JPH0339096A - High-performance liquid chromatography utilizing immobilized enzyme - Google Patents

Abstract

PURPOSE:To make it possible to simply and inexpensively measure the aimed substance in high sensitivity by measuring H2O2 produced by bringing a sample into contact with a specific immobilized enzyme. CONSTITUTION:A dehydrogenase (A) (e.g. 3alpha-hydroxysteroid dehydrogenase) each using an aimed substance such as bile acid as a substrate and oxidation type nicotic amide adenine dinucleotide phosphate [NAD(P)] as a coenzyme is immobilized on a carrier such as cellulose, etc., to afford bile acid dehydrogenase immobilized enzyme column 11. On the other hand, a reduction type NAD(P)H oxidase (B) derived from soil microorganism, etc., is immobilized on cellulose carrier, etc., to provide a NAD(P)H oxidase immobilized enzyme column 12. Then these columns 11 and 12 and pumps 8 and 10, etc., are arranged to prepare the high performance liquid chromatography (C) utilizing the immobilized enzyme. Then NAD(P)R produced by feeding transfer phases 1 and 2 and reaction liquids 7 and 9 to the column 11 is introduced into the column 12 and the produced H2O2 is detected through a reaction coil 13 with a chemical luminescence detecting device 14 to measure the aimed substance.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an analysis and measurement method, and more specifically to a method for accurately and easily determining a target substance using an immobilized enzyme in high-performance liquid chromatography. It concerns the method of measurement.

[Conventional technology]

High-performance liquid chromatography (HPLC) is widely used as a very useful method in the fields of biochemistry and clinical testing because it can simultaneously separate and quantify a large number of target substances in a sample.

In this HPLC, when measuring the target substance, the enzyme reaction involving the target substance is incorporated into the HPLC measurement system, and the substances produced by the enzyme reaction and the coenzymes involved in the enzyme reaction are measured, thereby achieving even higher sensitivity. and specific measurement is possible.

On the other hand, the coenzymes involved in many enzymatic reactions include nicotinamide adenine dinucleotide (the oxidized form is NAD and the reduced form is indicated by NADH) or nicotinamide adenine dinucleotide phosphate (the oxidized form is NADP and the reduced form is indicated by NADH). A method of quantifying the target substance concentration by measuring NADPH (expressed as NADPH) is widely used.

Combining these techniques, such immobilized enzyme column methods for HPLC have now been developed, and some have been put to practical use in certain types of measurements. For example, bile acid 1
In the case of 5-fraction measurement, after separating 15 fractions with a bile acid separation column, each bile acid is determined by fluorescence measurement of NADH produced by oxidation of bile acids by an immobilized enzyme reaction with immobilized 3α-hydroxysteroid dehydrogenase. It is known that concentration can be determined (Japanese Patent Application Laid-Open No. 59-513
No. 49).

As a method for quantifying NAD(P)H in HPLC,
The following methods (1) to (3) are known: (1)
A specific wavelength in the ultraviolet region of NAD(P)H (for example, NA
A method to directly measure the absorption at the maximum absorption wavelength of D(P)H (340 ns); (2) irradiate NAD(P)H with light of a specific excitation wavelength to optically excite it, and measure its unique fluorescence (The bile acid measurement method of JP-A No. 59-51349 mentioned above uses Honbo); (3) NAD(P)H is reacted with methylene blue to generate HzOt, and the H20 is used to generate potassium ferricyanide. A method for measuring chemiluminescence produced by oxidizing luminol in the presence of oxidation [Yuki Saeki et al.: Clinical Chemistry,
Volume 10, No. 4 286-293 (1981));
However, the methods (1) to (3) above have the following problems.

In method (1), NAD(P)H is measured in the ultraviolet region, so especially when analyzing biological samples such as blood or biological tissues, the influence of interfering substances contained in the biological samples must be avoided. It's easy to accept.

In method (2), since the fluorescence intensity of NAD(P)H is weak, even if an attempt is made to increase the sensitivity of the detector, the noise level will also increase at the same time, making it difficult to increase the sensitivity beyond a certain level.

In method (3), the generated H and O□ are chemically excited and emitted to perform fluorescence detection, so even the same fluorescence detection method (
2) Since it is hardly affected by fluctuations in the light from the light source, it is possible to perform high-sensitivity measurements with a wide dynamic range, but the reaction rate between NAD(P)H and methylene blue is slow. There are drawbacks.

As mentioned above, no matter which method is used, in the currently known method, the NA generated in the mobile phase by the enzyme column reaction is
There is no way to measure D(P)H stably and with high sensitivity, so it becomes impossible to measure when the concentration of the target component is low.For this reason, currently HPLC is trying to eliminate the influence of interfering substances. It is difficult to receive, does not require expensive equipment,
There is a need for the development of a new NAD(P)H quantitative method that is easy to operate and allows online measurement.

[Problem to be solved by the invention]

The present invention is intended to solve the above-mentioned conventional problems, and its purpose is to use NAD(P)H produced by an enzymatic reaction using NAD(P) as a coenzyme in a method for measuring a target substance by HPLC. It is an object of the present invention to provide a method for quantifying a target substance with high sensitivity, simply and inexpensively by quantifying it accurately, highly sensitively, simply and inexpensively.

[Means to solve the problem]

The present invention has been made to achieve the above object, and although various studies have been conducted on a system for directly quantifying NAD(P)H produced according to conventional methods, no success has been achieved. I became keenly aware of the need for fundamental changes.

Therefore, the present inventors investigated NAD(P)H in HPLC.
As a result of repeated research on methods for effectively quantifying
We found that it is advantageous to use a NAD(P)H oxidase-immobilized enzyme column in which @NAD(P)H oxidase is immobilized on a certain type of carrier.
) H oxidase was isolated by the present inventors from microorganisms in soil, is stable even in neutral to weak alkaline conditions, and acts specifically on NAD(P)H to produce hydrogen peroxide. We focused on NAD(P)H oxidase (Japanese Unexamined Patent Publication No. 63-251082), which has the property of producing .

We discovered that this NAD(P)H oxidase can coexist with dehydrogenase and can be immobilized.As a result of further investigation based on these new findings, we found that NAD(P)H oxidase exists in samples in HPLC or NAD (P) produced by a dehydrogenase reaction with (P) as a coenzyme
The present invention was completed by passing H through a hydrogen peroxide-generating NAD(P)H oxidase-immobilized enzyme column and successfully detecting the generated HtOt using an appropriate 820□ detection method.

That is, the present invention provides a method for measuring a target substance by high-performance liquid chromatography, in which the target substance is used as a substrate and N.
A dehydrogenase with AD(P) as a coenzyme and a hydrogen peroxide-generating NAD(P)H oxidase are immobilized, and a sample containing the target substance is brought into contact with the immobilized enzyme, and the target substance is produced by the enzymatic reaction. The present invention is a high performance liquid chromatography using immobilized enzymes characterized in that a target substance is measured by measuring hydrogen peroxide, thereby achieving the above object.

In the present invention, NAD (P
) as a coenzyme, any dehydrogenase reaction can be used as appropriate. Non-limiting examples include 3α-hydroxysteroid dehydrogenase, alcohol dehydrogenase, glucose dehydrogenase, lactate dehydrogenase, cholesterol dehydrogenase, and β-hydroxysteroid dehydrogenase. Substrate 3α-
Hydroxysteroids (bile acids, etc.), alcohol, glucose, lactic acid, cholesterol, 3β-hydroxysteroids, etc. can be measured.

In this way, in the first step of the present invention, NAD(P)H is generated by the action of dehydrogenase, and subsequently, in the second step, NAD(P)H oxidase is caused to act and measurement is performed. NAD(P)H oxidases are known to be of the )1201-generating type and H20-generating type, and the former type is used in the present invention.

Hydrogen peroxide-generating NAD (P) used in the present invention
The enzyme-chemical and physicochemical properties of H oxidase are shown below.

(1) Action and substrate specificity: specifically catalyzes the oxidation of NADH and/or NADPH to produce NAD and/or NADP and hydrogen peroxide.

(2) Optimum pH: Shows high activity at pH 6.5 to 8.0.

(3) pH stability: When kept at 30°C for 24 hours, the pH stability
More than 90% activity remains in H7-9.

(4) Molecular weight and structure: Subunit 2 with a molecular weight of approximately 50,000
Consists of individuals. Contains one molecule of FAD per subunit.

The action mechanism of this enzyme is shown by the following formula (1).

(1) As mentioned above, this NAD(P)H oxidase shows strong activity even in the neutral to alkaline range, so it uses NAD(P) as a coenzyme, which also has an optimal activity range in the neutral to alkaline range. Can be used in combination with dehydrogenase.

In addition, the Km value is 3.2 Xl0-'M, and the NAD (
It is thought that P)H is rapidly oxidized to generate hydrogen peroxide.

That is, NADLPJ tlT can be obtained by flowing the dehydrogenase substrate and NAD(P) through an immobilized enzyme column in which the present NAD(P)H oxidase and dehydrogenase are immobilized on a certain type of carrier simultaneously or separately. The amount of the substrate can be quantified by detecting the H and O generated as the reaction progresses using an appropriate Ht01 detection method.

H,0, detection methods include (1) electrochemical detection using a platinum electrode, (2) method of measuring chemiluminescence generated by oxidizing luminol in the presence of potassium ferricyanide (alkaline region). These methods allow for highly sensitive measurements compared to conventional NAD(P)H measurement methods. In particular, in the case of (1), the detection device is inexpensive and easy to use, and in the case of (2), high sensitivity measurement with a wide dynamic range is possible. There are no restrictions on the use of the NtO1 detection method.

In this way, this NAD(P)H oxidase-immobilized enzyme column method can be combined with almost any dehydrogenase that uses NAD(P) as a coenzyme, making it possible to perform measurements that conventionally lacked sensitivity. The present invention makes it possible to measure substances that were not previously available.

In the present invention, immobilized dehydrogenase and NAD(P)H oxidase are used, but conventional methods for immobilizing enzymes, microorganisms, and cells can be used as appropriate. can.

For example, (1) a method in which the enzyme is adsorbed onto a water-insoluble carrier and bound by ionic or covalent bonds; (2) a method in which the enzyme is confined in a microcapsule made of a semipermeable membrane or a lattice of gel particles; Comprehensive Method (3) Enzyme Protein Prize There is a method in which a cross-linking reagent is reacted between molecules to form cross-linked bonds and connect the molecules to form insoluble particles.

Further, as the immobilization carrier, cellulose, polyacrylamide gel, polystyrene, Sephadex (manufactured by Pharmacia), etc. can be used.

In the present invention, there is no limit to the number of immobilization methods and immobilization carriers that can be used.

Desirably, a method using a bonding method using cellulose, Sephadex, etc. with good alkali resistance and solvent resistance as a carrier is preferable in terms of enzyme stability and immobilization strength. 1) A method in which dehydrogenase and NAD(P)H oxidase are immobilized on the same carrier and used as an immobilized enzyme column, (2) Dehydrogenase and NAD(P))I oxidase are immobilized on separate carriers, (3) A first immobilized enzyme column on which dehydrogenase is immobilized and a second immobilized enzyme column on which NAD(P)H oxidase is immobilized. A possible method is to connect the two in series, but any of these methods can be used.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Fractional determination of bile acids (immobilization of NAD(P)H oxidase) The cyanogen bromide method using cellulose as a carrier was used for immobilization of the enzyme.

That is, CNBr-activated 5ep was prepared by dissolving 500 units of NAD(P)H oxidase in 0.1 M carbonate buffer (pH 8,3) 10I11 containing 0.5 M NaCl and sufficiently swelling with the same buffer.
Harose 4B (manufactured by Pharmacia) 10d was added to the gel suspension, and stirred and reacted at room temperature for 2 hours. After the zero reaction, the same buffer was removed by filtration, and the gel was suspended in 1M ethanolamine solution and allowed to stand at room temperature for 2 hours. Stir. After stirring, remove the ethanolamine solution by filtration, wash with carbonate buffer and acetate buffer (pH 4) containing 0,5 M NaCl, suspend again in carbonate buffer, and prepare NAD(P)H oxidase-immobilized enzyme gel. Make a suspension. This allows CNB
r-activated 5epharose 4B
30 units of NAD(P)H oxidase was immobilized per 1 d.

This immobilized enzyme gel suspension was immediately filled into a stainless steel column for HPLC (4, On+m 1.D,

[Fractional determination of bile acids]

Bile acids were fractionated and quantified using the bile acid measuring device shown in FIG. The device was composed of the following elements.

1. Mobile phase A; 2. Mobile phase B, 3. Liquid feed pump A, 4.
Liquid feed pump B, 5. Injector, 6° analytical column, 7
．． Enzyme reaction solution ■, 8. Enzyme reaction solution I liquid sending pump, 9. Enzyme reaction solution I [, 10, Enzyme reaction solution ■Liquid pump, 11
．． 3α-hydroxysteroid dehydrogenase immobilized enzyme column, 12. NAD(P)H oxidase immobilized enzyme column, 130 reaction coil, 14. Chemiluminescence detector.

In this device, the HPLC pump is an LC manufactured by Shimadzu Corporation.
-6A pump, the detector is JASCO Corporation's 825-CL chemiluminescence detector, the analytical column and 3α-hydroxysteroid dehydrogenase immobilized enzyme column are JASCO Corporation's B11epak-11C 4,6 mm CD, X 12
5IIIll) and Enzymepak-H2O (4
,6n+n+ 1. D, X 35au+) was used. As mobile phase A, 30mM ammonium acetate (pH 6,
8)/acetonitrile/methanol (60/20/20
), 30 mM ammonium acetate (
pH6,8)/acetonitrile/methanol (40/3
0/30), and 15 fractions of bile acids were eluted using a gradient elution method using mobile phases A and B. In addition, enzyme reaction solution I
The following measurements were performed using a 10 mM phosphoric acid deficient condition containing 5 mM β-NAD, 5 mM potassium ferricyanide, and 2 mM FAD, and the respective results were obtained.

FIG. 2 shows a calibration curve using sodium cholate at a known concentration, and good linearity was obtained. Figure 3 shows cholic acid,
Glycocholic acid, taurocholic acid, deoxycholic acid,
Glycodeoxycholic acid, taurodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, talalochenoboxycholic acid, lithocholic acid, glycolitocholic acid, taurolithocholic acid (CA, GCA, TCA, DCA.

GDCA, TDCA, UDCA, GUDCA, TUDC
A, CDCA, GCDCA, TCDCA.

This is a chromatogram obtained when 15 bile acid standard products (LCA, GLCA, TLCA) were dissolved in methanol to a concentration of 2 ng/μl, and 10 μl was injected. From this result, when S/N = 3 , the detection limit of LCA was 2 ng. Figure 4 shows methanol 2 in healthy human serum 0, 5-.
1011 of the methanol supernatant after mixing and centrifugation.
This is a chromatogram obtained by injecting Sample No. 1 into this measurement system.

As shown in FIG. 4, this system can be fully used for measuring 15 fractions of bile acids in the serum of healthy individuals.

[Comparative example]

Figure 5 shows the conventional method for measuring 15 fractions of bile acids (fluorometric measurement of the amount of NADH accumulated on a 3α-hydroxysteroid dehydrogenase immobilized enzyme column using an excitation wavelength of 349 nm and a measurement wavelength of 460 nm) using the above-mentioned bile acid standard solution. This is a chromatogram obtained when the same concentration of was injected, and at S/N-3, the LCA detection limit was 6 ng. As can be seen from Example 1 and Comparative Example, the present invention enables measurement with three times the sensitivity compared to the conventional method.

Example 2 Dehydroepiandrosterone and 17α-
Fractional determination of hydroxypregnenolone [Simultaneous immobilization of β-hydroxysteroid dehydrogenase and NAD(P)H oxidase] β-hydroxysteroid dehydrogenase and NAD(P)H
For simultaneous immobilization of oxidase, the 500 units NAD (P) H oxidase used in Example 1 was changed to 500 units NAD (P) H oxidase and 1000 units β-hydroxysteroid dehydrogenase (manufactured by Sigma). The procedure of Example 1 was followed.

[Fractional quantification of dehydroandrosterone and 17α-hydroxypregnenolone] Fractional quantification of dehydroandrostene and 17α-hydroxypregnenolone was carried out using the measuring device illustrated in FIG. The device was intercepted with the following elements.

15. Mobile phase; 16. Liquid pump, 17. Injector, 180 analytical column, 19. NAD(P)H oxidase and β-hydroxysteroid dehydrogenase co-immobilized enzyme column, 20. Electrochemical detector, 21° enzyme reaction liquid feed pump, 22. Enzyme reaction solution.

In this device, the HPLC pump is L manufactured by Shimadzu Corporation.
C-6^, the detector is an electrochemical detector E made by Block Chemical Industry Co., Ltd.
HzOz was detected using a platinum electrode using CD-120. In addition, the analytical column is 005-1 manufactured by Block Chemical Industry Co., Ltd.
22 (6,0ms 1.D, X150+u+),
As the mobile phase, 20mM phosphate buffer (pH 3,1)/
Fractionation was performed using methanol (40/60). In addition, 10+wM containing 5mM β-NAD was used as an enzyme reaction solution.
A phosphate buffer (pH 7.2) was used. FIGS. 7 and 8 are calibration curves for dehydroandrosterone and 17α-hydroxypregnenolone in this measurement system, and good linearity was obtained for both. Figure 9 is a chromatogram of a standard mixed solution of dehydroandrosterone (DHEA) and 17α-hydroxypregnenolone (17α-HP), and Figure 10 is a chromatogram of a standard mixed solution of dehydroandrosterone (DHEA) and 17α-hydroxypregnenolone (17α-HP).
This is a chromatogram obtained when 2 d of methanol was added to -, mixed and centrifuged, the methanol supernatant was dried and dissolved again in 20 μl of methanol, and 10 μl of the solution was injected. As shown in the figure, it is possible to simultaneously separate and quantify blood dehydrandrosterone and 17α-hydroxypregnenolone at normal human levels in a short period of time.

〔Effect of the invention〕

As described above, compared to the conventional method, that is, fluorescence detection of NADH produced by a dehydrogenase reaction, the present invention enables measurement with higher sensitivity. Furthermore, since the dehydrogenase and NAD (P)H oxidase are used immobilized, simple and low-cost measurement is possible.

As described above, according to the present invention, the target substance to be analyzed can be measured very advantageously.

[Brief explanation of drawings]

FIG. 1 shows the constituent countries of the bile acid measurement system according to the present invention. Figure 2 is a chromatogram showing the measurement results of sodium cholate test NvA, Figure 3 is a chromatogram showing the measurement results of bile acid standard solution, Figure 4 is a chromatogram showing the measurement results of bile acids in the serum of a healthy person, and Figure 5
The figure is a chromatogram showing the measurement results of a bile acid standard solution using a conventional fluorescence method. FIG. 6 shows dehydroandrosterone and 1 according to the present invention.
It is a member country of the 7α-hydroxypregnenolone measurement system. Figure 7 is the dehydroandrosterone calibration curve, Figure 8 is 1
7α-Hydroxypregnenolone calibration curve, FIG. 9 is a chromatogram showing the measurement results of a standard mixture of dehydroandrosterone and 17α-hydroxybregnenolone, and FIG. It is a chromatogram showing the measurement results of hydroxypregnenolone.

Claims (1)

[Claims] 1. In a method for measuring a target substance using high-performance liquid chromatography, a dehydrogenase that uses the target substance as a substrate and NAD(P) as a coenzyme and a hydrogen peroxide-generating NAD(P)H
A high-speed method using an immobilized enzyme, characterized in that the target substance is measured by immobilizing oxidase, contacting the immobilized enzyme with a sample containing the target substance, and measuring hydrogen peroxide produced by the enzymatic reaction. liquid chromatography.