JPH02276597A - Method for measuring calcium in human body - Google Patents

Abstract

PURPOSE:To accurately and simply measure calcium in a short time and high specificity by reacting alpha-amylase with a substrate of alpha-amylase and reagent containing a specific chelate agent. CONSTITUTION:A maltoligo sugar (A) such as 2,4-dichlorophenyl-alpha-D- maltotrioside having non-coloring source group on reducing terminal glucose is blended with a maltoligo sugar (B) such as 4-nitophenyl-alpha-D-maltotrioside in a concentration of 0.1-50mM/l at a ratio of 100-1:1 to provide a substrate (D) of alpha-amylase (C). Then a specimen (E) is blended with 1000-1000000U/l ingredient C derived from swine pancreas, ingredient D, 0.05-20mM/l glycol etherdiamine tetraacetic acid which is chelate agent specific to Ca and alpha- glucosidase and/or beta-glucosidase and the blend is reacted in a buffer of pH 5-8 and coloring source generated from the substrate is subjected to colorimetry to measure calcium in a body fluid.

Description

[Detailed description of the invention] Industrial applications The present invention relates to a method for measuring calcium in body fluids.

More specifically, the present invention relates to a method for measuring the amount of calcium in body fluids using the fact that α-amylase is activated by calcium.

BACKGROUND OF THE INVENTION Currently, the amount of calcium in various body fluids (serum, urine, etc.) is measured in hospital laboratories and used to diagnose diseases.

Calcium plays an important role in cell function in the body and blood coagulation function, but the amount in human plasma is said to be extremely tightly regulated, and abnormally low and high values are known. This has high diagnostic value.

For example, hypocalcemia includes hypoproteinemia,
Diseases such as hypophosphatemia, nephritis, nephrosis, vitamin D deficiency, hypoparathyroidism, and rickets, and hypercalcemia include bone tumors, Ajilan's disease, emphysema, hyperparathyroidism, renal failure, etc. There is a possibility of a disease, and it can be used to diagnose these diseases.

However, conventional methods have not been able to accurately and easily measure the amount of calcium.

Conventional techniques and their problems The following methods are known as methods for measuring calcium in body fluids.

(1) Titration method (2) Colorimetric method (3) Atomic absorption method (4) Tip analysis method (5) Electrode method (θ) Enzyme method The titration method of (1) uses oxalate or chelate. Although it is a chemical titration method, it has the disadvantage that it is complicated and that the measured values vary depending on the operator.

In the colorimetric method (2), o-CPC (ortho-CPC) is used as a coloring agent.
The method using cresol phthalein complexone) adds 8-hydroxyquinoline to eliminate the influence of Mg2~, and is currently most commonly used in hospital laboratories.

However, 1. Even if 8-hydroxyquinoline is added, there is still an effect of about a few percent of Mg2~on, 2. The absorbance changes depending on temperature and time, 3. There is an optimum pH range for color development. 4. It has drawbacks such as the fact that there is a region where the measured value is negative at low concentrations (calcium), which is particularly problematic when carried out manually.

Atomic absorption spectrometry (3) requires expensive equipment, requires skill, and requires dilution of the sample.

The flame color analysis method (4) also requires dilution of the specimen and has problems in specificity and reproducibility.

The electrode method (5) requires expensive equipment, is affected by pH, and requires complicated maintenance.

The enzymatic method of (6) includes l) a method using calmodulin [see JP-A-82-38199], and if) a method using phospholipase D1 choline oxidase [see JP-A-62-195297].

1) Although the method using calmodulin has excellent specificity, it is too sensitive and requires dilution of the specimen (100 to 100%).
1000 times) is required.

11) The method using phospholipase D1 choline oxidase has excellent specificity and does not require dilution of the specimen, but has the disadvantage that continuous measurement is not possible because the reaction takes time.

As mentioned above, all measurement methods have drawbacks, and none of them has been satisfactory.

Means for Solving the Problems The present inventors have conducted intensive studies to find a method for quantifying calcium using an enzyme that can be accurately and easily measured, and have found that the activity of α-amylase is proportional to the amount of calcium. I found that it can be improved.

It is known that α-amylases of various origins exist in an active form bound to calcium (Enzyme Handbook, edited by Tamiya and Maruo, Shokusen Shoten, p. 493). However, it is not known that there is a linear correlation between the activity strength and the concentration of calcium, and the present inventors have now demonstrated that calcium in body fluids can be measured using this correlation. This is the first time it has been discovered.

However, after repeated studies, it became clear that depending on the type of substrate used, it may not be possible to obtain a linear correlation or the measurement range may be too narrow to be put to practical use.

Therefore, the present inventors further investigated the possibility of (1) performing the enzymatic reaction on calcium in the presence of a more specific chelating agent, and/or (2) using its reducing terminal glucose as a substrate to form a coloring source. The inventors have discovered that these drawbacks can be solved by carrying out an enzymatic reaction using a combination of a maltooligosaccharide that does not have a group and a maltooligosaccharide that has a chromogenic group, and has completed the present invention.

It has long been known that in the presence of a chelating agent, α-amylase releases most of its own calcium and transforms into an inactive α-amylase [Eur, J.
, Biochem, 41.171 (1974)], it has become possible to quantitatively measure calcium for the first time in the presence of a chelating agent, and the fact that the measurement range has been expanded has been discovered for the first time. This is completely unexpected based on previously known facts.

Furthermore, the fact that the same effect can be obtained by using the two types of substrates shown in (2) is also discovered for the first time, and this is something that could not be expected from the prior art.

Accordingly, the present invention provides a method for treating a specimen with (1) α-amylase and a substrate for α-amylase in the presence of a chelating agent more specific for calcium, or (ii) treating a sample with α-amylase and α-amylase in the presence of a chelating agent more specific for calcium. Substrates for amylase include (a) maltooligosaccharides and maltooligosaccharides in which a non-chromogenic group is bound to the glucose at their reducing end; (b) maltooligosaccharides in which a chromogenic group is bound to the glucose at their reducing end and their reducing terminals. A specimen characterized by combining a substrate selected from malto-oligosaccharides in which a chromogenic group is bound to glucose and a substituent is bound to a non-reducing terminal glucose, and is made to act in the absence of a chelating agent more specific to calcium. Concerning a method for measuring calcium inside.

The α-amylase used in the present invention may be derived from microorganisms, plants, or animals, but is preferably derived from animal sources, such as commercially available α-amylase derived from porcine pancreas. α-amylase is 10
00-1000000 U/Ω, more preferably 500
It is used at a concentration of 0 to 500,000 U/R.

Substrates for α-amylase used in the present invention include polysaccharides such as starch and glycogen, and maltooligosaccharides, but maltooligosaccharides are advantageous in terms of simplification of measurement and shortening of measurement time.

Such maltooligosaccharides include (1) maltooligosaccharides such as maltoheptaoside and maltoheptaoside; (2) maltooligosaccharides in which a non-chromogenic group is bound to the reducing terminal glucose, such as 2,4-dichlorophenyl- α-D-maltotrioside, 2.4-dichlorophenyl-(α or β)-D-maltopentaoside, 2.4
-dichlorophenyl-(α or β)-D-maltoheptaoside, (3) a maltooligosaccharide with a chromogenic group attached to its reducing end glucose, e.g. 4-nitrophenyl-
α-D-maltotrioside, 4-nitrophenyl-(α
or β)-D-maltopentaoside, 4-nitrophenyl-(α or β)-D-maltoheptaoside, 2-
Chloro-4-nitrophenyl-α-D-maltotrioside, 2-chloro-4-nitrophenyl-(αmatahaβ)
-D-maltopentaoside, 2-chloro-4-nitrophenyl-(α or β)-D-maltoheptaoside, and (4) a chromogenic group is bonded to its reducing terminal glucose, and its Examples include maltooligosaccharides in which a substituent, such as a cyclic acetal, is bonded to a non-reducing terminal glucose.

Furthermore, (5) (a) a substrate selected from the aforementioned maltooligosaccharides and maltooligosaccharides in which a non-chromogenic group is bound to the reducing terminal glucose; Substrates selected from maltooligosaccharides and maltooligosaccharides in which a chromogenic group is bonded to the reducing terminal glucose and a substituent group is bonded to the non-reducing terminal glucose can be used in combination.

When the substrates shown in (1) to (4) above are used alone, the preferred concentration is 0.1 to 50 ++uaole/
, Q. In addition, when used in combination as shown in (5), the maltooligosaccharide without a chromogenic group and the maltooligosaccharide with a chromogenic group each have a 0.1
~50 mo+ole/! Concentration of I, 100:1-1
It is preferable to add in a ratio of :1.

When used in combination, due to competitiveness for the substrate, the reaction occurs when calcium is not present in the sample. is activated and the reaction proceeds. This is called a blank reaction) is suppressed, making measurement possible and broadening the measurement range.

More preferred α-amylase substrates include the above (3)
) or (5), more specifically 4-nitrophenyl-α-D-maltotrioside, 2
-chloro-4-nitrophenyl-β-D-maltopentaoside, or maltopentaose and 2-chloro-4-
A combination of nitrophenyl-β-D-maltopentaoside or 2,4-dichlorophenyl-β-D-maltopentaoside and 2-chloro-4-nitrophenyl-β-D
- maltopentaoside combinations.

More specific chelating agents for calcium include gelcol ether diaminetetraacetic acid and 1,2-bis(O-aminophenoxy)ethanetetraacetic acid (hereinafter referred to as GED).
It is abbreviated as TASBAPTA. ) etc. These chelating agents can be used alone or in combination. Chelating agent is 0.05-20 ++
Preferably, it is added at a concentration of n5 ole/l.

In the presence of a chelating agent, the calcium that increases enzyme activity (present in α-amylase and in the sample) is eaten away by the chelating agent, so even if the calcium concentration in the sample is actually high, It is thought that the enzyme activity will be suppressed to a low level, making measurement possible and widening the measurement range.

An equilibrium relationship exists between calcium that binds to the chelating agent and calcium that does not bind, and the enzyme activity decreases depending on the calcium concentration in the sample, and there is a linear relationship between the two. Even more conveniently,
Since a chelating agent that is more specific for calcium is used, measurements can be made without being affected by other metal ions, especially magnesium, present in the sample.

In addition, when two types of substrate groups are combined as shown in (5), it is possible to obtain a sufficiently wide measurement range either in the presence or absence of a chelating agent.

The detection system used in the present invention is that of a conventional α-amylase activity assay method. For example, when starch, a type of polysaccharide, is used as a substrate for α-amylase, the amount of reducing sugar produced can be measured using known methods such as Somogyi (S
omogyl) method or by a dye produced using pull starch.

When (1) of the above-mentioned malto-oligosaccharides is used as a substrate, α-glucosidase is conjugated, and the resulting glucose is conjugated with glucose oxidase/peroxidase/dye system or hekyrakinase/phosphoglucomutase/glucose-6. -Can be detected by directing to the phosphate dehydrogenase/NADH system. Also, among the above maltooligosaccharides as a substrate (2)
When using a non-chromogenic source, it can be calibrated by conjugating α-glucosidase and/or β-glucosidase, or by reacting the enzyme without conjugating it, and introducing the resulting non-chromogenic source into a chromogenic system. Furthermore, when (3) or (4) of the above maltooligosaccharides is used as a substrate, α-glucosidase and/or β-glucosidase
The glucosidase is conjugated or the enzyme is reacted without conjugation and the resulting chromogen, e.g. be able to.

In any case of (1) to (4), a conjugated enzyme is not required when a tricarbonate is used as a substrate, and the enzyme is required when a tetracarbonate to a heptose is used.

Among the above malto-oligosaccharides, when two types of substrates are used in combination as shown in (5), α-glucosidase and/or β-glucosidase may be conjugated, or the enzyme may not be conjugated. React (
It can be calibrated by directly colorimetrically measuring the chromogenic source generated from the substrates of group b).

The measurements according to the invention are carried out in a buffer solution that can maintain a pH of 5 to 8. Such buffers are well known, such as Gud's buffer [for example, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (hereinafter abbreviated as HEPES), piperazine-N, N '
-bis(2-ethanesulfonic acid) (hereinafter referred to as PI PE
It is abbreviated as S. '), N-2-hydroxyethylpiperazine-N'-2-hydroxypropane-3-sulfonic acid (hereinafter abbreviated as HEPPSO), phosphate buffer, Tris-HCl buffer, and the like. Buffer is 10
Preferably, it is added at a concentration of ~500 n+mole/1.

Furthermore, in the measurement system of the present invention, an alkali metal halide such as sodium chloride, potassium chloride, sodium bromide, and sodium iodide is added at a concentration of 1 to 400 a+mole/
It is preferable to add it at a concentration of .

The measurement according to the method of the present invention can be carried out not only manually but also by a conventional automatic analyzer for biochemical tests.

EXAMPLES The present invention will be explained in detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1: [Measurement of calcium using α-amylase using a combination of a maltooligosaccharide having a chromogenic group and a maltooligosaccharide without a chromogenic group as substrates] The following reagents were mixed to obtain a reagent solution. .

Porcine pancreatic α-amylase 8QQQU /Ω×100μg α-glucosidase 250000U/N X 100μg β-glucosidase 20000U/I -4-nitrophenyl-β-D-maltopentaoside 2 a+mole/N X 100 ttlPIPES
Buffer solution (pH 8,8) 500 + uole/N
280 μg total 980 μρ Calcium concentration 0 to 10 m
After adding 20 μg of an aqueous calcium acetate solution equivalent to g/clQ and reacting at 37° C., the increase in absorbance at 400 nil (37° C.) was measured over time to calculate each reaction rate. The obtained calibration curve is shown in FIG.

As can be seen from the figure, the calibration curve has a calcium concentration of 7.5 m
It showed linearity up to g/dρ.

Example 2: [Measurement of calcium by α-amylase in the presence of a chelating agent using a maltooligosaccharide having a chromogenic group as a substrate] The following reagents were mixed to obtain a reagent solution.

Porcine pancreatic α-amylase 8000U# X 100μg α-glucosidase 250000U/N X 100 tin β-glucosidase 20000U/N X 100! 112-chloro 4-nitrophenyl-β-D-maltopentaoside 2 On+mole/Rx 100 ttllAPTA 20 mmole/I N X 100μg purified water
280 μg total: 980 μg Measurement was performed in the same manner as in Example 1 using the reagent solution obtained above. The obtained calibration curve is shown in FIG.

As can be seen from the figure, the calibration curve is calculated at a calcium concentration of 10 mg/
It showed linearity up to d and Q.

Example 3: [Measurement of calcium by α-amylase in the presence of a chelating agent using a combination of maltooligosaccharides having a chromogenic group and maltooligosaccharides without a chromogenic group as substrates] The following reagents were mixed. A reagent solution was obtained.

Porcine pancreatic α-amylase 8000U/N x 100μg α-glucosidase 250000U/# x 100μg β-glucosidase 20000U/N Oshido 2 mmole/j? X 100 tllAPTA 20 u+ole/N X 100 tt! J phosphate buffer wave buffer 8.5) 500 mmole/, Q
180 μg Total: 980 μg Using the reagent solution obtained above, measurements were carried out in the same manner as in Example 1 (however, an aqueous calcium acetate solution with a calcium concentration of 0 to 30 mg/dΩ was used). The obtained calibration curve is shown in FIG.

As can be seen from the figure, the calibration curve is calculated at a calcium concentration of 30 mg/
Almost linearity was shown up to dff.

Example 4: [Measurement of calcium by α-amylase in the presence of a chelating agent using a maltooligosaccharide having a chromogenic group as a substrate] The following reagents were mixed to obtain a reagent solution.

Porcine pancreatic α-amylase 200000U/N x 100μg 4-nitrophenyl-α-D-maltotrioside 5
a+a+ole/N x 10 01t (IBAP
TA 1 +nole/l X100. u#G
EDTA 1.75 mmole/l×100μ
NHEPPSO buffer (pH 8,0) 500 mmole/N x 100 ttl Sodium chloride 500 a+mole/l x 100 ul purified water
390μg total 990μ
g Add a calcium concentration of 0 to 50 m to the reagent solution soaked above.
After adding 10 μg of a calcium acetate aqueous solution equivalent to g/d and ff and reacting at 37° C., the increase in absorbance at 4001 m (37° C.) was measured over time to calculate each reaction rate. The obtained calibration curve is shown in FIG.

As can be seen from the figure, the calibration curve is calculated at a calcium concentration of 50 mg/
Linearity was shown up to dJ2.

Comparative Example 1: [Influence of magnesium in the method of the present invention and the o-CPC method (conventional method)] As a specimen, 900 μg of serum and a magnesium concentration of O ~ 1
Magnesium acetate solution corresponding to 100 mg/dI
Calcium was measured by the method described in Example 3 using μg of the mixed solution.

On the other hand, a measurement kit for the o-CPC method [Calclum II
-11A Te5t Wako (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.)]
Calcium was measured in the same specimen using an automatic analyzer (Hitachi Model 705). Figure 5 shows the influence of magnesium in the two measurement methods.

As can be seen from the figure, the method of the present invention allows measurement with almost no influence of magnesium in the sample, but o-C
Although the PC method has been designed to suppress the influence of magnesium, it is still affected considerably.

Comparative Example 2: [Measured values by the method of the present invention] - Correlation with measurement values by CPC method (conventional method)] Calcium The amount was measured.

The correlation between the two is shown in FIG.

As can be seen from the figure, the regression line of the correlation is n-25, r =
0.944, y = 0.985 x + 1.552, and the two showed a good correlation. This shows that the method of the present invention can be fully used for routine testing.

Comparative Example 3: [Measured values by the method of the present invention] - Correlation with measurement values by CPC method (conventional method)] Calcium The amount was measured.

The correlation between the two is shown in FIG.

As can be seen from the figure, the regression line of the correlation is n=36, r −
0,923,y −1,0171+0.102,
Both showed good correlation. This shows that the method of the present invention can be fully used for routine testing.

Effects of the Invention Since the present invention uses an enzyme, it has higher specificity than other methods, and does not require a long reaction time compared to conventional enzyme-based methods, and is accurate and simple. This is a method for measuring calcium.

[Brief explanation of drawings]

1, 2, 3 and 4 show calcium calibration curves according to the method of the present invention, and FIG. 5 shows the calcium calibration curves according to the method of the present invention and
This is a graph comparing the influence of magnesium in the sample in the CPC method (conventional method), and Figures 6 and 7 show the correlation between the measured values by the method of the present invention and the o-CPC method (conventional method'). It is a graph showing a relationship. Patent applicant: Ono Pharmaceutical Co., Ltd.

Claims (1)

[Scope of Claims] 1) A sample is treated with (i) α-amylase and a substrate for α-amylase in the presence of a chelating agent more specific for calcium, or (ii) α-amylase and α-amylase are treated in the presence of a chelating agent more specific for calcium. Substrates for amylase include (a) maltooligosaccharides and maltooligosaccharides in which a non-chromogenic group is bound to the glucose at their reducing end; (b) maltooligosaccharides in which a chromogenic group is bound to the glucose at their reducing end and their reducing terminals. It is characterized by combining substrates selected from malto-oligosaccharides in which a chromogenic group is bound to glucose and a substituent is bound to the non-reducing terminal glucose, and is made to act in the absence of a chelating agent more specific to calcium. How to measure calcium in body fluids. 2) As a substrate for α-amylase and α-amylase, (1) maltooligosaccharide alone, (2) maltooligosaccharide with a non-chromogenic group bound to its reducing terminal glucose alone, or (3) A maltooligosaccharide in which a chromogenic group is bonded to the reducing terminal glucose alone, or (4) a maltooligosaccharide in which a chromogenic group is bonded to the reducing terminal glucose and a substituent group is bonded to the non-reducing terminal glucose, or (5) A substrate selected from (a) maltooligosaccharides and maltooligosaccharides in which a non-chromogenic group is bonded to the glucose at its reducing end, and (b) a maltooligosaccharide in which a chromogenic group is bonded to the glucose at its reducing end and its reducing end group. A claim characterized in that substrates selected from malto-oligosaccharides in which a chromogenic group is bound to the terminal glucose and a substituent is bound to the non-reducing terminal glucose are combined and made to act in the presence of a chelating agent more specific to calcium. The measuring method described in item 1. 3) As a substrate for α-amylase and α-amylase, (1) maltooligosaccharide with a chromogenic group attached to its reducing end glucose alone, or (2) (a) maltooligosaccharide and its reducing end glucose as a substrate for α-amylase. a substrate selected from malto-oligosaccharides to which a chromogenic group is bonded; (b) a malto-oligosaccharide to which a chromogenic group is bonded to its reducing terminal glucose; and a chromogenic group is bonded to its reducing terminal glucose, and the non-reducing terminal glucose is substituted. 3. The measuring method according to claim 2, wherein a combination of substrates selected from maltooligosaccharides to which groups are bonded is allowed to act in the presence of a chelating agent more specific for calcium. 4) As a substrate for α-amylase, (1) 4-nitrophenyl-α-D-maltotrioside, (2) 2-chloro-4-nitrophenyl-β-D-maltopentaoside, or (3) 4. The measuring method according to claim 3, characterized in that maltopentaose and 2-chloro-4-nitrophenyl-β-D-maltopentaoside are used in combination. 5) The measuring method according to claim 2, wherein the chelating agent is glycol ether diamine tetraacetic acid, 1,2-bis(o-aminophenoxy)ethanetetraacetic acid, or a mixture thereof. 6) As a substrate for α-amylase and α-amylase in the sample, (a) a substrate selected from maltooligosaccharides and maltooligosaccharides in which a non-chromogenic group is bound to the glucose at the reducing end; (b) a chromogenic source at the glucose at the reducing end; The absence of a chelating agent that is more specific for calcium is achieved by combining a maltooligosaccharide with a group attached thereto and a maltooligosaccharide with a chromogenic group attached to its reducing terminal glucose and a substituent attached to its non-reducing terminal glucose. The measuring method according to claim 1, characterized in that the measuring method is applied downwardly. 7) A combination of 2,4-dichlorophenyl-β-D-maltopentaoside and 2-chloro-4-nitrophenyl-β-D-maltopentaoside is used as a substrate for α-amylase. The measuring method according to claim 6.