JPH0612998B2 - Reagent for measuring enzyme activity - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention relates to a reagent used for measuring enzyme activity in serum, which is useful for medical purposes, clinical tests and the like.

(Prior Art) Conventionally, measurement of enzyme activity in serum has been widely used as an important role in medical diagnosis. As one of the measurement methods, a dye is bound to a certain substance. The amount of the dye that is released by the action of the enzyme that is dissociated by the action of the enzyme (substrate)
A method is used in which the enzyme activity is measured by measuring the absorbance with time and determining the rate of increase in absorbance per unit time at that time.

For example, as an enzyme in serum, for example, α-amylase or γ-glutamyl tranferase, for example, conventionally, to measure the activity of α-amylase or γ-glutamyl tranferase in serum, As its substrate, 4-nitrophenol, 2-chloro-4
A compound in which a polysaccharide or an amino acid is bound to a substance having an absorbance at 400 nm to 450 nm such as -nitrophenol, 4-nitroaniline, and 3-carboxy-4-nitroaniline is used.

The substrate used when measuring the activity of α-amylase is 2-chloro-4-nitro-phenyl-β-D.
-Maltoheptaoside (see the following formula a-1; hereinafter abbreviated as G7-CNP), 4-nitrophenyl-α-D-maltoheptaoside (see the following formula a-2; hereinafter abbreviated as G7-PNP) Do),
2-Chloro-4-nitrophenyl-β-D-maltopentaoside (see Formula a-3 below; hereinafter abbreviated as G5-CNP) is famous.

Further, as a substrate used for measuring the activity of γ-glutamyl transferase, L-γ-glutamyl-4-nitroanilide (see the following formula b-1; hereinafter Glu-4-
Abbreviated as NA) and L-γ-glutamyl-3-carboxy-4-nitroanilide (see the formula b-2 below; hereinafter Glu-CN).
Abbreviated as A) is famous.

Formula a-1: (G7-CNP) Formula a-2: (G7-PNP) Formula a-3: (G5-CNP) Formula b-1 (R = H): (Glu-4NA) Formula b-2 (R = COOH): (Glu-CNA) When measuring the activity of α-amylase or γ-glutamyl tranferase using these substrates, first, in the α-amylase activity measurement, G7-CNP, G7-PNP or G5
Of 2-chloro-4-nitrophenol or 4-nitrophenol generated from CNP (increased absorbance over time in the wavelength range of 400 to 450 nm, that is, increase rate of absorbance, α-amylase Calculate the activity value. In addition, in the measurement of γ-glutamyl transferase activity, the wavelength of 4-nitroaniline or 3-carboxy-4-nitroaniline generated from Glu-4-NA or Glu-CNA was measured.
The rate of increase in absorbance in the 400 to 450 nm region is calculated, and the γ-glutamyl transferase error value is calculated.

The method for obtaining such an enzyme activity is called a rate assay, and this rate assay will be described in detail as follows.

Method for calculating enzyme activity (U /) Change in absorbance over time (increase or decrease)
The method of measuring the
It is called Rate Atssei.

Enzyme activity 1 U / is defined as the amount of enzyme that catalyzes a reaction of 1 μmol / min in 1 minute, and in order to determine the enzyme activity from the change with time in absorbance (E), the change in absorbance with time (ΔE ), The change in absorbance per 4 minutes (ΔE / min) is calculated and further substituted into the following formula to obtain the enzyme activity (U /).

RV: Volume of reagent (solution) used for measurement SV: Volume of sample used for measurement ε: Molecular extinction coefficient of the substance whose absorbance is to be measured By using these methods, α-amylase or As a disadvantage in measuring the activity of γ-glutamyl tranferase, a phenomenon that a negative error occurs in the measured activity value of α-amylase or γ-glutamyl tranferase due to the hemolytic action that occurs when serum is prepared. (Japanese Journal of Clinical Laboratory Automation Vol. 9 Supplements P141, P145 (1984)).

This phenomenon is that hemoglobin and its derivatives produced by hemolytic action are gradually decomposed by light and heat in the measurement reagent, causing a decrease in absorbance with time in the wavelength range of 400 to 450 nm, and α-amylase and γ- It decreases the increase in absorbance over time in the wavelength range of 400 to 450 nm, that is, the rate of increase in absorbance, which is measured when measuring the activity of glutamyl transferase. As a result, a negative error will occur in the measured α-amylase and γ-glutamyl tranferase activity values.

(Problems to be Solved by the Invention) The present invention is based on the above-mentioned wavelength range of 400 to 4 in the prior art.
It is intended to prevent the phenomenon of decrease in the measured value of enzyme activity caused by hemoglobin and its derivatives caused by hemolytic action in the absorbance measurement at 50 nm.

DISCLOSURE OF THE INVENTION The present inventors have found that the phenomenon of decrease in the measured value of enzyme activity caused by hemoglobin and its derivatives caused by the hemolytic action can be prevented by using thiourea in the reagent.

The present invention was made based on such findings,
Therefore, the present invention is characterized by containing thiourea in the enzyme activity measuring reagent used in the method for measuring the absorbance in the region of 400 nm to 450 nm in order to measure the enzyme activity in serum. The present invention provides a reagent for measuring enzyme activity in serum.

The present invention will be described in detail below.

In the present invention, thiourea is used in the enzyme activity measuring reagent, but it is used at a concentration of 0.01% by weight or more in the solution of the reagent for measuring the enzyme activity. This concentration exerts an effect of preventing a negative error in the enzyme activity measurement value caused by hemolysis (hereinafter, this effect is referred to as an anti-hemolytic effect). As the concentration of thiourea increases, the anti-hemolytic effect also increases, but if the concentration of thiourea increases more than necessary, it will inhibit the activity of the enzyme, so it is usually 0.2 to 0.25% by weight. It is desirable to use it at a concentration of.

Regarding the concentration of thiourea to be used, any optimum concentration may be appropriately determined depending on the conditions of serum as a sample, the type of substrate used, and other various factors under the use conditions, and is not particularly limited. Absent.

The preparation of the reagent of the present invention and the method of measuring the enzyme activity using the reagent are carried out according to the conventional conventional technique except that thiourea is used.

Hereinafter, the present invention will be described more specifically with reference to examples of the present invention.

In each of the following Examples and Comparative Examples, as a reference serum, α-amylase 100 U / was contained, and as a result of measuring hemoglobin in the serum by the cyanmethemoglobin method, the serum containing no hemoglobin (S (Abbreviated as ₀ ) is used.

Examples 1 to 5 (A) Preparation of Reagent: Thiourea was dissolved in 0.05M phosphate buffer (pH 7.0) containing 0.05M potassium chloride and 0.1% by weight sodium azide to obtain 0.2. Make up a wt% solution. 70 KU of α-glucosidase and 10 KU of β-glucosidase were added and dissolved in this solution 1, and G7-CNP was further added and dissolved so as to be 2.5 mM to obtain a reagent for measuring α-amylase activity (solution A).

(B) Preparation of hemoglobin-containing serum: The serum S ₀ was divided into 5 equal parts, hemoglobin produced by hemolysis was added to each serum, and the hemoglobin concentration contained in each serum was 0.1%, 0.2%, 0.3. %, 0.4%, 0.
Lyse to 5% and add those sera to S1, S2, S3, S
4 and S5.

Example 1 3.0 ml of the above-mentioned solution A was mixed with 0.05 ml of S1 as a sample, and the increase in absorbance with time at a wavelength of 405 nm was measured at 37 ° C., and the degree of increase in absorbance per minute was determined.
The α-amylase activity was calculated by substituting the obtained value of the degree of increase per minute into the following equation.

Example 2 Using S2 as a sample, the α-amylase activity was measured in the same manner as in Example 1.

Example 3 Using S3 as a sample, the α-amylase activity was measured in the same manner as in Example 1.

Example 4 Using S4 as a sample, the α-amylase activity was measured in the same manner as in Example 1.

Example 5 Using S5 as a sample, the α-amylase activity was measured in the same manner as in Example 1.

In FIG. 1 of the accompanying drawings, the results of Examples 1 to 5 are as follows:
◯ − is shown with an example number attached.

Comparative Examples 1 to 5 A solution (solution A ′) having a composition in which only thiourea was removed from the components of the solution A used in Examples 1 to 5 was prepared and used as an α-amylase activity measuring reagent described below. Used in Comparative Examples 1-5.

Comparative Example 1 S1 0.05 ml was mixed as a sample with 3.0 ml of solution A ′,
The increase in absorbance with time at a wavelength of 405 nm was measured at 37 ° C., and α-amylase activity was determined by the same method as in Example 1.

Comparative Example 2 Using the above S2 as a sample, α-
Amylase activity was measured.

Comparative Example 3 Using the above S3 as a sample, α-
Amylase activity was measured.

Comparative Example 4 Using the above S4 as a sample, α-
Amylase activity was measured.

Comparative Example 5 Using S5 as a sample, α-
Amylase activity was measured.

The result of each comparative example is shown as a comparative example number as-●-in the attached FIG.

As is clear from FIG. 1, in the activity measurement of α-amylase using G7-CNP as a substrate, it is shown that the present invention eliminates the influence of hemoglobin due to hemolysis.

Examples 6-10 (A) Preparation of Reagents: 0.1 M phosphate buffer (pH 7.0) containing 0.05 M sodium chloride, 1.5 mM G7-PNP, 0.1% sodium azide and 50 KU / α-glucosidase. ) Is added to thiourea to adjust the concentration to 0.2% to prepare an α-amylase activity measuring reagent (solution B).

(B) Hemoglobin-containing serum S1, S2, S3, S4, S5 described in Examples 1-5 and (B) above
To use.

Example 6 3.0 ml of the above solution B was used as a sample in Example 1.
S1 (0.05 ml) was mixed, the increase in absorbance with time at a wavelength of 405 nm was measured at 37 ° C., and the increase in absorbance per minute was determined. The α-amylase activity was calculated by substituting the obtained value of the degree of increase per minute into the following equation.

Example 7 Using the S2 used in Example 2 as a sample, the α-amylase activity was measured in the same manner as in Example 6.

Example 8 Using the S3 used in Example 3 as a sample, the α-amylase activity was measured in the same manner as in Example 6.

Example 9 Using the S4 used in Example 4 as a sample, the α-amylase activity was measured in the same manner as in Example 6.

Example 10 Using S5 used in Example 5 as a sample, the α-amylase activity was measured in the same manner as in Example 6.

The results of Examples 6 to 10 are shown in FIG.
Are shown with the example numbers.

As is clear from FIG. 2, the present invention also shows that the effect of hemoglobin due to hemolysis is eliminated by the present invention even in the measurement of α-amylase activity using G7-PNP as a substrate.

Examples 11 to 15 (A) Preparation of Reagent: Glycylglycine 20 g was dissolved in 800 ml of distilled water,
The pH is adjusted to 7.9 by adding a 1N-sodium hydroxide aqueous solution. To this solution, 2.08 g of Glu-CNA, 1 g of sodium azide and 2.5 g of thiourea were added and dissolved, and distilled water was added to bring the total amount to 1, which was used as a reagent for measuring the activity of γ-glutamyl tranferase (solution C ).

(B) Preparation of hemoglobin-containing serum containing γ-glutamyl transferrorase 100U / as a standard serum, the cyanmethemoglobin method, as a result of measuring hemoglobin in the serum, serum that did not contain hemoglobin (T (Abbreviated as ₀ ) was used. T ₀
Into 5 equal parts, add hemoglobin produced by hemolysis to each serum, and dissolve so that the hemoglobin concentration in each serum becomes 0.1%, 0.2%, 0.3%, 0.4%, 0.5% , Each serum was designated as T1, T2, T3, T4, T5.

Example 11 To 3.0 ml of the above solution C, 0.05 ml of T1 was mixed as a sample,
The increase in absorbance with time at a wavelength of 415 nm was measured at 37 ° C, and the degree of increase in absorbance per minute was determined. The obtained value of the degree of increase per minute was substituted into the following formula to determine the γ-glutamyl tranferase activity.

Example 12 Using T2 as a sample, the γ-glutamyl transferase activity was measured in the same manner as in Example 11.

Example 13 Using T3 as a sample, the γ-glutamyl transferase activity was measured in the same manner as in Example 11.

Example 14 Using T4 as a sample, the γ-glutamyl transferase activity was measured in the same manner as in Example 11.

Example 15 Using T5 as a sample, the γ-glutamyl transferase activity was measured in the same manner as in Example 11.

The respective results of Examples 11 to 15 are shown as Example numbers in the attached FIG.

Comparative Examples 6 to 10 A solution (solution C ′) having a composition in which only thiourea was removed from the components of the solution C used in Examples 11 to 15 was prepared and used as a reagent for measuring γ-glutamyl tranferase activity. Was used in Comparative Examples 6 to 10 below.

Comparative Example 6 3.0 ml of the solution C ′ was mixed with 0.05 ml of T1 as a sample, and the increase in absorbance with time at a wavelength of 415 nm at 37 ° C. was measured. The same procedure as in Example 11 was used to determine the γ-glutamyl transferase. The activity was sought.

Comparative Example 7 Using T2 as a sample, the same operation as in Comparative Example 6 was carried out.
Glutamyl transferase activity was measured.

Comparative Example 8 Using T3 as a sample, the same operation as in Comparative Example 6 was carried out.
Glutamyl transferase activity was measured.

Comparative Example 9 Using T4 as a sample, the same operation as in Comparative Example 6 was carried out.
Glutamyl transferase activity was measured.

Comparative Example 10 Using the above T5 as a sample, the same operation as in Comparative Example 6 was carried out.
Glutamyl transferase activity was measured.

The results of Comparative Examples 6 to 10 are shown in FIG.
It is shown with a comparative example number as −.

Since Comparative Example 10 showed a negative value, it is not shown in the figure.

As is clear from FIG. 3, the present invention also shows that the effect of hemoglobin due to hemolysis can be eliminated by measuring the activity of γ-glutamyl transferase using Glu-CNA as a substrate.

[Brief description of drawings]

The attached drawings and FIG. 1 are graphs showing the results of measuring α-amylase activity in serum according to Examples 1 to 5 of the present invention and Comparative Examples 1 to 5 described above. FIG. 2 is a graph showing the measurement results of α-amylase activity in serum according to Examples 6 to 10 of the present invention described above. FIG. 3 is a graph showing the measurement results of γ-glutamyl tranferase activity in serum according to Examples 11 to 15 of the present invention and Comparative Examples 6 to 9 described above.

Claims (3)

[Claims] 1. To measure the enzyme activity in serum, 400
A reagent for measuring enzyme activity in serum, which comprises thiourea in the reagent for measuring enzyme activity used in the method for measuring absorbance in the range of nm to 450 nm. 2. The enzyme activity measuring reagent according to claim 1, wherein the enzyme is α-amylase. 3. The reagent for measuring enzyme activity according to claim 1, wherein the enzyme is γ-glutamyl transferase.