JPWO2006051986A1 - Method for measuring aromatase activity and measurement kit used therefor - Google Patents

Abstract

Provided is a method for measuring aromatase activity which is excellent in measurement accuracy and reliability, does not require special equipment and devices, is easy to operate, and can be easily applied to a biological sample. After reacting a pentahalogenated benzyl compound or a pentahalogenated benzoyl compound with an estradiol derivative produced from a substrate such as 2α-bromoandrostenedione by aromatase under alkaline conditions, 1-lower alkyl-2-halogenated pyridine And the pentahalogenated benzyl or pentahalogenated benzoyl and 1-lower alkyl-2-pyridinium derivatives of estradiol or estradiol derivative contained in the obtained reaction mixture are measured by LC-MS.

Description

  The present invention relates to a novel and useful method for measuring aromatase activity and a measurement kit used therefor.

  Aromatase is an enzyme that converts androgen to estrogen at the final stage of the steroid hormone biosynthetic pathway, and is also called cytochrome P-450 arom. Aromatase has a molecular weight of 55 kD and is encoded by the CYP19 gene. Aromatase is widely distributed in the living body, and is present, for example, in placenta, ovarian granulosa cells, Sertoli cells, Leydig cells, brain, muscle, hair, and the like. In tissues, it exists in the microsomal membrane.

  Conventionally, the relationship between aromatase and disease has been studied. For example, it has been found that aromatase is closely involved in estrogen-dependent diseases such as breast cancer and uterine cancer. Recently, it has been reported that aromatase is expressed with endometriosis, uterine adenomyosis, uterine fibroids, and the like (see Non-Patent Document 1). In addition, the relationship between aromatase and hair growth / hair loss has been reported (see Patent Document 1). In addition, the relationship between aromatase and endocrine disrupting substances (so-called environmental hormones) has attracted attention. Under such circumstances, a method for measuring the enzyme activity of aromatase is important.

As a method for measuring aromatase activity, for example, the following methods have been developed, and some are actually used.
(1) In vitro measurement method using animal cells or yeast (see Patent Document 2, Non-Patent Document 2 and Non-Patent Document 3)
(2) Tritium water release assay (see Non-Patent Document 4)
(3) Fluorescence method (see Non-Patent Document 5)
(4) Liquid chromatography method (see Non-Patent Document 6)
(5) Measurement method using mRNA (see Patent Document 3)
Japanese National Patent Publication No. 10-508828 Japanese National Patent Publication No. 4-502261 JP 2003-207509 A Kitawaki, J. et al. , Et. al. Biol. Reprod. , Vol. 57, 514-519 (1997) Schenkel et. al. , J .; Steroid Biochem, Vol. 33, pp125-131 (1989) Ponpon D. et. al. , Molecular Endocrinology Vol. 3, pp 1477-1487 (1989) Bellino, F.M. L. et. al. , J .; Clin. Endocrinol. Metab. , Vol. 44, pp699 (1977) Stresser, D.M. M.M. , Et. al. , Anal. Biochem. , Vol. 284, pp 427-430 (2000) Taniguchi, H .; , Et. al. , Anal. Biochem. , Vol. 181, pp 167-171 (1989)

  The method (1) using animal cells and the method (5) using mRNA do not directly measure aromatase activity, and thus have problems in measurement accuracy and reliability. The tritium water release assay method (2) is widely used as a general method. However, since a radioactive compound is used, special equipment and equipment are required, and the operation is complicated. The fluorescence method (3) and the liquid chromatography method (4) have a problem that the operation is complicated. And these conventional measuring methods have the problem that it is difficult to apply as it is to the measurement of aromatase in the state which existed in biological samples, such as menstrual blood.

  Therefore, the present invention is a method for measuring aromatase activity that is excellent in measurement accuracy and reliability, does not require the use of special equipment and devices, is simple in operation, and can be easily applied to a biological sample, and is used therefor The purpose is to provide a measurement kit.

前記一般式（１）及び（２）において、Ｘ_１は、ハロゲン原子を表し、Ｌ及びＬ’は、それぞれ脱離基を表す。
（Ｃ）工程：前記（Ｂ）工程で生成したペンタハロゲン化ベンジル化合物又はペンタハロゲン化ベンゾイル化合物による誘導体を、１−低級アルキル−２−ハロゲン化ピリジンと反応させる工程。
（Ｄ）工程：前記（Ｃ）工程において生成した１−低級アルキル−２−ピリジニウム誘導体を測定し、この測定値からアロマターゼ活性を求める工程。In order to achieve the above object, the measurement method of the present invention is a method for measuring aromatase activity, and includes the following steps (A), (B), (C) and (D). .
(A) Process: The process of producing an estradiol or its derivative (s) by making aromatase act on the substrate androstenedione or its derivative (s).
Step (B): Under alkaline conditions, the estradiol produced in step (A) or a derivative thereof is converted to a pentahalogenated benzyl compound represented by the following general formula (1) or a pentahalogen represented by the following general formula (2). Reacting with a benzoyl fluoride compound.

In the general formulas (1) and (2), X ₁ represents a halogen atom, and L and L ′ each represent a leaving group.
Step (C): A step of reacting the pentahalogenated benzyl compound or the pentahalogenated benzoyl compound derivative produced in the step (B) with 1-lower alkyl-2-halogenated pyridine.
(D) Step: A step of measuring the 1-lower alkyl-2-pyridinium derivative produced in the step (C) and determining aromatase activity from this measured value.

  The measurement kit of the present invention is a kit used for measuring the aromatase activity of the present invention, and is a pentahalogenated benzyl compound represented by the general formula (1) or a pentahalogenated benzoyl compound represented by the general formula (2). It is a measurement kit containing.

  Thus, in the method for measuring aromatase activity of the present invention, estradiol or a derivative thereof, which is a product of an aromatase enzymatic reaction, is directly measured. For this reason, the measurement method of the present invention is a simple method that is excellent in measurement accuracy and reliability and does not require any special equipment or apparatus because no radioactive substance is used. In addition to these, the measurement method of the present invention is a method that can be easily applied to biological samples (particularly body fluid samples such as menstrual blood).

  The measuring method of the present invention is characterized by measuring steps (steps (B) to (D)) of estradiol or a derivative thereof produced by an enzyme reaction of aromatase. In the conventional measurement method, the level was usually 20 pg / mL (detection limit was 5 pg / mL). However, in the measurement method of the present invention, a small amount of estradiol or a derivative thereof was 2 pg / mL (lower limit of quantification). Can be quantified at the level of Like the measurement method of the present invention, pentahalogenated benzyl ether or pentahalogenated benzoyl ester and 1-lower alkyl-2-halogenated pyridine are used in combination to derivatize all hydroxyl groups in estradiol or estradiol derivatives. This suppresses the generation of divalent ions at the time of liquid chromatography-mass spectrometer (LC-MS) measurement, thereby enabling measurement with higher sensitivity.

  In the measurement method of the present invention, the measurement of the derivative in the step (D) is preferably performed by LC-MS.

  The substrate is not particularly limited, and examples of the androstenedione derivatives include 2-substituted androstenedione, 6-substituted androstenedione, 19-substituted androstenedione, and 6,19-substituted androstenedione. It is done.

  Examples of the 2-substituted androstenedione include 2-haloandrostenedione and 2-alkoxyandrostenedione.

  Examples of the 2-haloandrostenedione include 2α-chloroandrostenedione, 2β-chloroandrostenedione, 2α-bromoandrostenedione, and 2β-bromoandrostenedione. Androstenedione is preferred. Examples of the 2-alkoxyandrostenedione include androstenedione substituted at the 2-position with alkoxy having 1 to 6 carbon atoms, and among them, 2α-methoxyandrostenedione, 2β-methoxyandrostenedione. Dione, 2α-ethoxyandrostenedione and 2β-ethoxyandrostenedione are preferred.

  Examples of the 6-substituted androstenedione include 6-methylandrostenedione, among which 6α-methylandrost-4-en-3,17-dione is preferable.

  Examples of the 19-substituted androstenedione include 19-oxoandrostenedione and 19-hydroxyandrostenedione.

  Examples of the 6,19-substituted androstenedione include 6-alkyl-19-oxoandrostenedione and 6-alkyl-19-hydroxyandrostenedione. Examples of the alkyl group include the alkyl group having 1 to 6 carbon atoms, which may be linear or branched, and examples of such an alkyl group include, for example, methyl , Ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl and tert-pentyl. Examples of the 6,19-substituted androstenedione include 6-methyl-19-oxoandrostenedione, 6-ethyl-19-oxoandrostenedione, 6-methyl-19-hydroxyandrostenedione and 6-ethyl. -19-hydroxyandrostenedione and the like. Among them, 6-methyl-19-oxoandrostenedione is preferable, and 6α-methyl-19-oxoandrostenedione is more preferable.

  In the present invention, the aromatase is preferably an aromatase in a sample. The sample is not particularly limited, but is preferably a biological sample. The biological sample is not particularly limited, and examples thereof include biological tissues, body fluids, cultured tissues, cultured cells, and the like, among which biological tissues and body fluids are preferable. The biological tissue is preferably at least one of a lesioned tissue and a non-lesioned tissue related to an estrogen-dependent disease described later. Examples of the living tissue include a placenta. As the body fluid, for example, blood (whole blood), serum, plasma, lymph fluid, intestinal fluid, urine, spinal fluid, saliva, menstrual blood, nasal discharge and tears are preferable.

  In the method for measuring aromatase activity of the present invention, the amount of estradiol (E2) in the sample is further measured, and the ratio (E2 / Arom or Arom / E2) of this to the aromatase activity (Arom) in the sample is determined. It is preferable to obtain. That is, by measuring the amount of estradiol in the sample in addition to the aromatase activity and determining the ratio, a more reliable diagnosis can be made in the diagnosis of an estrogen-dependent disease described later.

前記一般式（１）及び（２）において、Ｘ_１は、ハロゲン原子を表し、Ｌ及びＬ’は、それぞれ脱離基を表す。
（Ｆ）工程：前記（Ｅ）工程で生成したペンタハロゲン化ベンジル化合物又はペンタハロゲン化ベンゾイル化合物による誘導体を、１−低級アルキル−２−ハロゲン化ピリジンと反応させる工程。
（Ｇ）工程：前記（Ｆ）工程において生成した１−低級アルキル−２−ピリジニウム誘導体を測定し、その値から前記試料中のエストラジオールの量（Ｅ２）を求める工程。As described above, the measurement method of the amount of estradiol (E2) is preferably a measurement method including the following steps (E), (F) and (G) from the viewpoint of measurement sensitivity and the like.
Step (E): Under alkaline conditions, estradiol or a derivative thereof in the sample is reacted with a pentahalogenated benzyl compound represented by the following general formula (1) or a pentahalogenated benzoyl compound represented by the general formula (2). Process.

In the general formulas (1) and (2), X ₁ represents a halogen atom, and L and L ′ each represent a leaving group.
(F) Process: The process with which the derivative by the pentahalogenated benzyl compound or pentahalogenated benzoyl compound produced | generated at the said (E) process is made to react with 1-lower alkyl-2-halogenated pyridine.
Step (G): A step of measuring the 1-lower alkyl-2-pyridinium derivative produced in Step (F) and determining the amount (E2) of estradiol in the sample from the value.

  The method for measuring the amount of estradiol (E2) is substantially the same as the steps (B), (C) and (D) of the method for measuring aromatase activity of the present invention. Substances (reagents, etc.), measurement procedures, measurement conditions, etc., can be the same as the method for measuring the enzyme activity of aromatase of the present invention.

  The method for measuring aromatase activity of the present invention is not particularly limited in its use, but is preferably used for diagnosis of estrogen-dependent diseases. Examples of the estrogen-dependent diseases include dysmenorrhea, infertility, miscarriage, endometriosis, adenomyosis, uterine fibroids, ovarian chocolate cysts, ovarian cancer, breast cancer, endometrial cancer, cervical cancer, PMS (Syndrome that causes psychosomatic modulation with the menstrual cycle), coronary heart disease, lipid metabolism abnormality and thyroid function abnormality.

  The measurement kit of the present invention preferably further contains 1-lower alkyl-2-halogenated pyridine. Moreover, the measurement kit of the present invention preferably further contains the androstenedione or a derivative thereof. Examples of the androstenedione derivative include those described above, and preferred ones are also the same. The measurement kit of the present invention can be used, for example, for diagnosis of the estrogen-dependent disease.

  Next, the diagnostic method of the present invention is a method for diagnosing an estrogen-dependent disease, a measurement step for measuring aromatase activity, and comparing the measured value with that of a normal biological sample, whereby an estrogen-dependent disease is obtained. Including the determination step of determining the likelihood of morbidity, and the measurement step is the aromatase activity measurement method of the present invention. The diagnosis includes, for example, determination of whether or not the subject suffers from the disease, determination of whether or not there is a risk of suffering from the disease in the future, and risk of recurrence of the disease after treatment Determination of whether or not a sex exists, and determination of the degree of risk and the risk of the disease.

  Next, the present invention will be described in more detail.

  In the measurement method of the present invention, the substrate is androstenedione or a derivative thereof, and the derivative is as described above. Moreover, these substrates may use commercially available products, or may be prepared in-house by a known method.

  In the present invention, examples of the “estradiol derivative” include estriol, 2-hydroxy-3-methoxyestradiol, 4-hydroxy-3-methoxyestradiol, 2-methoxyestradiol and 4-methoxyestradiol. These derivatives exist in the living body, for example. In addition, as described above, when an androstenedione derivative is used as a substrate, an “estradiol derivative” derived from the androstenedione derivative is generated by an enzymatic reaction with aromatase.

  In the measurement method of the present invention, examples of the “LC-MS” include LC-MS / MS (liquid chromatography-tandem mass spectrometer), LC-ESI-MS / MS (liquid chromatography-electrospray). An ionization tandem mass spectrometer) and LC-APCI-MS / MS (liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometer) can be exemplified, and LC-ESI-MS / MS is particularly preferable.

In the pentahalogenated benzyl compound represented by the general formula (1), X ₁ represents a halogen atom, and L represents a leaving group. X ₁ specifically represents a fluorine atom, a bromine atom, a chlorine atom, or an iodine atom. Examples of the leaving group L include a leaving group such as a halogen atom, tosyloxy, mesyloxy, or trifluoromethanesulfonyloxy group, and a preferred example is a halogen atom. Examples of the pentahalogenated benzyl compound include pentafluorobenzyl chloride, pentafluorobenzyl bromide, pentabromobenzyl bromide, and pentabromobenzyl bromide. Among them, pentafluorobenzyl bromide is preferable.

In the pentahalogenated benzoyl compound represented by the general formula (2), X ₁ represents a halogen atom, L ′ represents a leaving group, and preferred examples include a halogen atom. Examples of the pentahalogenated benzoyl compound include pentafluorobenzoyl chloride, pentafluorobenzoyl bromide, pentabromobenzoyl chloride, and pentabromobenzoyl bromide. Among these, pentafluorobenzoyl bromide is preferable.

  As these pentahalogenated benzyl compounds and pentahalogenated benzoyl compounds, known compounds (commercial products) may be used, or they may be synthesized from known compounds by a known method.

  In the “1-lower alkyl-2-halogenated pyridine” according to the present invention, lower alkyl means a linear or branched alkyl group having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms. Examples of such lower alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, tert-butyl, n-pentyl, isopentyl and tert-pentyl. Preferred examples include methyl, ethyl, and n-propyl. In the “1-lower alkyl-2-halogenated pyridine”, the halogen specifically means a fluorine atom, a bromine atom, a chlorine atom, or an iodine atom. Examples of the “1-lower alkyl-2-halogenated pyridine” include 1-methyl-2-fluoropyridine, 1-ethyl-2-fluoropyridine, 1-propyl-2-monofluoropyridine, 1-methyl-2- Examples thereof include chloropyridine, 1-methyl-2-bromopyridine, 1-ethyl-2-chloropyridine and 1-ethyl-2-bromopyridine. Among these, 1-methyl-2-fluoropyridine is particularly preferable. This “1-lower alkyl-2-halogenated pyridine” may also be a known compound (commercial product) or may be synthesized from a known compound by a known method.

  Next, the method for measuring aromatase activity of the present invention will be described below with specific examples.

  In the method for measuring aromatase activity of the present invention, the aromatase activity is measured by converting the substrate androstenedione or a derivative thereof into estradiol or a derivative thereof by aromatase, and measuring the amount produced.

  The aromatase may be in a form existing in a sample, or may be extracted from the sample and purified. For example, when a biological sample such as blood, menstrual blood, or placenta is used, it may be used as an enzyme as it is, or may be diluted with a buffer solution and used as an enzyme solution. As the substrate, those described above can be used, and among them, 2α-bromoandrostenedione and 6α-methyl-19-oxoandrostenedione are preferably used. Measurement sensitivity is further improved by using such a derivative.

The conditions for the enzyme reaction are not particularly limited, but the temperature is, for example, in the range of 20 to 70 ° C, preferably in the range of 20 to 50 ° C, more preferably in the range of 30 to 45 ° C. It is about 37 ° C., and the pH is, for example, in the range of pH 4.5 to 9.0, preferably in the range of pH 6.5 to 8.0, more preferably in the range of pH 7.0 to 7.8. Is about pH 7.2. The reaction is preferably carried out in a buffer solution, for example, phosphate buffer, Good's buffer, etc. In addition, oxygen is required for the reaction of aromatase, and oxygen may be used which is naturally supplied from the air or liquid. Moreover, since NADPH is required for the reaction of aromatase, it is preferable to add this to the reaction system. NADPH may be in the form of a salt such as sodium salt or potassium salt. The amount of NADPH added is, for example, 0.1 to 10 mg, preferably 0.5 to 5 mg, more preferably 0.8 to 2 mg, and most preferably about 1 mg with respect to the entire reaction solution. . The total amount of the reaction solution is, for example, about 1 mL or about 1 g. For stopping the enzyme reaction, a general method can be applied. For example, a method of inactivating aromatase with a high temperature or a drug, or a method of adjusting the reaction solution to a pH deviating from the optimum pH can be applied. And aromatase activity can be calculated | required by measuring the estradiol or its derivative produced | generated by the enzyme reaction. In the enzyme reaction of aromatase, in addition to estradiol or a derivative thereof, estrone or a derivative thereof is also generated. Therefore, it is preferable to treat with NaBH ₄ . For the treatment, for example, a NaBH ₄ methanol solution can be used.

  Next, a method for measuring the produced estradiol or a derivative thereof will be described with a specific example.

Preparation of pentahalogenated benzyl derivative or pentahalogenated benzoyl derivative The above enzyme reaction solution is dissolved in an inert solvent such as acetonitrile, and reacted with the pentahalogenated benzyl compound or pentahalogenated benzoyl compound under alkaline conditions. The estradiol or estradiol derivative in the sample prepared in step 1 is converted into a pentahalogenated benzyl derivative or a pentahalogenated benzoyl derivative (an example of estradiol is shown in the following Step-1 (formula (3))).

（前記式（３）中、Ｘ_１は、ハロゲン原子を表し、Ｌ及びＬ’は、それぞれ脱離基を表す）

(In the formula (3), X ₁ represents a halogen atom, and L and L ′ each represent a leaving group)

  This reaction can be carried out, for example, at a temperature within the range of −10 to 60 ° C., and preferably within the range of 5 to 40 ° C. Further, the use ratio of the pentahalogenated benzyl compound or the pentahalogenated benzoyl compound is not particularly limited. For example, it can be 0.2 to 20 mg with respect to 1 mL or 1 g of a biological sample, and is preferable. 1 to 4 mg is suitable. Furthermore, the alkaline condition means that the pH of the reaction solution is in the range of 8 to 13, preferably 9 to 11. For example, from among bases such as potassium hydroxide, sodium hydroxide and potassium carbonate, A kind and amount of base sufficient to bring the reaction solution into the above pH range can be appropriately selected and used. Here, since this reaction is carried out under alkaline conditions, the phenolic hydroxyl group among the hydroxyl groups in estradiol and / or estradiol derivatives is selectively pentahalogenated benzyl etherified or pentahalogenated benzoyl esterified. The 1-lower alkyl-2-pyridinium derivative thus obtained can be measured with high sensitivity using LC-MS.

  Examples of the pentahalogenated benzyl derivative or pentahalogenated benzoyl derivative prepared by the same method as in Step 1 above include estradiol-3-pentafluorobenzyl ether, estradiol-3-pentafluorobenzoyl ester, and 2-methoxyestradiol. -3-pentafluorobenzyl ether, 2-ethoxyestradiol-3-pentafluorobenzyl ether, 2-chloroestradiol-3-pentafluorobenzyl ether, 2-bromoestradiol-3-pentafluorobenzyl ether, 3-methoxyestradiol-2 -Pentafluorobenzyl ether, 4-methoxyestradiol-3-pentafluorobenzyl ether, 3-methoxy-4-hydroxyestradi It can be exemplified ol-4-pentafluorobenzyl ether, 6-methyl-estradiol-3-pentafluorobenzyl ether and 6-ethyl-estradiol-3-pentafluorobenzyl ether.

Preparation of pyridinium derivative Estradiol or pentahalogenated benzyl derivative or pentahalogenated benzoyl derivative of estradiol derivative prepared by the method of Step-1 above is dissolved in an inert solvent such as dichloromethane and then a basic catalyst such as triethylamine is present. Below, it can be converted to a pyridinium derivative by reacting with 1-lower alkyl-2-halogenated pyridine (an example for estradiol is shown in Step-2 below (Formula (4))).

（前記式（４）中、Ｘ_１及びＸ_２は、それぞれ独立に、ハロゲン原子を表し、Ｒは、低級アルキル基を表す。）

(In the formula (4), X ₁ and X ₂ each independently represent a halogen atom, and R represents a lower alkyl group.)

  This reaction can be carried out, for example, at a temperature within the range of −10 to 60 ° C., and preferably within the range of 5 to 40 ° C. Moreover, as a reaction solvent, a general inert organic solvent, for example, chloroform, a dichloromethane, acetonitrile, ethyl acetate, etc. can be used.

  The ratio of 1-lower alkyl-2-halogenated pyridine to estradiol or the pentahalogenated benzyl derivative or pentahalogenated benzoyl derivative of estradiol derivative is not particularly limited. For example, a sample (enzyme reaction solution) ) It can be 0.2-20 mg per 1 mL or 1 g, preferably 1-4 mg is suitable.

Examples of the pyridinium derivative prepared by the same method as in Step-2 above include estradiol-3-pentafluorobenzyl ether 17-O-methylpyridinium,
Estradiol-3-pentafluorobenzoyl ester 17-O-methylpyridinium,
2-methoxyestradiol-3-pentafluorobenzyl ether 17-O-methylpyridinium,
2-ethoxyestradiol-3-pentafluorobenzyl ether 17-O-methylpyridinium,
2-chloroestradiol-3-pentafluorobenzyl ether 17-O-methylpyridinium,
2-bromoestradiol-3-pentafluorobenzyl ether 17-O-methylpyridinium,
3-methoxyestradiol-2-pentafluorobenzyl ether 17-O-methylpyridinium,
4-methoxyestradiol-3-pentafluoropentyl ether 17-O-methylpyridinium,
3-methoxy-4-hydroxyestradiol-4-pentafluorobenzyl ether 17-O-methylpyridinium,
6-methylestradiol-3-pentafluorobenzyl ether 17-O-methylpyridinium,
Examples thereof include 6-ethylestradiol-3-pentafluorobenzyl ether 17-O-methylpyridinium.

  The LC-MS measurement in the measurement method of the present invention can be performed by a general method.

  And by this measurement, the quantity of the produced | generated estradiol or its derivative (s) can be calculated | required, and the enzyme activity of aromatase can be calculated | required from this. The expression method of the enzyme activity is not particularly limited, and for example, there is an expression shown in Examples described later. A calibration curve may be used when determining enzyme activity.

  For example, when measuring the amount of estradiol or a derivative thereof in a biological sample such as a serum sample, menstrual blood sample, or placenta sample, it may be used as it is, or the sample is processed to prepare a measurement sample May be. As the treatment method, for example, a treatment for diluting with a buffer solution or a general preparation method for separation and purification by simple column chromatography can be appropriately selected and used.

  Next, examples of the present invention will be described together with comparative examples. However, the present invention is not limited to the following examples.

  In this example, 2α-bromoandrostenedione was used as a substrate and the enzyme activity of aromatase was measured.

(1) Aromatase enzyme reaction (preparation of 2-bromoestrogens)
As an aromatase enzyme solution, a placental microsomal phosphate buffer solution (pH 7.2, protein amount 50 μg / 500 μL) was prepared. To this solution, a nicotine adenine dinucleotide reduced aqueous solution (NADPH, 1 mg / 450 μL) and a 2α-bromoandrostenedione methanol solution (1.5 μg / 50 μL) are added to make a total volume of 1.0 mL, while shaking at 37 ° C. Incubate for 15 minutes to allow enzyme reaction.

(2) Preparation of 2-bromoestradiol After the enzyme reaction, the reaction vessel was cooled under ice cooling. Since both 2-bromoestrone and 2-bromoestradiol were produced by the enzymatic reaction, 200 μL of NaBH ₄ methanol solution (15 mg / mL) was added and allowed to stand at room temperature for 10 minutes in order to convert the former into 2-bromoestradiol. Subsequently, the reaction was stopped with 0.05N aqueous hydrochloric acid (1 mL), 200 pg of deuterium labeled estradiol was added as an internal standard substance, the steroid fraction was extracted with ether, and the solvent was distilled off.

(3) Preparation of 2-bromoestradiol-3-pentafluorobenzyl ether The obtained residue was dissolved in 50 μL of acetonitrile, and then 50 μL of 0.8% potassium hydroxide in ethanol was added to adjust the pH of the reaction solution. 11, 50 μL of 5% pentafluorobenzyl bromide solution was added, and the mixture was heated at 50 ° C. for 1 hour. The reaction mixture was diluted with water and extracted with ether, and the solvent was evaporated.

(4) Preparation of 2-bromoestradiol-3-pentafluorobenzylether 17-O-methylpyridinium 200 μL of 2% 1-methyl-2-fluoropyridine in dichloromethane and 5% triethylamine in dichloromethane 30 μL was added and left at room temperature for 90 minutes. Next, after the solvent was distilled off, the residue was dissolved in 1.25 mL of a 25% aqueous methanol solution and loaded onto a Bond Elut C ₁₈ column prepared beforehand with 6 mL of methanol and 6 mL of water. After sequentially washing with 1 mL of water, 3 mL of 3% aqueous ammonia, 2 mL of methanol and a 0.01% formic acid / methanol mixture (1: 1), elution was performed with 3.5 mL of a 10% formic acid / acetonitrile (1: 4) mixture. The eluate was distilled off under reduced pressure, and then dissolved in 100 μL of a 0.05% formic acid / acetonitrile (1: 1) mixture.

(5) Measurement by LC-MS / MS method From the liquid obtained in the step (4), 10 μL was sampled and used for LC-MS / MS measurement. LC-MS / MS was measured by ESI method using a mass spectrometer (manufactured by Japan Applied Biosystems) API4000 connected to a liquid chromatography apparatus (Azident 1100 type). The measurement conditions for LC and MS are as shown below. In this measurement, the peak area of the pyridinium derivative of 2-bromoestradiol produced from 2α-bromoandrostenedione was measured under the enzyme reaction conditions, and the peak area ratio between it and the internal standard was calculated. -The bromestradiol concentration was determined. The measurement was performed 3 times, and the average value was calculated. The results are shown in Table 1 below. The enzyme activity of aromatase was expressed as the amount (pg) of estradiol produced during the incubation time (15 minutes) in the placental microsome as 100 mg of protein. Therefore, the unit of enzyme activity of aromatase is “pg / 15 minutes / 100 mg protein”.

(Measurement condition)
(1) LC condition column: Docosil (inner diameter 2 mm × length 100 mm, manufactured by Senshu Kagaku)
Flow rate: 0.4 mL / min (2) MS condition model: API4000
Mode: ESI + (SRM)
Declustering potential: 65V
Enforcement Potential: 8V
Collison energy: 40V
Collison cell exit potential: 10V
Measured ion parent ion (M / Z): 624.2 (I.S. M / Z 547.0)
Daughter ion (M / Z): 110.0 (I.S. M / Z 109.8)

(Comparative Example 1)
In the aromatase enzyme reaction, the enzyme reaction was performed without adding the substrate and NADPH. Except for this, the enzyme activity of aromatase was measured in the same manner as in the above Examples. The results are shown in Table 1 below.

(Comparative Example 2)
In the aromatase enzyme reaction, the enzyme reaction was performed without adding NADPH. Except for this, the enzyme activity of aromatase was measured in the same manner as in the above Example. The results are shown in Table 1 below.

  As can be seen from Table 1, in Example 1, a large amount of estradiol was produced by the enzyme reaction of aromatase, and thereby the enzyme activity of aromatase could be measured. In contrast, in Comparative Examples 1 and 2, the amount of estradiol was very small.

  In this example, 6α-methylandrost-4-ene-3,17-dione (MeAD) was used as a substrate, and the enzyme activity of aromatase in a sample was measured.

(1) Preparation of calibration curve 6α-methyl-1,3,5 (10) -estraditriene-3,17β-diol (0, 1, 3, 10, 30, 100 and 300 pg) was added to a phosphate buffer ( pH 7.2, 1/15 mol / L) 1 mL and internal standard (1,3,5 (10) estradien-3-ol-17-one-2,4,16,16-d ₄ (E1-d ₄ ) (CDN Isotopes)) 200 pg was added and extracted with diethyl ether. The solvent was dried and a 6α-methylestradiol-3-pentafluorobenzyloxy-17β-2-methylpyridinium ether (MeE2-PFBZPY) derivative was prepared in the same manner as (3) and (4) described below, and LC -Measured by MS / MS.

(2) Aromatase enzyme reaction First, menstrual blood was collected from a subject, mixed with an equal amount of phosphate buffer (1/15 mol / L, pH 7.2), and homogenized to prepare a sample. Next, human placental microsomes are dissolved in a phosphate buffer solution (pH 7.2, 1/15 mol / L) containing 0.5% BSA, and the amount of human placental microsomes added is adjusted to 0.5 μg. The enzyme solution was prepared by adding to 400 μL of the sample. To 0.8 mL of the enzyme solution, 0.1 mL of a substrate (MeAD, 20 μmol / L, 50% aqueous methanol solution) and 0.1 mL of a coenzyme (β-NADPH (Oriental Yeast), 1 mg, phosphate buffer) are added. The total volume was 1.0 mL, and the mixture was incubated at 37 ° C. with shaking (60 minutes) to allow enzyme reaction. After the enzyme reaction, the reaction vessel was cooled under ice cooling, and in that state, the internal standard substance (1,3,5 (10) estraditrien-3-ol-17-one-2,4,16,16-d ₄ (E1-d ₄ ) (CDN Isotopes) 200 pg was added, the steroid fraction was extracted with diethyl ether, and the solvent was dried.

(3) Preparation of 6α-methylestradiol-3-pentafluorobenzyloxy-17β-2-methylpyridinium ether (MeE2-PFBZPY) NaBH ₄ (2.5 mg / 0.25 mL methanol solution) was added to the obtained residue After standing at room temperature for about 10 minutes, 1 mL of purified water was added and loaded onto Bond Elut C ₁₈ (200 mg, manufactured by Varian). After washing with 1 mL of purified water and 2 mL of 30% acetonitrile aqueous solution, elution was performed with 2.5 mL of 70% acetonitrile aqueous solution.

  The eluate was evaporated to dryness, the residue was dissolved in 0.05 mL of AcN, 0.05 mL of pentafluorobenzyl bromide / acetonitrile (1:19) solution and 0.05 mL of 0.8% KOH ethanol solution were added, and 52 It was made to react at -53 degreeC for 1 hour. After the solvent was dried, 0.2 mL of 2% 2-Fluoro-1-methylpyridinium p-Toluenesulfate in dichloromethane and 0.03 mL of 10% triethylamine in dichloromethane were added and reacted at room temperature for 1.5 hours.

After the reaction solution was evaporated to dryness, the residue was dissolved in 0.25 mL of methanol, and 1 mL of purified water was added to load Bond Elut _C18 . Purified water (1 mL), 0.3% aqueous ammonia (3 mL), MeOH (3 mL), and 0.01% formic acid aqueous solution / MeOH (1: 1) mixed solution (3 mL) were sequentially washed, and then 10% formic acid aqueous solution / AcN (1: 9) mixed solution 2 Elute with 5 mL.

(4) Measurement by LC-MS / MS method After elution of the eluate obtained in the step (2) was evaporated to a solvent, the mobile phase (0.05% formic acid solution / (acetonitrile: methanol = 10: 3)) was adjusted to 100 μL. It melt | dissolved and 10 microliters was extract | collected as a sample and this was used for LC-MS / MS measurement. LC-MS / MS was measured by an ESI method using a mass spectrometer (manufactured by Japan Applied Biosystems) API4000 (trade name) connected to a liquid chromatography apparatus (trade name: Agilent 1100 type). The measurement conditions for LC and MS are as shown below. In this measurement, the peak area of MeE2-PFBZPY was measured under the enzyme reaction conditions, the peak area ratio between the peak area and the internal standard substance was calculated, and the MeE2 concentration was determined therefrom. The measurement was performed twice, and the aromatase enzyme activity was calculated for each. The results are shown in Table 1 below together with the measured values. The enzyme activity of aromatase was expressed as the amount (pmol) of MeE2 (Mw286.4) produced during the incubation time (60 minutes) in the placental microsome as 1 mg of protein. Therefore, the unit of enzyme activity of aromatase is “pmol / 60 minutes / 1 mg protein”.

(Measurement condition)
(1) HPLC condition column: trade name XTerra MS C ₁₈ (3.5 μm, inner diameter 2.1 mm × length 100 mm, manufactured by Waters)
Mobile phase: 0.05% formic acid solution / (acetonitrile: methanol = 10: 3)
0.0-0.5 min; 35: 65 → 0: 100
0.5-2.5 minutes; 0: 100
2.5-6.0 minutes; 35:65
Column temperature: 40 ° C
Flow rate: 0.4 mL / min Injection volume: 10 μL
Run Time: 6.0 minutes (2) MS requirement Model: API4000
Ionization method: ESI (Positive)
CAD: 4 psi
CUR: 10 psi
Gas1: 30 psi
Gas2: 65 psi
IonSpray Voltage: 5000V
Ion power supply temperature: 500 ° C
Declustering potential: 65V
Enforcement Potential: 9.0V
Collison energy: 45V
Collison cell exit potential: 13V
IQ1: -9.0
Measurement ion (m / z): 558.3 / 353.1

  As shown in Table 2, according to the measurement method of the present invention, the enzyme activity of aromatase can be measured stably and with excellent accuracy.

  This example is an example showing the measurement accuracy and sensitivity of the measurement method of the present invention. As a comparative example, the same measurement was performed by a tritium water release assay.

  Except that the amount of human placental microsome added was 0.0.01, 0.02, 0.05 and 0.10 μg, and the concentration of the substrate (MeAD) was 2 μmol / L, the same as in Example 2 above. The amount of MeE2 is measured, the enzyme activity of aromatase is calculated from the measured value, and the average value is shown in Table 3 below together with the standard error and the coefficient of variation (CV).

(Comparative Example 3)
0.5 mL of enzyme solution and 0.1 mL of substrate (1β- ³ H-Androst-4-ene-3, 17-dione ( ³ H-AD) (manufactured by PerkinElmer; 0.5 μCi) and coenzyme (β-NADPH 1 mg) 0.1 mL was added and incubated for 60 minutes at 37 ° C. Then, 2 mL of ethanol was added to stop the reaction, allowed to stand for 15 minutes under ice-cooling, and then centrifuged (4 ° C., 3000 rpm, 10 minutes). 1 mL of purified water was added to the liquid, and the aqueous layer was washed twice with 2 mL of chloroform (4 ° C., 3000 rpm, 5 minutes), then about 100 mg of activated carbon was added and left at room temperature for 20 minutes. Liquid scintillation count by adding 4 mL of scintillator (Clearsol II, manufactured by Nacalai Tesque) to 0.85 mL of supernatant at 3000 rpm for 10 minutes Chromatography.; Were measured radioactivity (manufactured by Aloka trade name LSC2000) to measure the MeE2 amount in each human placental microsomes amounts shown in Table 3 below, and the results are shown in Table 3 below.

As shown in Table 3, the measurement method of the present invention was superior in accuracy and sensitivity to the method of Comparative Example 3. In addition, the measurement method of the present invention and the method of the comparative example showed a good positive correlation (R ² = 0.9645). Therefore, it can be said that the measurement method of the present invention is excellent in measurement accuracy and reliability.

  This example is an example showing the daily reproducibility of the measurement method of the present invention.

  Except that the measurement date was changed three times, the amount of MeE2 was measured in the same manner as in Example 2, and the enzyme activity of aromatase was calculated using it. The results are shown in Table 4 below.

  As shown in Table 4, since the daily fluctuation of the enzyme activity of aromatase was 20% or less, the measurement method of the present invention can stably measure the enzyme activity of aromatase with excellent accuracy.

  In this example, the enzyme activity of aromatase was measured using samples stored at various temperatures.

  Examples were used except that samples previously stored at 4 ° C. for 0.5 or 2 hours, room temperature for 2 hours, and 70 ° C. for 1 or 3 weeks were used, and the amount of the sample was 0.15 mL. Measurement was performed in the same manner as in 2. The results (n = 3) are shown in Table 5 below together with the relative values when the activity value when stored at 4 ° C. for 0.5 hour is defined as 100.

  As shown in Table 5, even with samples stored under various conditions, the enzyme activity of aromatase could be measured with high accuracy according to the measurement method of the present invention.

  The present example is an example in which menstrual blood of an estrogen-dependent disease patient and a healthy person is used and the enzyme activity of aromatase is used.

  The enzyme activity of aromatase was measured in the same manner as in Example 2 for the estrogen-dependent disease patient group (n = 7) and healthy subjects (n = 5). The results are shown in Table 6.

  As shown in Table 6, since the measurement method of the present invention can accurately measure the enzyme activity of aromatase without false positives, it can be applied to diagnosis of estrogen-dependent diseases such as endometriosis.

  The method for measuring the enzyme activity of aromatase of the present invention is not limited in its use, and can be applied to a wide range of fields such as biology, medicine, agriculture, pharmacy, biochemistry, etc. , Miscarriage, endometriosis, adenomyosis, uterine fibroids, ovarian chocolate cyst, ovarian cancer, breast cancer, endometrial cancer, cervical cancer, PMS (syndrome that changes in mind and body with menstrual cycle), coronary heart It can be preferably applied to diagnosis of estrogen-dependent diseases such as diseases, lipid metabolism abnormalities and thyroid function abnormalities.

Claims (22)

A method for measuring aromatase activity, comprising the following steps (A), (B), (C) and (D).
(A) Process: The process of producing an estradiol or its derivative (s) by making aromatase act on the substrate androstenedione or its derivative (s).
Step (B): The pentahalogenated benzyl compound represented by the following general formula (1) or the pentahalogenated compound represented by the general formula (2) is converted from the estradiol produced in the step (A) or a derivative thereof under alkaline conditions. Reacting with a benzoyl compound.

In the general formulas (1) and (2), X ₁ represents a halogen atom, and L and L ′ each represent a leaving group.
Step (C): A step of reacting the pentahalogenated benzyl compound or the pentahalogenated benzoyl compound derivative produced in the step (B) with 1-lower alkyl-2-halogenated pyridine.
(D) Step: A step of measuring the 1-lower alkyl-2-pyridinium derivative produced in the step (C) and determining aromatase activity from this measured value.   The measurement method according to claim 1, wherein in the compounds represented by the general formulas (1) and (2), L or L ′ represents a halogen atom.   The measurement method according to claim 1, wherein in the step (D), the measurement of the 1-lower alkyl-2-pyridinium derivative is measurement by LC-MS.   The measurement method according to claim 3, wherein ionization of MS is performed by ESI in the LC-MS measurement.   As the substrate, at least one androstenedione derivative selected from the group consisting of 2-substituted androstenedione, 6-substituted androstenedione, 19-substituted androstenedione and 6,19-substituted androstenedione is used. The measurement method according to claim 1.   The measurement method according to claim 5, wherein the 2-substituted androstenedione is at least one of 2-haloandrostenedione and 2-alkoxyandrostenedione.   The measurement according to claim 6, wherein 2-haloandrostenedione is at least one of 2α-chloroandrostenedione and 2α-bromoandrostenedione, and 2-alkoxyandrostenedione is 2α-methoxyandrostenedione. Method.   The measurement method according to claim 5, wherein the 19-substituted androstenedione is at least one of 19-oxoandrostenedione and 19-hydroxyandrostenedione.   6. The measuring method according to claim 5, wherein the 6,19-substituted androstenedione is 6α-methyl-19-oxoandrostenedione.   The measurement method according to claim 1, wherein the aromatase is aromatase in a sample.   Furthermore, the measuring method of Claim 10 which measures the quantity (E2) of the estradiol in the said sample, and calculates | requires ratio of this and the aromatase activity (Arom) in the said sample. The measuring method according to claim 11, wherein the measuring method of the amount (E2) of estradiol includes the following steps (E), (F) and (G).
Step (E): Under alkaline conditions, estradiol or a derivative thereof in the sample is reacted with a pentahalogenated benzyl compound represented by the following general formula (1) or a pentahalogenated benzoyl compound represented by the general formula (2). Process.

In the general formulas (1) and (2), X ₁ represents a halogen atom, and L and L ′ each represent a leaving group.
(F) Process: The process with which the derivative by the pentahalogenated benzyl compound or pentahalogenated benzoyl compound produced | generated at the said (E) process is made to react with 1-lower alkyl-2-halogenated pyridine.
Step (G): A step of measuring the 1-lower alkyl-2-pyridinium derivative produced in Step (F) and determining the amount (E2) of estradiol in the sample from the value.   The measurement method according to claim 10, wherein the sample is a biological sample.   The measurement method according to claim 13, wherein the biological sample is a sample derived from at least one of a biological tissue and a body fluid.   The measurement method according to claim 14, wherein the biological tissue is a biological tissue derived from at least one of a lesioned tissue and a non-lesioned tissue.   The measurement according to claim 14, wherein the body fluid is at least one selected from the group consisting of blood (whole blood), serum, plasma, lymph, intestinal fluid, urine, spinal fluid, saliva, menstrual blood, nasal discharge and tears. Method.   The measurement method according to claim 1, wherein the aromatase activity measurement method is a measurement method used for diagnosis of an estrogen-dependent disease.   The estrogen-dependent disease is dysmenorrhea, infertility, miscarriage, endometriosis, adenomyosis, uterine fibroid, ovarian chocolate cyst, ovarian cancer, breast cancer, endometrial cancer, cervical cancer, PMS (menstrual cycle The measurement method according to claim 17, wherein the measurement is at least one disease selected from the group consisting of a syndrome that causes physical and mental modulation, coronary heart disease, lipid metabolism abnormality, and thyroid function abnormality.   A measurement kit for use in a method for measuring aromatase activity, wherein the method for measuring aromatase activity is the method for measuring aromatase activity according to claim 1, wherein the pentahalogenated benzyl compound represented by the general formula (1) Or the measurement kit containing the pentahalogenated benzoyl compound shown by General formula (2).   The measurement kit according to claim 19, further comprising 1-lower alkyl-2-halogenated pyridine.   The measurement kit according to claim 19, further comprising androstenedione or a derivative thereof.   A method for diagnosing an estrogen-dependent disease, a measurement step for measuring aromatase activity, and a determination for comparing the measured value with that of a normal biological sample, thereby determining the susceptibility to estrogen-dependent disease A diagnostic method comprising a step, wherein the measuring step is the measuring method according to claim 1.