JPH012598A - How to measure calcium - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for indirectly measuring calcium using an enzyme.

[Conventional technology]

Calcium In particular, there are practical standard methods for static analysis of minute amounts of calcium in serum, such as atomic absorption spectrometry and ion chromatography.
The so-called o-cpc method, based on the method of narty-Br igg@, is most commonly used.

However, in the O-CPC method, coexisting ions, especially magnesium, give a positive error to the calcium measurement value.
Although this influence has been eliminated by adding oxyquinoline and strictly preparing the -region, this is not a completely satisfactory measurement method. In other words, if the amount of 8-oxyquinoline added as a magnesium inhibitor increases,
Although the influence of magnesium can be suppressed, if it becomes excessive, the coloration of the target calcium will be considerably reduced. In order to obtain an accurate amount of calcium, the ideal range is pH 11.0 ± 0.05.
I4I! i requires delicate manipulation. Furthermore o-
Since the cpc method is affected by the oxidation quality, it requires removal and removal operations before measurement, making the process complicated.

[Problems to be solved by the present invention]

In this way, especially when measuring trace amounts of calcium in serum, the O-CPC method requires the addition of 8-oxyquinoline after the detanning operation to eliminate the influence of coexisting ions, especially magnesium. Requires strict optimal settings. However, it is by no means a satisfactory measurement method for determining the exact amount of calcium, as the presence of excess 8-oxyquinoline reduces the coloration of calcium, and the amount of calcium outside the optimal range. A method that requires a reduction in calcium and a strict general definition is not suitable for daily routine work.The present invention aims to provide an accurate and easy-to-operate method for measuring calcium that solves the above-mentioned problems. It is.

[Means for solving problems]

As a result of intensive research to avoid the problems of the above measurement method, especially the influence of magnesium, the present inventors developed an enzymatic method for measuring the amount of oxalate using oxalate oxidase, which oxidizes the oxalate that precipitates calcium. When this method was applied to the measurement of calcium, particularly calcium in serum, it was discovered that the problems caused by magnesium in the conventional method could be solved at once, and the present invention was completed.

That is, the present invention precipitates calcium as oxalate, oxidizes excess oxalate with oxalate oxidase, generates hydrogen peroxide, develops a chromogen in the presence of peroxidase, and performs colorimetric measurement. This is a calcium measurement method that is characterized by determining the amount of calcium.

The present invention will be explained in detail below.

In the present invention, a certain amount of excess oxalic acid or a calcium salt or a highly ionized water-soluble oxalate is added to a sample such as serum containing calcium ions, and the calcium ions are precipitated as an insoluble calcium oxalate salt. The amount of calcium is measured indirectly by measuring the amount of oxalic acid remaining in the aqueous solution. Note that oxalic acid here includes oxalic acid that exists as a water-soluble oxalate. To quantify oxalic acid, a colorimetric method is used in which hydrogen peroxide is generated by oxalate oxidase and a chromogen develops color in the presence of peroxidase.

The reaction system of the present invention is as follows.

Ca"+Na2C204-* CaC2O4↓10 excess 020 m' -+ 2N a "Oxalate oxidase C2O4"+2H++0 2CO2+H
2O2 base oxidase H2O2 + chromogen chromogen + H2゜The water-soluble oxalate used in the present invention is sodium oxalate, ammonium oxalate, preferably JIS-secondary standard, purity. Sodium oxalate is used because of its reliability.

When the invention is practiced on serum, the oxalic acid is preferably 10 to 40 m of sodium oxalate.
mol/A! The particularly preferred concentration is 30 mmol/J.The colorimetric measurement of oxalic acid in the present invention is performed using Cl1n.

Ch@n, 29 Nia 00-702.1983, etc., and the chromogens, measurement operations, etc. that are commonly used can be used.

Examples of reagents used for colorimetric measurement of oxalic acid include the following: Oxalic acid measurement reagent A Urine, oxalic acid measurement kit “Oxalic acid “Asuka Sigma” # ( (manufactured by Asuka Tsumugi Co., Ltd.) f: was used, and the reagent was prepared to have the following concentration.

3-(dimethylamino)benzoic acid:
3.0mmol peroxidase
:5000U/l oxalate oxidase
: 100U/J3-methyl-2-benzothiazolinone hydrazone : 0.2mmol Tris buffer : 0
．． 1 mmol (measurement wavelength 590 nm) [Example] Hereinafter, a more specific explanation will be given based on an example.

Example 1 - Magnesilla Al #13.415.10$15*100 mmol in calcium standard solution (10,019/di)
/j! A sample was prepared so that the calcium content was quantified using the following measurement method.

Add 50μ of 30 mmol/l sodium oxalate reagent to 200μ each of sample solution, purified water (for blind testing), and standard solution.
l' and left at room temperature for 60 minutes. The optimal reaction time between sodium oxalate test solution and calcium is [1] at room temperature.
Although the reaction was almost completed in 0 minutes, it was set to 60 minutes to improve accuracy. (See Figure 1. In the diagram, A, C, C, and B indicate the sodium oxalate concentration, respectively. A: 10 mm
ol/l, oral: 20 mmol/l.

C: 30 mmol/l, 2: 40 mmol//
This is a graph of ) Calcium concentration is 10 m9/dl
I'll go. After centrifugation, 0.5 mjt of the oxalic acid measurement reagent A was added to 20 μE of the supernatant, and the mixture was heated at 37° C. for 40 minutes. Next, 5 d of purified water was added, and colorimetry was performed at 590 nm using the purified water as a control to examine the effect of coexisting magnesium. As shown in Table 1, there was no effect at all.

Table 1 Coexisting Mg concentration (mmo 1/l) Calcium quantitative value (1t) 1 1
0.042 10.053
10.004
9.975
9.9610 1
0.0515 9.9910
0 9.98 - Example 2 - According to Example 1, calcium in serum was measured. For comparison, measurements were also made using the conventional O-CPC method. The results are shown in Table 2.

Table 2 As shown in Table 2, a good correlation was observed between the mainstream measurement values and the conventional method measurement values.

(y = 1.0183!-0,10-r=0.984)
[Effects of the Invention] The method for quantifying calcium of the present invention simply adds a reagent to a sample containing the calcium and performs colorimetric measurement after the completion of the reaction, thereby eliminating the effects of sputum removal and magnesium in conventional methods. Problems such as the reduction in calcium coloring due to the addition of a preservative and the complexity of optimal settings have been resolved, and it has become possible to quantify calcium with high accuracy.

[Brief explanation of drawings]

Figure 1 is a graph showing the relationship between the reaction time of sodium oxalate solution and calcium depending on the concentration of sodium oxalate.

Claims (1)

[Claims] Calcium is precipitated as oxalate, excess oxalate is oxidized with oxalate oxidase, the generated hydrogen peroxide develops a chromogen in the presence of peroxidase, and the amount of calcium is determined by colorimetric measurement. A method for measuring calcium, characterized by the following.