mg standard solution to 3 ml of reagent preheated at 30 ° C for 3 minutes in a cuvette with an optical path length of 1 cm, and measuring the absorbance at 340 nm for 3 minutes, and then using manganese as the concentration in the sample. The effect was investigated by adding 0.4 mM.

For comparison, a manganese ion addition test was performed using the above reagent containing no GEDTA.

The results are shown in FIG. FIG. 1 shows the amount of change in absorbance for 3 minutes on the vertical axis, and the manganese concentration (mM) added on the horizontal axis. In the figure, the ○ marks indicate Example 1 and the ● marks indicate Comparative Example 1. There is.

From Fig. 1, it is clear that when the chelating agent GEDTA is not used, a positive error is apparently caused by the addition of manganese ion, but this error is avoided by the addition of GEDTA.

This indicates that the quantitative determination of magnesium ions in the presence of manganese ions leads to a dramatic improvement in accuracy by the addition of the chelating agent GEDTA.

Example 2, Comparative Example 2 All were the same as Example 1, Comparative Example 1 except that HK was replaced with GlcK (derived from Bacillus stearothermophilus, purchased from Seikagaku Corporation) and GEDTA was replaced with HIDA. Using a reagent, we investigated the effect of divalent iron ions.

The results are shown in FIG. FIG. 2 shows the amount of change in absorbance for 3 minutes on the vertical axis, and the divalent iron concentration (mM) added on the horizontal axis. In the figure, the circles show Example 2 and the circles show Comparative Example 2. Is shown. From Fig. 2, the effect of divalent iron ion was not observed in the reagent to which the chelating agent HIDA was added, but a positive error was clearly observed in the reagent without HIDA.

Example 3, Comparative Example 3 Glycerose 5 mM, GlyK (derived from genus Gandida, purchased from Boehringer Mannheim Yamanouchi Co., Ltd.) 0.25 u / ml, GOD (derived from microorganism, purchased from Boehringer Mannheim Yamanouchi Co., Ltd.) 5 u / ml, .POD (Derived from horseradish, purchased from Oriental Yeast Co., Ltd.) 1u / ml, ATP 0.3mM, 4AA 1mM, HDCBS 0.5m
A reagent for determination of magnesium ion consisting of M and BAPTA 0.5 mM was prepared.

In order to investigate the effect of calcium ion using this reagent, a calcium ion addition test was conducted. The measurement was carried out by adding 0.01 ml of the sample to 3 ml of reagent preheated at 37 ° C. for 3 minutes in a cube having an optical path length of 1 cm, and examining the change in absorbance at 510 nm for 3 minutes.

For comparison, the same addition test was performed with reagents that did not contain BAPTA.

The results are shown in FIG.

FIG. 3 shows the amount of change in absorbance for 3 minutes on the vertical axis and the calcium concentration (mM) to be added on the horizontal axis. In the figure, ○ indicates Example 3 and ● indicates Comparative Example 3. ing.

From this experimental result, it was confirmed that the chelating agent is effective in avoiding the influence of calcium ions on the measurement of magnesium.

(Effects of the Invention) The reagent for quantifying magnesium ions of the present invention uses a chelating agent having an affinity for divalent metal ions other than magnesium in order to avoid the influence of divalent metal ions other than magnesium. It does not affect the quantification and can eliminate measurement errors due to other metal ions.

[Brief description of drawings]

FIG. 1 to FIG. 3 are diagrams showing the effects of metal ions on the reagent of the present invention. In each figure, the vertical axis represents the amount of change in absorbance for 3 minutes, and the horizontal axis represents the metal added. Shows the concentration of.

Front Page Continuation (72) Inventor Takashi Ochi 1144 Owada Nitta, Yachiyo-shi, Chiba, inside the Yatron Yachiyo Factory (72) Inventor Hiroshi Suzuki 1144 Owada Nitta, Yachiyo, Chiba Inspector, Yatron Yachi Co., Ltd. Akira Ito

Claims (1)

[Claims] 1. A reagent for quantifying magnesium ion, which comprises a chelating agent in a reagent for quantifying magnesium ion as a complex with adenosine 5'-triphosphate, which comprises a chelating agent.