JPH0659240B2 - Method for quantifying magnesium and composition therefor - Google Patents

Description

The present invention relates to a method for quantifying magnesium and a composition therefor. More specifically, it relates to a method for quantifying magnesium, which comprises reacting a sample with two types of enzymes and allowing a chelating agent to be present in a system for quantifying magnesium, and to improve a composition for the method.

BACKGROUND OF THE INVENTION At present, various amounts of magnesium in body fluids and urine are frequently measured in hospital laboratories. Known causes of hypomagnesemia and magnesium deficiency include malabsorption in the intestinal tract, endocrine disorders, and long-term administration of diuretics. In addition, renal dysfunction causes hypermagnesemia and excess magnesium. Measuring the amount of magnesium in body fluids and urine helps in the diagnosis of these diseases, but conventional measurement methods do not provide accurate and simple measurement values, and therefore better measurement methods can be found. is necessary.

[Prior Art and Its Problems] Currently, the following 5 methods are available for quantifying magnesium in body fluids.
Two methods are known. That is, the atomic absorption method,
Flame photometric method, xylidine blue method, enzymatic method using hexokinase and glucose-6-phosphate dehydrogenase (JP-A-61-124398), enzymatic method using glycerol kinase and glycerophosphate (Clin. Chem., 32 (4)
629 (1986)).

The method and method can be said to be a standard method, and an accurate measurement value can be obtained, but they have the drawbacks that the equipment is expensive, the measurement requires skill, the sample needs to be diluted, and it takes time.

The method is a measurement method currently used in hospital laboratories,
Although it is a colorimetric method using xylidine blue, it has low specificity for magnesium and causes large variations in the obtained values. However, there are some problems such as the fact that it is necessary to appropriately dilute the sample if a high value is expected.

The law avoids the drawbacks of the law. However,
Since the enzymatic reaction is stopped and the ultraviolet absorbance at 340 nm is obtained, there are drawbacks such that the sample is easily affected by the cloudiness and the reaction takes a relatively long time.

The method is a visible colorimetric method, and although the drawbacks of the method have been solved, glycerol kinase has a high affinity for magnesium, so only a sample with a low magnesium concentration can be quantified. Therefore, it is necessary to dilute and quantify a urine sample having a high magnesium concentration.

As described above, all the methods have drawbacks and cannot be said to be satisfactory quantitative methods.

[Means for Solving the Problem] The inventors of the present invention have earnestly studied to find a method for quantifying magnesium up to a high concentration range in a short time without being affected by the turbidity of a sample by a magnesium quantification method using an enzyme. As a result, the inventors have found that the object can be achieved by the presence of a chelating agent in the method, and have completed the present invention.

Generally, when a chelating agent is present in the reaction system of a metal-dependent enzyme, the metal is trapped and the enzymatic reaction is stopped. Surprisingly, the glycerol kinase used in the present invention is present in the presence of the chelating agent. However, it was found that the activity was proportional to the amount of magnesium and that it could be quantified even in a high concentration range which could not be quantified in the past. This is completely unexpected from the relationship between conventional metal-dependent enzymes and chelating agents.

[Constitution of the Invention] Accordingly, the present invention measures hydrogen peroxide produced by reacting a glycerol kinase and glycerophosphate oxidase in the presence of adenosine-3-phosphate (hereinafter abbreviated as ATP) and glycerol in a sample. Thus, the present invention relates to a method for quantifying magnesium, characterized in that a chelating agent having a concentration of 50 mM or less is present in a system for quantifying magnesium in a sample, and a composition therefor.

The most characteristic feature of the present invention is that a chelating agent is present in the reaction system. By adding a chelating agent,
ATP and a chelating agent are competitively antagonized via magnesium, and ATP-M which is a substrate of glycerol kinase
By appropriately suppressing the formation of the g ⁺⁺ complex, the reaction rate of the enzyme linearly increases even with a high concentration of magnesium that cannot be measured by the conventional method.

The sample used for the measurement can be any sample as long as it contains magnesium, but is preferably a biological sample such as plasma, serum or urine. In the present invention, pretreatment such as deproteinization of the sample is unnecessary.

As the chelating agent used in the present invention, any chelating agent may be used so long as it chelate magnesium and inhibit the activity of glycerol kinase. Examples of such chelating agents include ethylenediaminetetraacetic acid (hereinafter referred to as E
Abbreviated as DTA. ), Diethylenetriaminepentaacetic acid, 8-
Hydroxyquinoline-5-sulfonic acid may be mentioned,
Preferred is EDTA. The concentration of the chelating agent used is 50 mM or less, preferably 1 to 20 mM.

Glycerol kinase used in the present invention is a soluble fraction of rat, pigeon liver, kidney, small intestine and adipose tissue, bovine sperm, insect muscle granular fraction, Escherichia coli (Escherichia coli).
It is present in many bacteria such as coli) and yeast and may be derived from any origin, but a purified commercial product is preferable.
For example, those derived from Cellulomonas are commercially available from Toyobo. Glycerophosphate oxidase is known to exist in various bacteria, but since purified ones are commercially available, it is preferable to use them. For example, those derived from Pdioococcus are commercially available from Toyobo.

The amount of enzyme used is 0.1-5 U / ml of glycerol kinase,
The combination of glycerophosphate oxidase is 1 to 10 U / ml.

The concentrations of ATP and glycerol, which are substrates for glycerol kinase, are appropriately 0.1 to 5 mM and 0.1 to 10 mM, respectively.

When calcium ether and glycol ether diamine tetraacetic acid (hereinafter, abbreviated as GEDTA), which is a chelating agent with high specificity for calcium, are added to this reaction system, the time until the reaction rate becomes constant becomes shorter, This is advantageous for analytical applications, and the measured value is less susceptible to calcium from the sample. The concentration used is calcium
GEDTA is 1-10 mM at 0.1-2 mM. Of course, the object of the present invention can be sufficiently achieved even if the enzyme reaction is carried out in the absence of calcium and GEDTA. The order of adding the sample, substrate and enzyme is not particularly limited.

The generated hydrogen peroxide is measured using a known hydrogen peroxide detection system. A typical system is 4-aminoantipyrine or 3-aminochromin as a chromogen in the presence of peroxidase.
Oxidative condensation product of methyl-2-benzothiazolinone-hydrazone hydrochloride with a derivative of aniline, anisidine or toluidine [eg, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine] There is a system using. The oxygen reaction is carried out by a known method.

In practice, the invention includes a composition of the above, in a buffer solution.
A sample containing magnesium is added to a liquid adjusted to pH 6 to 9, preferably pH 6.5 to 7.5, reacted at 37 ° C for 2 to 10 minutes, and then measured by a spectrophotometer.

The buffer used in the above reaction may be any as long as it can be adjusted to pH 6 to 9, for example, Zerenzen (SΦre
Examples thereof include an nsen buffer solution, a Good buffer solution, and a McIlvaine buffer solution. Buffers are used at concentrations ranging from 10 to 200 mM.

The measurement according to the method of the present invention can be performed not only by a manual method but also by an ordinary automatic analyzer for biochemical examination.

[Examples] The present invention is described in more detail below with reference to Examples, but the present invention is not limited to these Examples.

Example (magnesium ion calibration curve in the presence of chelating agent) ATP (1 mM), glycerol (0.7 mM), glycerol kinase (0.7 U / ml), glycerophosphate oxidase (5 U / ml), peroxidase (2 U / ml),
4-aminoantipyrine (0.2 mM), calcium chloride (1 mM), EDTA (5 mM), GEDTA (5 mM), and n-ethyl-N-sulfopropyl-m-toluidine (1
mM) to Hepes buffer (5), which is one of the Good's buffers.
0 mM, pH 7.0) was added to adjust the concentration in parentheses. To this mixture (990 μl) was added 10 μl containing magnesium chloride (1-20 mM) similarly adjusted with Hepes buffer, and the increase in absorbance from 2 minutes to 5 minutes after the reaction was measured at 37 ° C. with a spectrophotometer at a wavelength of 546 nm. Then, the absorbance increased per minute was determined.

The obtained results are shown in FIG. The vertical axis represents the absorbance that increases per minute, and the horizontal axis represents the magnesium concentration.

As can be seen from the figure, in the presence of EDTA, dilution linearity was recognized up to 20 mM. The upper limit of the magnesium concentration of the concentrated urine sample is about 7 mM, and therefore, the present invention can be sufficiently used in a hospital laboratory.

Comparative Example 1 (Conventional method without adding a chelating agent) Of the above Examples, EDTA, GEDTA, and CaCl ₂ were removed, and glycerol kinase was set to 0.02 U / ml (oxygen amount that is usually used in the conventional method), and other The results when the conditions are the same are shown in FIG.

In the conventional method, linearity can be obtained only up to 5 mM.

Comparative Example 2 (Correlation between the present invention and the xylidine blue method) The same as the actual measurement value when a urine sample was measured as a raw solution by the method of the example and the xylidine blue method currently used in a hospital laboratory. The correlation of the actual measurement values when the urine sample was diluted 5-fold and measured is shown in FIG. 3 (however, the X-axis value is the actual measurement value by the xylidine blue method multiplied by 5). With 1 mM of magnesium chloride as a standard, in the xylidine blue method, magnesium in various urine samples was measured by an automatic analyzer Hitachi 705 type using a magnesium measuring kit, Magnesium-HATest wako (Wako Pure Chemical Industries, Ltd.).

Both measured values showed a good correlation passing through the origin. That is, it was found that the method of the present invention can be sufficiently used as an ordinary daily inspection.

[Effects of the Invention] The method of the present invention is an excellent quantification method capable of quantifying magnesium to a higher concentration range than conventional quantification methods without the need to dilute the sample without being affected by the turbidity of the sample. . By using this quantification method, the measured value can be obtained accurately and in a short time, and the determination in renal dysfunction, endocrine system disease, etc. can be made more accurate.

[Brief description of drawings]

FIG. 1 shows a magnesium calibration curve according to the method of the present invention,
FIG. 2 shows a magnesium calibration curve by the conventional method without adding a chelating agent. FIG. 3 shows the correlation between the method of the present invention and the xylidine blue method.

Claims (3)

[Claims] 1. A system for quantifying magnesium in a sample by reacting a sample with glycerol kinase and glycerophosphate oxidase in the presence of adenosine-3-phosphate and glycerol, and measuring the produced hydrogen peroxide, A method for quantifying magnesium, characterized in that a chelating agent having a concentration of 50 mM or less is present. 2. The quantitative method according to claim 1, wherein the chelating agent is ethylenediaminetetraacetic acid. 3. Adenosine-3-phosphate, glycerol,
A magnesium quantitative composition comprising glycerol kinase, glycerophosphate oxidase and a chelating agent.