JPH07132098A - Calcium assay in body fluid - Google Patents

Abstract

PURPOSE:To quickly, accurately and easily assay the subject calcium by making a combination of alpha-amylase, a substrate such as a maltooligosaccharide or maltooligosaccharide with its reduced terminal glucose bearing a non-color developing anlage and another substrate, a maltooligosaccharide with its reduced terminal glucose bearing a color developing anlage act on a specimen. CONSTITUTION:The calcium in a body fluid is assayed accurately and easily in a short time by making a combination of alpha-amylase, (A) as a substrate of the alpha-amylase, a maltooligosaccharide or maltooligosaccharide with its reduced terminal glucose bearing a non-color developing anlage (e.g. 2,4-dichlorophenyl-beta- D-maltopentaoside) and (B) another substrate such as a maltooligosaccharide with its reduced terminal glucose bearing a color developing anlage or with its reduced terminal glucose bearing a color developing anlage and non-reduced terminal glucose bearing a substituent (e.g. 2-chloro-4-nitrophenyl-betaD- maltopentaoside) act on a specimen consisting of a body fluid such as serum or urine.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for measuring calcium in body fluids. More specifically, it relates to a method for measuring the amount of calcium in a body fluid, which utilizes the fact that α-amylase is activated by calcium.

[0002]

BACKGROUND OF THE INVENTION At present, calcium levels in various body fluids (serum, urine, etc.) are measured in a laboratory of a hospital and used for diagnosis of diseases. Calcium plays an important role in cell function in vivo and blood coagulation function, but it is said that the amount in human plasma is regulated very strictly, and it is known that abnormally low and high levels. It has a high diagnostic value. For example, hypocalcemia includes hypoproteinemia, hypophosphatemia, nephritis, nephrosis, vitamin D deficiency, hypoparathyroidism, and diseases such as Kurdish disease, and hypercalcemia includes bone tumor and addison. Disease, pulmonary emphysema, hyperparathyroidism, renal insufficiency, and other diseases, which can be used for diagnosis. However, the amount of calcium could not be measured accurately and simply by the conventional method.

[0003]

2. Description of the Related Art The following methods are known as methods for measuring calcium in body fluids. (1) Titration method (2) Colorimetric method (3) Atomic absorption method (4) Flame photometric method (5) Electrode method (6) Enzymatic method

The titration method (1) is a chemical titration method using an oxalate salt or a chelate, but it has the drawback that it is complicated and that the practitioner has a difference in the measured value. Among the colorimetric methods of (2), the method of using o-CPC (ortho-cresolphthalein complexone) as a color former is to add 8-hydroxyquinoline to eliminate the influence of Mg ²⁺ ions. Currently, it is most commonly used in hospital laboratories. However, even if 8-hydroxyquinoline is added, the influence of Mg ²⁺ ion of about several% is still present, and the absorbance changes with temperature and time,
It has drawbacks such as an optimum pH range for color development, a low concentration (calcium), and a negative measured value region, and there are many problems particularly when the method is practiced. The atomic absorption method (3) requires expensive equipment, requires skill, and requires dilution of the sample.

The flame color analysis method (4) also requires dilution of the sample, and has problems in specificity and reproducibility. In the electrode method of (5), the equipment is expensive, it is affected by pH, and maintenance of the equipment is complicated. In the enzymatic method of (6),
There are i) a method using calmodulin [see JP-A-62-36199], and ii) a method using phospholipase D and choline oxidase [see JP-A-62-195297]. i) The method using calmodulin has excellent specificity, but the sensitivity is too high, so the sample is diluted (100 to 1000 times).
Is required. ii) The method using phospholipase D and choline oxidase has excellent specificity and does not require dilution of the sample, but it has a drawback that the reaction takes time and continuous measurement is not possible. As described above, all of the measuring methods have drawbacks, and there has been no satisfactory measuring method.

[0006]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies to find a method for quantitatively determining calcium using an enzyme that can be accurately and easily measured. It was found that it can be increased proportionally. It is known that α-amylases of various origins exist as active forms bound to calcium (Enzyme Handbook, edited by Tamiya and Maruo, Asakura Shoten, 493).
page). However, it is not known at all that there is a linear correlation between the activity intensity and the concentration of calcium, and the fact that calcium in body fluids can be measured by utilizing this is now shown by the present inventors. It was discovered for the first time. However, through repeated investigations, it was found that depending on the type of substrate used, a linear correlation could not be obtained, or the measurement range was narrow, so that it could not be put to practical use.

Therefore, the present inventors have made further studies,
(1) Carrying out an enzymatic reaction in the presence of a chelating agent more specific to calcium, and / or (2) As a substrate, a maltooligosaccharide having no chromogenic group at its reducing terminal glucose and a chromogenic source group. It has been found that these drawbacks can be solved by carrying out an enzymatic reaction using a combination of maltooligosaccharides having ## STR4 ## and completed the present invention.

It has been known for a long time that α-amylase releases most of the calcium possessed by the enzyme itself in the presence of a chelating agent and changes to an inactive α-amylase [Eur. J. Biochem., 41 , 171 (1974)], the fact that it became possible to quantitatively measure calcium for the first time in the presence of a chelating agent, and that the measurement range widened, was discovered for the first time. Yes, and it is completely unpredictable from the previously known facts. Furthermore, the fact that the same effect can be obtained by using the two types of substrates shown in (2) was also found for the first time this time, and is not predicted from the prior art.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates particularly to the latter of the findings of (1) and (2) above. 1) The sample contains α-amylase and (a) maltooligosaccharide as a substrate for α-amylase And a substrate selected from maltooligosaccharides having a non-chromogenic source group bound to their reducing terminal glucose, and (b) a maltooligosaccharide having a chromogenic source group bound to its reducing terminal glucose and a chromogenic source group bound to its reducing terminal glucose, and A method for measuring calcium in a body fluid, which comprises operating a substrate selected from maltooligosaccharides having a substituent bonded to non-reducing terminal glucose, 2) 2,4-dichlorophenyl-β as a substrate for α-amylase -D-maltopentaoside and 2-chloro-4
-Nitrophenyl-β-D-maltopentaoside is used in combination, and 3) the malto-oligosaccharide in which a chromogenic group is bonded to the reducing terminal glucose is 2-chloro-4. -Nitrophenyl-α-D-
The measurement method according to the above 1, wherein the measurement method is maltotrioside.

The α-amylase used in the present invention may originate from any of microorganisms, plants or animals, but is preferably of animal origin. For example, commercially available porcine pancreatic α-amylase is Used. α-amylase is 1000 to 100000 U / l, more preferably 5000
Used at a concentration of ~ 500,000 U / l.

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

As such maltooligosaccharides,
(1) Maltooligosaccharides, for example, maltopentaose, maltoheptaose, (2) Maltooligosaccharides having a non-chromogenic source group bonded to the reducing terminal glucose thereof, such as 2,4-dichlorophenyl-α-D-maltotrioside, 2, 4-dichlorophenyl- (α or β) -D-maltopentaoside, 2,4-dichlorophenyl- (α or β) -D-
Malt heptaoside,

(3) Malto-oligosaccharide having a chromogenic group bonded to its reducing terminal glucose, for example, 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- (α or β)
-D-maltopentaoside, 2-chloro-4-nitrophenyl- (α or β) -D-maltoheptaoside, and

(4) Malto-oligosaccharide having a reducing terminal glucose bound to a chromogenic source group and a non-reducing terminal glucose bound to a substituent, for example, 3-ketobutylidene-2-
Chloro-4-nitrophenyl- (α or β) -D-maltopentaoside, ethylidene-4-nitrophenyl-
(Α or β) -D-maltoheptaoside, benzyl-
4-nitrophenyl- (α or β) -D-maltopentaoside, benzylidene-4-nitrophenyl- (α or β) -D-maltopentaoside and the like can be mentioned.

(A) a substrate selected from the above-mentioned maltooligosaccharides represented by (1) and (2) and a reducing oligosaccharide having a non-chromogenic source group bonded to glucose, and (b)
As described in (3) and (4) above, a malgo-oligosaccharide in which a chromogenic source group is bound to its reducing terminal glucose and a maltooligosaccharide in which a chromogenic group is bound to its reducing terminal glucose and a substituent group is bound to a non-reducing terminal glucose. Used in combination with a substrate selected from sugars.

When used in combination, the reaction in the absence of calcium in the sample due to the competitiveness with respect to the substrate (since the enzyme originally has calcium, calcium does not exist in the sample if the substrate is present). However, the enzyme is activated and the reaction proceeds. This is called a blank reaction.) It is thought that measurement becomes possible and the measurement range becomes wider.

The maltooligosaccharide having no chromogenic group shown in (a) and the maltooligosaccharide having a chromogenic group shown in (b) each have a concentration of 0.1 to 50 mmole / l and a concentration of 100: 100. It is preferably added in a ratio of 1 to 1: 1.

Specific examples of the preferable α-amylase represented by the above (b) include 4-nitrophenyl-ammonium in the above (3).
α-D-maltotrioside and 2-chloro-4-nitrophenyl-β-D-maltopentaoside, the above (4)
Among these is 3-ketobutylidene-2-chloro-4-nitrophenyl-β-D-maltopentaoside. Further, a preferable combination of (a) and (b) is maltopentaose and 2-chloro-4-nitrophenyl-β-D.
A combination of -maltopentaoside or a combination of 2,4-dichlorophenyl-β-D-maltopentaoside and 2-chloro-4-nitrophenyl-β-D-maltopentaoside.

In general, the substrate group represented by the above (4) has a small change in blank value with time. On the other hand, in the presence of the chelating agent, calcium (which exists in α-amylase and in the sample) that increases the enzyme activity is eaten by the chelating agent, so that the calcium concentration in the sample is actually high. However, it is considered that the enzyme activity is suppressed to a low level, the measurement becomes possible, and the measurement range becomes wide. However, when two kinds of substrate groups are combined as in the present invention, it is possible to obtain a sufficiently wide measurement range even in the absence of a chelating agent.

As the detection system in the present invention, the one used in the usual α-amylase activity measuring method is used. For example, when starch, which is one of the polysaccharides, is used as a substrate for α-amylase, the amount of reducing sugar produced is calibrated using a known method, for example, the Somogyi method, or it is brewed starch. It can be calibrated by the dye produced.

A color-developing source produced from the substrate of group (b), which is reacted with α-glucosidase and / or β-glucosidase coupled or without the enzyme being coupled,
For example, 4-nitrophenol or 2-chloro-4-nitrophenol can be calibrated by direct colorimetry.

The measurement according to the present invention is carried out in a buffer solution capable of maintaining a pH of 5-8. Such buffers are well known, for example Good's buffer [eg N-
2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (hereinafter abbreviated as HEPES), piperazine-N, N'-bis (2-ethanesulfonic acid) (hereinafter,
Abbreviated as PIPES. ), N-2-hydroxyethylpiperazine-N′-2-hydroxypropane-3-sulfonic acid (hereinafter abbreviated as HEPPSO), etc.], a phosphate buffer solution, a Tris-hydrochloric acid buffer solution and the like. The buffer solution is preferably added at a concentration of 10 to 500 mmole / l.

Further, in the measuring system of the present invention, an alkali metal halide such as sodium chloride, potassium chloride, sodium bromide or sodium iodide is used in an amount of 1 to 400 m.
It is preferable to add it at a concentration of mole / l.

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

[0025]

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

Example 1 [Measurement of calcium by α-amylase using a combination of maltooligosaccharides having a chromogenic group and maltooligosaccharides having no chromogenic group as a substrate] The following reagents were mixed to prepare a reagent solution. Got

[0027]

[Table 1] BUTTER Pancreatic α-Amira-8000U / l × 100 μl α-Glucosidase-2500000 U / l × 100 μl β-Glucosidase-2000 0 U / l × 100 μl 2,4-Dichlorophenyl-β-D-maltopentaocito 20mmole / L × 100 μl 2-chloro-4-nitrophenyl-β-D-maltopentaside 2 mmole / l × 100 μl PIPES buffer (pH 6.8) 500 mmole / l × 100 μl sodium chloride 500 mmole / l × 100 μl Purified water 280 μl Total 980 μl

20 μ of an aqueous calcium acetate solution having a calcium concentration of 0 to 10 mg / dl is added to the reagent solution obtained above.
(1) was added and reacted at 37 ° C., then 400 nm (37
The increase in absorbance at (° C.) Was measured with time, and the reaction rate of each was obtained. The calibration curve obtained is shown in FIG. As can be seen from the figure, the calibration curve showed linearity up to a calcium concentration of 7.5 mg / dl.

Reference Example 1: [α in the presence of a chelating agent using a maltooligosaccharide having a chromogenic group as a substrate]
-Measurement of Calcium by Amylase] The following reagents were mixed to obtain a reagent solution.

[0030]

[Table 2] Butter Pancreatic α-Amylla 8000U / l × 100 μl α-Glucosidase-250 0000 U / l × 100 μl β-Glucosidase-2000 0 U / l × 100 μl 2-Chloro-4-nitrophenyl-β-D-maltopenta Osid 2mmole / l × 100μl BAPTA 20mmole / l × 100μl Phosphate buffer (pH 6.5) 500mmole / l × 100μl Sodium chloride 500mmole / l × 100μl Purified water 280μl Total 980μl

Example 1 using the reagent solution obtained above
The measurement was performed in the same manner as in. The calibration curve obtained is shown in FIG. As can be seen from the figure, the calibration curve shows a calcium concentration of 10
It showed linearity up to mg / dl.

Reference Example 2: [Measurement of calcium by α-amylase in the presence of a chelating agent, using a combination of a maltooligosaccharide having a chromogenic group and a maltooligosaccharide having no chromogenic group as a substrate] Were mixed to obtain a reagent solution.

[0033]

[Table 3] Butter Pancreatic α-Amilla-8000U / l × 100 μl α-Glucosidase-250 0000 U / l × 100 μl β-Glucosidase-20000U / l × 100 μl Maltopentao-s 20mmole / l × 100 μl 2-Chloro-4- Nitrophenyl-β-D-maltopentaside 2mmole / l × 100μl BAPTA 20mmole / l × 100μl Phosphate buffer solution (pH 6.5) 500mmole / l × 100μl Sodium chloride 500mmole / l × 100μl Purified water 180μl 980 μl in total

Example 1 using the reagent solution obtained above
Same as above (however, calcium concentration 0 to 30 mg / dl
A corresponding aqueous solution of calcium acetate was used. ) The measurement was performed. The calibration curve obtained is shown in FIG. As you can see from the figure,
The calibration curve showed almost linearity up to a calcium concentration of 30 mg / dl.

Reference Example 3: [α in the presence of a chelating agent using a maltooligosaccharide having a chromogenic group as a substrate]
-Measurement of Calcium by Amylase] The following reagents were mixed to obtain a reagent solution.

[0036]

[Table 4] Butter Pancreatic α-Amylase 200000U / l × 100 μl 4-Nitrophenyl-α-D-maltotrioside 5 mmole / l × 100 μl BAPTA 1 mmole / l × 100 μl GEDTA 1.75 mmole / l × 100 μl HEPPSO buffer ( pH 8.0) 500mmole / l × 100μl Sodium chloride 500mmole / l × 100μl Purified water 390μl Total 990μl

10 μl of an aqueous calcium acetate solution having a calcium concentration of 0 to 50 mg / dl was added to the reagent solution obtained above.
(1) was added and reacted at 37 ° C., then 400 nm (37
The increase in absorbance at (° C.) Was measured with time, and the reaction rate of each was obtained. The calibration curve obtained is shown in FIG. As can be seen from the figure, the calibration curve showed linearity up to a calcium concentration of 50 mg / dl.

Reference Example 4: [Using α-amylase in the presence of a chelating agent using a maltooligosaccharide having a chromogenic group bound to its reducing terminal glucose and a substituent bound to its non-reducing terminal glucose as a substrate Measurement of Calcium] The following reagents were mixed to obtain a reagent solution.

[0039]

[Table 5] Butter Pancreas α-Amyla-8000U / l × 100 μl α-Glucosidase-2500000 U / l × 100 μl β-Glucosidase-20000 U / l × 100 μl 3-Ketobutylidene-2-chloro-4-nitrophenyl-β- D-maltopentaside 20mmole / l × 100 μl BAPTA 20mmole / l × 100 μl Phosphate buffer solution (pH 6.5) 500 mmole / l × 100 μl Sodium chloride 500 mmole / l × 100 μl Purified water 280 μl Total 980 μl

Example 1 using the reagent solution obtained above
Same as above (however, calcium concentration is 0-100mg /
An aqueous solution of calcium acetate corresponding to dl was used. ) The measurement was performed. The calibration curve obtained is shown in FIG. As can be seen from the figure, the calibration curve showed linearity up to a calcium concentration of 100 mg / dl.

Comparative Example 1: [Method of Reference Example 2 and o-CPC
Effect of Magnesium in Method (Conventional Method)] As a sample, serum 900 μl and magnesium concentration 0 to 1
100 μl of magnesium acetate solution corresponding to 00 mg / dl
Using the mixed solution of, the calcium was measured by the method described in Reference Example 2. On the other hand, using an o-CPC method measurement kit [Calcium II-HA Test Wako (manufactured by Wako Pure Chemical Industries)], calcium of the same sample was measured by an automatic analyzer (Hitachi 705 type). The influence of magnesium on the two measuring methods is shown in FIG. As can be seen from the figure, the method of Reference Example 2 can be measured with almost no influence of magnesium in the sample, but the o-CPC method has been devised to suppress the influence of magnesium, It has been affected significantly.

Comparative Example 2: [Correlation between the value measured by the method of Reference Example 2 and the value measured by the o-CPC method (conventional method)] For 25 serum samples, the method described in Reference Example 2 and o-C
The amount of calcium was measured by the PC method (using the measuring kit described in Comparative Example 1). Figure 7 shows the correlation between the two.
Shown in. As can be seen from the figure, the regression line of the correlation is n = 2
5, r = 0.944, y = 0.985 χ + 1.552, and both showed a good correlation. From this, it can be seen that the method of Reference Example 2 can be sufficiently used for daily inspection.

Comparative Example 3: [Correlation between the value measured by the method of Reference Example 3 and the value measured by the o-CPC method (conventional method)] For 36 serum samples, the method described in Reference Example 3 and o-C were used.
The amount of calcium was measured by the PC method (using the measuring kit described in Comparative Example 1). Figure 8 shows the correlation between the two.
Shown in. As can be seen from the figure, the regression line of the correlation is n = 3
6, r = 0.923, y = 1.017 χ + 0.102, and both showed a good correlation. From this, it can be seen that the method of Reference Example 3 can be sufficiently used for daily inspection.

Comparative Example 4: [Correlation between the value measured by the method of Reference Example 3 and the value measured by the o-CPC method (conventional method)] For 40 urine specimens, the method described in Reference Example 3 and o-CP
The amount of calcium was measured by the C method (using the measurement kit described in Comparative Example 1). The correlation between the two is shown in FIG. As can be seen from the figure, the regression line of the correlation is n = 40,
r = 0.994 and y = 0.845 χ + 0.32, indicating a good correlation between the two. From this, it can be seen that the method of Reference Example 3 can be sufficiently used for daily inspection.

[0045]

EFFECTS OF THE INVENTION Since the present invention is a method using an enzyme, it has a higher specificity than other methods, and does not require a long reaction time as compared with a conventional method using an enzyme, and it is accurate and This is a simple method for measuring calcium.

[Brief description of drawings]

FIG. 1 is a calibration curve according to the calcium measuring method of the present invention.

FIG. 2 is a calibration curve according to the calcium measurement method of Reference Example 1.

FIG. 3 is a calibration curve according to the calcium measurement method of Reference Example 2.

FIG. 4 is a calibration curve according to the calcium measurement method of Reference Example 3.

FIG. 5 is a calibration curve according to the calcium measurement method of Reference Example 4.

FIG. 6 is a graph comparing the effect of magnesium in a sample between the method of Reference Example 2 and the o-CPC method (conventional method).

FIG. 7 is a graph showing the correlation between the measured value by the method of Reference Example 2 and the measured value by the o-CPC method (conventional method).

FIG. 8 is a graph showing the correlation between the measured value of the serum sample by the method of Reference Example 3 and the measured value by the o-CPC method (conventional method).

FIG. 9 is a graph showing the correlation between the measurement value of the urine sample according to the method of Reference Example 3 and the measurement value according to the o-CPC method (conventional method).

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Hiroshi Shimizu 3-1-1 Sakurai, Shimamoto-cho, Mishima-gun, Osaka Prefecture Ono Pharmaceutical Co., Ltd. Minase Research Laboratory

Claims (3)

[Claims] 1. A sample containing α-amylase, a substrate selected from (a) a maltooligosaccharide and a maltooligosaccharide in which a non-chromogenic source group is bound to glucose at its reducing terminal, and (b) a reduction thereof. Characterized by combining a malto-oligosaccharide having a chromogenic group attached to the terminal glucose and a substrate selected from malto-oligosaccharide having a chromogenic group attached to the reducing terminal glucose and a substituent attached to the non-reducing terminal glucose in combination. Method for measuring calcium in body fluids. 2. As a substrate for α-amylase, 2,4-
Dichlorophenyl-β-D-maltopentaoside and 2-
The measuring method according to claim 1, wherein chloro-4-nitrophenyl-β-D-maltopentaoside is used in combination. 3. The measuring method according to claim 1, wherein the maltooligosaccharide having a chromogenic group bonded to the reducing terminal glucose is 2-chloro-4-nitrophenyl-α-D-maltotrioside.