JPH0536038B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel method for quantifying biological components. More specifically, the present invention provides a method for quantifying biological components, which is carried out by causing an oxidizing enzyme to act on a biological component or a substance produced by the action of an enzyme on a biological component, and measuring the generated hydrogen peroxide. A reagent consisting of a tetravalent titanium compound and 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol or/and a salt thereof (hereinafter referred to as Ti-PAPS reagent). This invention relates to a novel method for quantifying biological components using Measuring biological components, such as body fluid components such as blood and urine, is essential for diagnosing diseases, elucidating pathological conditions, and determining the course of treatment, as their fluctuations are strongly related to diseases. It's summery. For example, methods have been developed to measure a wide variety of trace components, including cholesterol, triglycerides, glucose, uric acid, phospholipids, bile acids, and monoamine oxidase in the blood. As is well known. Currently, the method for measuring serum components is to use an enzyme that specifically acts on the target component, and if the target component is an enzyme, use an enzyme that acts specifically on the target component. Using a compound,
The so-called "enzyme method" in which the target component amount is determined by carrying out an enzymatic reaction and measuring the resulting product is generally widely used. Among them, H ₂ O ₂ generating enzyme,
For example, oxidase is activated to generate H ₂ O ₂ corresponding to the target component, and this is converted into peroxidase,
Along with the development of oxidizable coloring reagents, the method of determining the amount of the target component by introducing the oxidizable coloring reagent, which is a coloring component, into a coloring system and quantifying it colorimetrically is increasing. . For example, H ₂ O ₂ generated by a combination of cholesterol-cholesterol oxidase, triglyceride-lipoprotein lipase-glycerol oxidase, uric acid-uricase, etc. is converted into peronosidase (POD),
In this method, an oxidizable coloring reagent is introduced into a coloring system, and the amount of the target component is determined by measuring the absorbance of the coloring. Typical examples of the oxidizable coloring reagent used in this method include oxidizable color reagents that are a combination of 4-aminoantipyrine and a phenol compound or an N,N-disubstituted aniline compound. color reagent, combination reagent of 3-methylbenzothiazolinone hydrazone (MBTH) and aniline compound, 2,2'-azinobis(3
-ethylbenzothiazoline-6-sulfonic acid)
(ABTS), triphenylmethane-based leuco dyes, etc., but all of these are H ₂ O ₂ −POD
The system develops oxidative color as a result of the dehydrogenation reaction from the oxidizable coloring reagent. Therefore, when ascorbic acid reducing substances coexist in the sample, negative errors often occur in the measured values due to their influence. As a method to avoid such interference with reducing substances, a method using ascorbic acid oxidase (Japanese Patent Publication No. 56-39198
(No. Publication), a method using iodic acid or its salt,
Alternatively, methods using periodic acid or salts thereof (Japanese Patent Application Laid-open No. 109595/1982, 151358/1982, 107161/1982) have been disclosed and practiced so far. . However, all of these methods have advantages and disadvantages, and are not necessarily completely satisfactory. In view of this situation, the present inventors are in the process of conducting extensive research into a method for quantifying biological trace components that does not require the above-mentioned method for removing reducing substances, that is, is less susceptible to the effects of coexisting reducing substances. A soluble tetravalent titanium compound and 4-(2-pyridylazo)-resorcinol (hereinafter abbreviated as PAR).
Alternatively, they discovered a method using a reagent consisting of 1-(2-pyridylazo)-2-naphthol (hereinafter abbreviated as PAN), and have previously filed a patent application (Japanese Patent Laid-Open Publication No. 45197/1983). That is, this method captures hydrogen peroxide produced by an enzymatic reaction by forming a complex, and by measuring the absorbance of this complex,
It is possible to quantify biological components almost unaffected by reducing substances. However, this
All of the complexes produced in the method using PAR or PAN have slightly small molecular extinction coefficients (ε = 36000 in the case of the Ti-PAR-H ₂ O ₂ ternary complex,
For the Ti-PAN-H ₂ O ₂ ternary complex, ε = 12000), and the maximum absorption wavelengths for both are around 500 nm, which cannot necessarily be said to be on the sufficiently long wavelength side (Ti-PAR-
In the case of H ₂ O ₂ ternary complex, λ _nax = 508 nm), sensitivity and accuracy are important when quantifying biological fluid components such as serum that coexist with impurities (bilirubin, hemoglobin, etc.) that have absorption around 400 nm. However, I was still not completely satisfied with it. As a result of further research into a reagent capable of forming a ternary complex with higher sensitivity and absorption on the longer wavelength side, and a method for quantifying biological components using this, the inventors discovered that tetravalent titanium Compound and 2-
(5-Bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol or/and a salt thereof, it has been found that the object can be achieved, and the present invention reached. That is, the present invention provides a method for quantifying biological components, which is carried out by applying an oxidizing enzyme that specifically acts on a biological component or a substance produced by the action of an enzyme on a biological component, and measuring the produced hydrogen peroxide. The resulting hydrogen peroxide contains a tetravalent titanium compound and 2-
Reacting a reagent comprising (5-bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol or/and a salt thereof to produce a dye, and measuring the dye colorimetrically. This is a method for quantifying biological components characterized by: PAPS of the Ti-PAPS reagent used in the present invention has the following structural formula: Examples include 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol or/and its mono- or di-sodium salt, potassium salt, ammonium salt, Each may be anhydrous or hydrated. As the tetravalent titanium compound used in the present invention,
Examples include water-soluble titanium compounds such as titanium tetrachloride, titanium tetrabromide, and titanium sulfate, but titanium tetrachloride is usually more preferably used. The Ti-PAPS reagent used in the present invention is usually prepared, for example, as follows. Preparation example (Ti-PAPS reagent) (1) Reagent solution Dissolve 24ml of titanium tetrachloride in 4N hydrochloric acid and dilute the entire volume.
The volume should be 500ml. Next, this is diluted with water to obtain a solution with a titanium tetrachloride concentration of 1×10 ⁻³ mo/. (2) Test solution 2-(5-bromo-2-pyridylazo)-5-
Dissolve 0.0617 g of (N-propyl-N-sulfopropylamino)phenol disodium dihydrate in water to make 100 ml and give a concentration of 1 x 10 ^-3 mo/. (3) Ti-PAPS reagent If you take 40ml each of the test solution and test solution and add water to make the total volume 100ml, Ti()4×10 ^-4 M,2-
A hydrochloric acid acidic solution of Ti-PAPS containing 4×10 ⁻⁴ of (5-bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol is obtained. The amount of Ti-PAPS reagent used in the quantitative method of the present invention is usually Ti()4×10 ^-4 M, PAPS4×10 ^-4 M.
1/20 to 3/10, preferably 1/10 to 2/10 of the total volume of the measurement system is used as an acidic hydrochloric acid solution (PH3↓) containing . In the method for quantifying biological components of the present invention using the Ti-PAPS reagent, the optimal pH is 6 to 8, and the buffer to maintain the pH within this range is not particularly limited, but usually phosphate buffer is used. A liquid is more preferably used. The method for quantifying biological components of the present invention using the Ti-PAPS reagent is performed by causing an oxidizing enzyme to act on a biological component or a substance produced by the action of an enzyme on a biological component, and quantifying the hydrogen peroxide produced thereby. This is done indirectly, and the quantitative determination is made by measuring the absorbance of the color of the hydrogen peroxide-titanium ()-PAPS ternary complex.
Therefore, the measuring means itself is the same as that of the ordinary spectrophotometric assay, and the same measuring device as used in the ordinary spectrophotometric assay is used. The quantitative method of the present invention is usually carried out in two steps: treating a sample with an oxidizing enzyme to produce hydrogen peroxide, and then reacting this hydrogen peroxide with a Ti-PAPS reagent, or using an enzyme. This can be done effectively even in one step by using a large amount. The quantitative method of the present invention does not utilize a color reaction based on a redox reaction, but rather measures color based on a complex formation reaction. Almost no effect of reducing substances is observed. Therefore, there is no need to remove reducing substances coexisting in the biological sample by the above-mentioned method or the like. Below is a table showing the results of an investigation into the effects of coexisting substances on the determination of glucose by this method.

[Table] Note: Add 2U/ml of glucose oxidase and 3 _{× 10} ^-4 of NaN3 to 0.5ml of 0.01M phosphate buffer (PH5.6).
Glucose standard solution containing each coexisting substance (final concentration 1.00 ×
10 ^-5 M) was added and heated at 37°C for 15 minutes. Then, Ti prepared according to the above preparation
-0.5 ml of PAPS reagent, 0.5 ml of water, and 0.5 ml of 0.2 M phosphate buffer (PH7.0) were added in sequence, and the mixture was further heated at 37°C for 10 minutes. Cool the reaction solution to room temperature and scan at 539 nm.
The absorbance was measured at As is clear from the table, in the method of the present invention,
It can be seen that there is almost no influence from coexisting substances. In the measurement method of the present invention, Ti-PAPS reagent and H ₂
The Ti-PAPS-H ₂ O ₂ ternary complex formed by O ₂ has a maximum absorption wavelength λ _nax of 539 nm and Ti-PAR-H ₂
Since it is on the longer wavelength side compared to that of the O ₂ complex,
It is less susceptible to the effects of coexisting substances such as bilirubin and hemoglobin, and the molecular extinction coefficient ε is 5.7×10 ⁴
Because of its large size, it is more suitable for quantifying trace components in biological samples. The composition of the Ti-PAPS-H ₂ O ₂ ternary complex was determined by a continuous change method and was found to be 1:1:1. The quantitative method of the present invention is suitable for quantifying trace components in biological fluids such as serum and urine, such as uric acid, glucose, free fatty acids, cholesterol, and choline, but is not limited to these. It can be used to quantify any biological component that uses an oxidase that acts to produce hydrogen peroxide and quantifies the produced hydrogen peroxide. The measurement method of the present invention can be applied both manually and to a method using an automatic analyzer. As described above, the present invention provides a novel and effective biological trace component that is hardly affected by coexisting reducing substances such as ascorbic acid, has high sensitivity, and is hardly affected by serum components such as bilirubin and hemoglobin. This method provides a method for quantifying the amount of carbon dioxide, making it an enormous contribution to this industry. Examples are shown below, but the present invention is not limited to these Examples. Example 1 Determination of glucose in serum (1) Sample preparation Glucose concentrations (×10 ³ M) were 1.25, 2.5, and 2.5, respectively.
Standard serum of 3.75, 5.0, 7.5, 10.0, and 15.0 was prepared. (2) Preparation of Ti-PAPS reagent Contains Ti()4×10 ^-4 M and PAPS4×10 ^-4 M in the same manner as in the preparation example (Ti-PAPS reagent) above.
A hydrochloric acid acidic solution of Ti-PAPS was prepared. (3) Measurement procedure Add glucose oxidase to 0.5 _ml of 0.01 M phosphate buffer (PH5.6) at 2 U/ml and _NaN3 at 3×10 ^-4 M.
10μ of each of the standard serum prepared in (1) above was added thereto, and each was heated at 37°C for 15 minutes. Next, 0.5 ml of Ti-PAPS reagent, 3.5 ml of water
ml and 0.5 ml of 0.2M phosphate buffer (PH7.0) were added sequentially, and the mixture was further heated at 37°C for 10 minutes. The reaction solution was cooled to room temperature, and the absorbance at 539 nm was measured.
The calibration curve connecting the absorbance plotted against the glucose concentration (concentration in the final coloring solution) of each standard serum is a straight line connecting the origin, as shown in Figure 1.
The calibration curve shows good quantitative properties. Example 2 Determination of glucose in serum (addition recovery rate) A serum sample with a glucose concentration of 72.0 to 120 mg/dl was used. Add glucose to this at 25mg/dl or 50mg/dl
The absorbance was measured in the same manner as in Example 1, the glucose concentration was determined from the calibration curve obtained in Example 1, and the glucose addition recovery rate was calculated. The results are shown in Table 2.

[Table] Example 3 Determination of glucose in serum (correlation) [1] This method (Y) Using 50 serum samples, the absorbance was measured in the same manner as in Example 1. Glucose concentrations were determined from the obtained calibration curves. [2] Conventional method (X) Using the same sample as in [1], a commercially available glucose measurement reagent, Kitt Glucose B-Test Wako (Wako Pure Chemical Industries, Ltd.), was used to perform "glucose oxidase-peroxidase-4". -Aminoantipyrine-phenol method' was used to measure glucose concentration. The correlation between [] and [] is shown in Figure 2. As is clear from FIG. 2, this method shows good correlation with the conventional method. (Y ⁼ 0.92
Standard serum of 1.0, 1.5, 2.0, 2.5, and 3.0 was prepared. (2) Preparation of Ti-PAPS reagent In the same manner as in (2) of Example 1, Ti()4×10 ^-4
A hydrochloric acid acidic solution of Ti-PAPS containing 4×10 ⁻⁴ M of M, PAPS was prepared. (3) Measurement procedure Dissolve uricase in 0.6ml of 0.01M phosphate buffer (PH5.6) to a concentration of 100mU/ml, add 50μ each of the standard serum prepared in (1) above,
Each was heated at 25°C for 15 minutes. Next, add 0.5 ml of Ti-PAPS reagent, 3.5 ml of water, and 0.2 M phosphate buffer (PH7.0).
0.4 ml was added one after another and heated at 37°C for 10 minutes. The reaction solution was cooled to room temperature, and the absorbance at 539 nm was measured. The calibration curve connecting the absorbance plotted against the uric acid concentration of each standard serum (concentration in the final coloring solution) is a straight line connecting the origin, as shown in Figure 3, and the calibration curve shows good quantitative properties. There is. Example 5 Quantification of uric acid in serum (addition recovery rate) 5.0 mg/dl or 10.0 mg/dl of uric acid was added to a serum sample, and the absorbance was measured in the same manner as in Example 4. The uric acid concentration was determined from the calibration curve obtained, and the uric acid addition recovery rate was calculated. Table 3 shows the results.
Shown below.

【table】 [Brief explanation of the drawing]

Figure 1 shows the calibration curve obtained in Example 1, connecting the points obtained by plotting the absorbance (OD) obtained for each glucose concentration (x10 ⁵ M) on the horizontal axis along the vertical axis. It is something. Figure 2 shows the correlation between the glucose concentration measurements obtained by the present method in Example 3 and the glucose concentration measurements obtained by the conventional method, and the horizontal axis X represents the glucose concentration measurement values obtained by the conventional method. The vertical axis Y represents the measured glucose concentration value (mg/dl) in this method. Third
The figure shows the calibration curve obtained in Example 4, connecting the points obtained by plotting the absorbance (OD) obtained for each uric acid concentration (x10 ⁵ M) on the horizontal axis along the vertical axis. It is.

[Claims]

1 (1) A step of introducing a sample solution containing estrogen into a separation column and separating and eluating the estrogen into each component of estrone, estradiol, and estriol using an eluent with a pH of 4.0 to 10.5, (2 ) a step of introducing the mixture in which the eluate contains nicotinamide adenine dinucleotide and whose pH is adjusted to 6.5 to 11.0 to a column filled with the immobilized enzyme 17β-hydroxysteroid dehydrogenase; and (3) the immobilized enzyme column. A method for quantifying estrogen, comprising the step of measuring fluorescence of reduced nicotinamide adenine dinucleotide flowing out from. 2. The method of claim 1, wherein the eluent contains 10 to 85% by weight of a water-soluble organic solvent. 3. The method according to claim 2, wherein the water-soluble organic solvent is nitrile or alcohol.