JPH0759596A - Determination of magnesium and reagent therefor - Google Patents

Abstract

PURPOSE:To determine the magnesium concentration in body fluid using an enzymatic method having high specificity. CONSTITUTION:A specimen is reacted with phosphoenolpyruvate carboxylase in the presence of phosphoenolpyruvic acid salt and a bicarbonate, the produced oxaloacetic acid is reacted with malate dehydrogenase in the presence of NADH and the decrease of NADH is measured to determine the magnesium concentration.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to
In particular, the present invention relates to a method for measuring magnesium in body fluid and its reagent.

[0002]

2. Description of the Related Art Magnesium is one of the essential metals in the living body and is known as an activator of various enzymes. Clinically, it has been clarified that it is associated with various cardiovascular diseases such as hypertension, heart disease, diabetes, and renal stones, and its existence has been attracting attention. To measure magnesium,
Conventionally, atomic absorption method, colorimetric method represented by xylidine blue, etc. have been generally used, but recently, several enzymatic methods with high specificity have been developed and their utility value as a highly practical method. Increasing (inspection and technology vol.19 n
See o16 p507). For example, hexokinase glucose
The -6-phosphate dehydrogenase method produces glucose-6- because the reaction rate of hexokinase in the presence of D-glucose and ATP depends on the magnesium concentration.
This is a method for measuring magnesium in a sample by allowing glucose-6-phosphate dehydrogenase to act on phosphoric acid and measuring the decrease of NADH which is a coenzyme. This method utilizes that a conjugate of magnesium and ATP serves as a substrate for the enzyme. Other known measurement methods based on the same principle include the glycerol kinase-glycerophosphate oxidase method and the phosphoglucomutase-glucose-6-phosphate dehydrogenase method that utilizes magnesium as an activator rather than a substrate. ing.

[0003]

The above-mentioned enzyme method with high specificity is becoming widespread as a method for measuring magnesium in clinical tests as an alternative to the conventional chemical method using xylidine blue. It is known that the stability of enzymes and substrates used for the measurement is insufficient, the time required for the measurement is longer than that of the chemical method, and the quantification range is narrow.

[0004]

[Means for Solving the Problems] The present inventors have studied various magnesium measuring methods for solving the above problems,
The present invention has been accomplished by finding a new measuring method using phosphoenolpyruvate carboxylase (PEPC) in which magnesium acts as an activator of an enzymatic reaction.

That is, according to the present invention, a sample containing magnesium is reacted with phosphoenolpyruvate carboxylase in the presence of phosphoenolpyruvate (PEP) and bicarbonate, and oxaloacetate produced is subjected to NADH.
In the presence of the above, a magnesium measuring method is characterized in that the magnesium in the sample is measured by causing malate dehydrogenase (MDH) to act and measuring the decreasing NADH.

The measurement principle of the present invention is as follows.

[0007]

[Chemical 1]

[0008]

[Chemical 2]

The present invention also provides (a) phosphoenolpyruvate, (b) bicarbonate, (c) phosphoenolpyruvate carboxylase, (d) NADH, (e) malate dehydrogenase and (f) buffer. It is a magnesium measuring reagent characterized by containing.

The phosphoenolpyruvate carboxylase used in the present invention can be obtained from many plant tissues and bacteria, and any source can be used. However, the known enzyme is not stable in plant origin and cannot be stored in a liquid state for a long time. On the other hand, many of the bacterial origins are expensive activators such as acetyl coenzyme A in addition to magnesium. Is known to be necessary for the reaction. An example suitable for the purpose of the present invention without these drawbacks is phosphoenolpyruvate carboxylase derived from acetic acid bacteria.

The malate dehydrogenase used in the present invention may be derived from any source, and examples thereof include those derived from pig heart, those derived from Thermus, and those derived from Bacillus. Examples of the phosphoenolpyruvate include potassium salt, sodium salt, tricyclohexyl ammonium salt, monocyclohexyl ammonium salt and the like. As the bicarbonate, potassium salt, sodium salt,
Examples thereof include ammonium salts.

The buffer used in the present invention may be any buffer as long as it is maintained at a pH suitable for the reaction of phosphoenolpyruvate carboxylase and malate dehydrogenase, but a pH of 7-9 is usually used. For example, phosphate buffer, Tris buffer, Tricine buffer and the like can be mentioned. The concentration of the buffer solution is not particularly limited,
Usually, it is about 10 to 100 mM.

If necessary, a surfactant, a preservative, a stabilizer, an enzyme activator and the like may be added to the reagent of the present invention.
The surfactant, preservative, stabilizer and enzyme activator are not particularly limited. As the surfactant, a nonionic surfactant is preferably used. As a preservative, N
AN ₃ , chelating agents and antibiotics are preferably used. The stabilizer and the enzyme activator are not particularly limited as long as they exhibit the effect. Examples thereof include albumin, acetyl coenzyme A, manganese ion and the like.

The general composition of the reagent of the present invention is shown below. Weak alkaline buffer 10-50 mM Phosphoenolpyruvate 2-5 mM Bicarbonate 5-20 mM NADH 0.1-0.3 mM Phosphoenolpyruvate carboxylase 0.02-0.2 U / ml Malate dehydrogenase 1-10 U / ml

An example of a more specific composition is shown below. Tris-HCl buffer 10-50 mM phosphoenolpyruvate potassium salt 2-5 mM sodium carbonate 5-20 mM NADH 0.1-0.3 mM phosphoenolpyruvate carboxylase from acetic acid bacteria 0.02-0.2 U / ml malate dehydrogenase 1-10 U / ml

Further, in order to suppress the interfering reaction of lactate dehydrogenase and pyruvate in the sample, it is desirable to add lactate inhibitor such as oxamate to about 1 to 20 mM. The present invention measures the amount of magnesium in a sample by utilizing the fact that magnesium acts as an activator of phosphoenolpyruvate carboxylase and the amount of magnesium is proportional to the reaction rate of phosphoenolpyruvate carboxylase under specific conditions. The method for measuring magnesium is characterized by Therefore, in order to weaken the rapid binding of magnesium and phosphoenolpyruvate carboxylase in the sample, EDT
It is also possible to freely adjust the measurement sensitivity by adding an appropriate amount of a chelating agent such as A.

Preliminary heating of 3.0 to 10 ml of the above reagent at 30 ° C. for about 5 minutes, and then a sample containing magnesium 0.1
After mixing ml, react at 30 ° C for 1 to 10 minutes, NAD
The decrease of the H concentration is, for example, 340 nm, 360 nm, 38
It is measured from the absorbance such as 0 nm.

[0018]

EXAMPLES Next, the present invention will be specifically described with reference to Examples. Example 1 Acetobacter hansenii
Using the phosphoenolpyruvate carboxylase derived from IFO14820, the following magnesium measuring reagents were prepared. Tris-HCl buffer (pH 8.0) 50 mM phosphoenolpyruvate potassium salt 3.5 mM sodium carbonate 10 mM NADH 0.14 mM phosphoenolpyruvate carboxylase 0.1 U / ml malate dehydrogenase 1 U / ml

After preliminarily heating 3.0 ml of the above reagent at 30 ° C. for 5 minutes, a sample 0.1 containing magnesium 0-2 mM was prepared.
ml was added, the mixture was reacted at 30 ° C. for 5 minutes, and the decrease in absorbance at 340 nm was measured. The relationship between the magnesium concentration in the sample and the amount of decrease in the absorbance per minute is shown in FIG. For magnesium 0 to 2 mM, a linear relationship was established with the change in absorbance.

Example 2 Acetobacter pasteu
rianus) IFO14814-derived phosphoenolpyruvate carboxylase was used to prepare a magnesium measuring reagent shown below. Tris-hydrochloric acid buffer solution (pH 8.0) 50 mM phosphoenolpyruvate potassium salt 3.5 mM sodium carbonate 10 mM magnesium sulfate 30 mM NADH 0.14 mM phosphoenolpyruvate carboxylase 0.05 U / ml malate dehydrogenase 1 U / ml

After preliminarily warming 3.0 ml of the above reagent at 30 ° C. for 5 minutes, 0.1 m of a sample containing 0-1 M magnesium
1 was added, and the mixture was reacted at 30 ° C. for 5 minutes and the decrease in absorbance at 340 nm was measured (the decrease amount when the magnesium concentration in the sample was 0 mM was reduced as a blank). The relationship between the concentration of magnesium in the sample and the amount of decrease in absorbance per minute is shown in FIG. A linear relationship was established with the change in absorbance between 0 and 1.2 M of magnesium.

[0022]

According to the present invention using phosphoenolpyruvate carboxylase, compared with the conventional enzymatic method,
The stability of the enzyme and substrate used for measurement is sufficient, the time required for measurement is shorter than that of the chemical method, and the quantification range is wide. Moreover, it is possible to inexpensively and rapidly measure magnesium while making use of the advantage of the enzyme method of high specificity, and it is particularly useful for magnesium measurement performed for prevention / diagnosis of cardiovascular diseases.

[Brief description of drawings]

FIG. 1 shows the dilution linearity of a 2 mM aqueous magnesium sulfate solution.

FIG. 2 shows the dilution linearity of a 1M magnesium sulfate aqueous solution.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoshihisa Kawamura 10-24 Toyomachi, Tsuruga City, Fukui Prefecture Toyobo Co., Ltd.

Claims (3)

[Claims] 1. A method for measuring magnesium, which comprises measuring magnesium in a sample by reacting a sample containing magnesium with phosphoenolpyruvate carboxylase. 2. A sample containing magnesium is reacted with phosphoenolpyruvate carboxylase in the presence of phosphoenolpyruvate and bicarbonate, and malate dehydrogenase is allowed to act on the produced oxaloacetate in the presence of NADH. A method for measuring magnesium, which comprises measuring magnesium in a sample by measuring decreasing NADH. 3. (a) phosphoenolpyruvate,
A magnesium measuring reagent containing (b) bicarbonate, (c) phosphoenolpyruvate carboxylase, (d) NADH, (e) malate dehydrogenase and (f) buffer.