JPS58146299A - Selective high-sensitive detection method of steroid hormone intermediate - Google Patents

Abstract

PURPOSE:To detect steroid hormone intermediates having a hydroxyl group at the C3 position economically, easily, quickly, in high sensitivity, and accurately. CONSTITUTION:Various kinds of steroid hormone intermediates having a hydroxl group at the C3 position in a specimen are separated into each of components using preferably high-speed liquid chromatography, an elute containing each intermediate component separated successively is mixed with a reaction solution containing nicotinamide adenine nucleotide, the prepared mixed solution is then passed through an immobilized enzyme column maintaining 3alpha-hydroxy steroid dehydrogenase and/or 3beta-hydroxy steroid dehydrogenase, so that each intermediate component is successively reacted with nicotinamide adenine nucleotide. Each steroid hormone intermediate component is detected by measuring the fluorescence of a fluorescent substance obtained by the reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for the selective and sensitive detection of nutroid hormone substitute products.

Hormones are broadly classified into nutroid hormones and non-steroid hormones, and the former is responsible for the development and maintenance of nutroid hormones.The main hormones include male hormone tenu1, nuterone, female hormones estradiol and progeneuterone I know. Adrenocortical hormones are responsible for regulating water and salt metabolism as well as sugar, protein, and lipid metabolism, and the main hormones include aldosterone, cortisol, and cortisone.

Most of these hormones are bound to plasma proteins in the blood, and are inactivated by being reduced in the liver or conjugated with glucuronic acid or sulfuric acid, and then excreted in the urine. This is what happens. The steroid hormones excreted in the urine are mainly aldosterone, 2-thiocola/n, dehydro-andro-nuterone (also known as dehydro-isoandrosterone), and 03-position steroid hormone (also known as iso-andro-nuterone). It is a metabolite that has a hydroxyl group in the first two, particularly the first two have a 3α hydroxyl group, and the latter two have a 3β hydroxyl group.

By the way, Addison (A
In ddiSon) disease, the amount of the above-mentioned metabolites excreted in the urine decreases, while in Cushing's disease,
On the contrary, in Cushing syndrome, the amount of excretion increases. In addition, the amount of steroid hormone metabolites excreted decreases in cases of decreased gonadal function and liver disease, and increases in gonadal tumors and adrenal gland tumors. Therefore, it is important to measure all the metabolites of steroid hormones that have 3α or 3β hydroxyl groups.

The liver's function is not diagnosed.
-ing

Here, as a result of intensive studies in view of the above circumstances, the present inventors have determined that the above-mentioned 3α, 3
The present invention was completed by discovering the fact that steroid hormone metabolites having a β hydroxyl group can be detected very effectively and selectively.

That is, the object of the present invention is to economically develop nutroid hormone metabolites having a hydroxyl group at the C3 position.

The object of the present invention is to provide a method that can be easily and quickly measured, and can detect even minute concentrations with high sensitivity and accuracy.

In order to achieve the object as described above, in the present invention, various steroid hormone metabolites in a sample having a hydroxyl group at the 03 position are separated into their respective components by liquid chromatography, and then the components are sequentially separated. mixed with a reaction solution containing nicotinamide adenine nucleotides containing each steroid hormone metabolite component to be mixed;
This mixture is then passed through a column containing immobilized 3α-hydroxysteroid dehydrogenase and/or 3β-hydroxysteroid dehydrogenase, thereby converting each steroid hormone metabolic product component to the nicotinamide adenine nucleotide. After sequentially reacting the two steroid hormones, the substances produced by the reaction are subjected to fluorescence measurement to detect each steroid hormone metabolite component.

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

First, in FIG. 1 showing a system diagram of an apparatus suitable for carrying out the present invention, ■ is an eluent reservoir, which sequentially elutes each steroid hormone metabolite in a biological sample such as urine retained in a column. It contains an eluent as a mobile phase, such as a methanol/'water mixture. The eluent in ml of eluent is continuously supplied by a liquid feed pump 2 to a separation column 3 completely filled with a packing material used in ordinary liquid chromatography. Moreover, between the liquid sending pump 2 and the separation column 3,
A sample injector 4 is provided, and a sample to be analyzed such as urine (
A biological sample) is injected from the sample injector 4 and guided to the separation column 8. Therefore, within the separation column s, various steroid hormone metabolites in the injected sample are retained and then sequentially eluted by the eluent that is passed through the column s. In addition, liquid chromatography is used to rapidly separate various steroid hormone metabolites into each component in sequence.
In particular, high performance liquid chromatography is preferably employed.

In addition, 5 is nicotinamide adenine dinucleotide (N
A reaction solution reservoir containing a reaction solution containing AD), the reaction solution in this reaction solution reservoir 5 is sent out at a predetermined rate by a reaction solution pump 6, and the eluate flowing out from the separation column 3 is It is mixed into the eluate through a reaction liquid flow path 8 connected to the flowing eluate flow path 7.

Furthermore, an immobilized enzyme column 9 is provided on the flow path downstream of the connection point of the eluate flow path 7 with the reaction liquid flow path 8, and the immobilized enzyme column 9 is provided with A mixed solution of the exudate fluid and the reaction solution containing each steroid hormone metabolite component is allowed to flow in.

In this immobilized enzyme column 9, 8α-hydroxynutheroid dehydrogenase (
3α-H', 8D) and/or 3β-hydroxynuteroid dehydrogenase (3β-H, 81)), and this 8a-H, 81].

Due to the catalytic action of 3β-148D, the steroid hormone metabolites contained in the eluate are released.

The substance having a 3β hydroxyl group and NΔ1] are reacted sequentially. This reaction is 3θ-
Due to the catalytic action of f(8D, 3β-H, and 8D, the hydroxyl groups at the 3α and 3β positions of the basic skeleton of the nutroid hormone are oxidized to the keto form, and at the same time, the N A
When D is reduced, the fluorescent substance NADH is produced. Further, this reaction is an equilibrium reaction, and proceeds in the direction of producing N A D Hi under neutral or alkaline conditions, so the reaction solution is adjusted to be neutral or alkaline.

The immobilized enzyme column 9 containing 3α-H8D and 3β-HSD is prepared by immobilizing 3α-1-fS on a suitable support.
I), 8β-I-I SD is immobilized by adsorption, covalent bonding, etc., and its support is packed in a column (C), or when a tube is used as a column, the tube is It can be obtained by immobilizing 8a-1(Sr) and 8β-H-SD on its inner surface according to the above-mentioned method, but in particular, glass beads are used as the support and alkylamine is bonded to the surface. 8 (Z HsD) in the presence of a coupling agent such as gluculaldehyde.

A method of chemically immobilizing 3β-]-I S I) on the surface is preferably employed.

Then, the N A D H produced by the reaction within the immobilized enzyme column 9 flows out from the column 9,
And a fluorescence detector 10 provided on the downstream side of the column 9
It will be detected and analyzed. In addition, 11
is a recorder.

In this way, the 5 immobilized in the immobilized enzyme column 9
a-H8D, sβ-H, S
3α-1i8D, 3β
-Since H8D is immobilized on a suitable support, unlike a system in which it is continuously supplied and consumed, factors that destabilize the reaction, such as changes in concentration in the supply solution and supply rate, are removed. Furthermore, once immobilized, it can be used repeatedly tens to hundreds of times, so the analysis cost is low and it can be advantageously employed as a general-purpose analytical means.

In addition, N 4 DI-1 produced by such a reaction shows a maximum absorption spectrum near 865 nm and a maximum fluorescence spectrum near 465 nm, so the excitation wavelength: 865 nm and the fluorescence wavelength :465n
By performing fluorescence analysis with a setting of
In other words, if there is even a very small amount of
, 3β) If a steroid hormone metabolite having a hydroxyl group exists, it becomes possible to detect and analyze it.
-9- It was.

In particular, steroid hormone metabolites with hydroxyl groups present at the 3α and 3α positions have extremely short wavelengths of around 20 On rn, and weak absorption, but relatively large ultraviolet absorption, and have never been analyzed using high-performance liquid chromatography. However, according to the method of the present invention, since steroids having a hydroxyl group at the C3 position are detected, for example, androsterone has a high IR of 41g, and dehydroepiandronuterone has a high IR of xOng (S/N = 2). This made it possible to analyze biological samples using high-performance liquid chromatography.

-t, Lte, 3'a H,' 8 D to3 p
H, S D Qp] By using an immobilized enzyme column with constant immobilization, a nuteroid hormone metabolite component having a hydroxyl group at the 3α position and a nucleoid hormone metabolite component having a hydroxyl group at the 3α position can be simultaneously synthesized. It can be measured.

Incidentally, in order to confirm the effects of the present invention, (C, androstylo: / (And, ): l 25 ngl dehydroeviandronuterone (1) E A ): 12
Standard sample containing 0 +1 g 1. We analyzed C and examined their quantitative range.

The analysis conditions are as follows.

Mobile phase: methanol)y/water = 60/40 Liquid pump: JA8C(Ji'RIROi
"A R [manufactured by JASCO Corporation] Transport phase flow liver: 1 ml Z fraction separation column: Finepak SIL C
+ [Manufactured by JASCO Corporation: silica gel with chemically bonded methyl groups] Reaction solution: 1mM EDTA, 0.005% 2-Melf-1butethanol f Contains b 10 rn MK l
(2PO< Solution with 0.3mM NAD+ added. pH, = 7.80 Reaction liquid pump: JASCOS P-024-1
[Manufactured by JASCO Corporation] Reaction liquid flow meter: 1nrl/'min Temperature = 20°C Immobilized enzyme power 7mm: H8D-Gradell (manufactured by Sigma) (including 3β form and 3β form) is chemically immobilized Amine-propylated porous glass beads around 10011
c filling.

2.5tb Fluorescence detector: JASCOFP-110C JASCO Corporation ■
Made]. Excitation wavelength: 365 nm.

Fluorescence wavelength: 470 nm In addition, hydroxynuteroid dehydrogenase (1-(
8D) was immobilized by the following method. That is, first, ami/bromolized porous glass y, y as a support: 20
Add 2 ynl k of glutaraldehyde to 0 mQ, degas at room temperature in a desiccator for 30 minutes, and then
The reaction was continued for an additional 30 minutes at normal pressure and washed with distilled water. Next, the porous glass beads that have been subjected to such treatment are preliminarily treated with the above-mentioned hydroxynuteroid hydrogenase 5.
Add to 1 ml of 10 m M potassium phosphate buffer (1) II7) obtained by diluting 1 ml of the solution, and add to 1 ml of 10 m M potassium phosphate buffer (1) II7),
After degassing for 1 minute, cooling with water and reacting at normal pressure for 30 minutes, immobilization was completed by washing with distilled water.

The chromatogram obtained by analyzing the standard sample is
Figure (C1 and A n d,, I) l! ;Ao
The calibration curves obtained from the analysis results obtained by changing the injection amount are shown in FIG. 3.

From these results, And, which is a steroid hormone metabolite having a hydroxyl group at the C3 position, and D1! : It is understood that A can be very clearly separated and detected, and the minimum detection sensitivity is S/'N-2 and IQng
(DEA) and 4βg (And), and furthermore, it was confirmed that both components had linearity up to 1200 ng or more.

In addition, in place of the above standard sample, I) EA 120
The results obtained using human urine containing approximately +1g, And, and approximately 125ng are shown in Figure 4.
It has been shown that 13-d, 13- and IJEA can be effectively detected.

[Brief explanation of drawings]

) Figure 1 is a system diagram showing an example of an analytical device suitable for carrying out the present invention, Figure 2 is a chromatogram obtained according to the method of the present invention, Figure 3 is a calibration curve, and Figure 4 is a diagram showing a chromatogram obtained according to the method of the present invention. Fig. 2 shows another chromatogram obtained according to the method of the present invention. 1: Bath separation liquid reservoir 2: Liquid feed pump 3: Separation column 4: Sample injector 5: Reaction liquid reservoir
6: Reaction liquid pump 7: Eluate flow path 8: Reaction liquid flow path 9: Immobilized enzyme column 10: Fluorescence detector 11: Recorder applicant JASCO Corporation 14- No. 30 Concentration- Fig. 41 Time □

Claims (1)

[Scope of Claims] After separating all the components of various steroid hormone metabolites in a sample that have a hydroxyl group at the C3 position by liquid chromatography, nicotine is added to the effluent of each steroid hormone metabolite component that is sequentially separated. By mixing a reaction solution containing amide adenine nucleotides and then passing this mixture through a column containing immobilized 3α-hydroxysteroid dehydrogenase and H/' or 3β-hydroxysteroid dehydrogenase, each nucleotide was Nutroid hormone metabolism characterized by sequentially reacting hormone metabolite components and the nicotinamide adenine nucleotide, and then performing fluorescence measurement of the substances produced by the reaction to detect each nuteroid hormone 7th generation mll product component. Selective and sensitive detection method for products. 1-