JP7292058B2 - Method for preparing or quantifying steroid hormone - Google Patents

Description

The present invention relates to a method for preparing or quantifying steroid hormones, and analysis of skin characteristics or blood steroid hormones using the method.

Steroid hormones are important endocrine factors for maintaining the functions of various organs throughout the body. Steroid hormones include types such as glucocorticoids such as cortisol, mineralocorticoids such as aldosterone, male hormones androgens, female hormones estrogen, and progesterone.

It has been reported that steroid hormones affect the physiological functions of various parts of the skin, such as the epidermis, sebaceous glands, dermis, and hair. In postmenopausal women, collagen fibers and elastin fibers in the skin decrease as blood estrogen levels decrease, resulting in induction of skin aging such as wrinkles and sagging (Non-Patent Documents 1 and 2). In addition, it has been reported that application of estrogen to the skin of postmenopausal women not only increases collagen fibers and improves wrinkles, but also improves moisture retention and suppresses sebum secretion (non-patent literature). 3-5). It has been reported that application of progesterone, a progesterone, to the skin of premenopausal and postmenopausal women improves skin elasticity (Non-Patent Document 6). Androgens, male hormones, are deeply involved in hair growth. It has been reported that hair growth over the whole body, excluding eyebrows and eyelashes, depends on androgens (Non-Patent Document 7). While testosterone promotes the growth of body hair such as armpit hair and pubic hair, it has also been reported that dihydrotestosterone suppresses the growth of hair on the head (Non-Patent Document 8). Furthermore, dihydrotestosterone is closely related to androgenetic alopecia, and inhibitors of the enzyme 5α-reductase that produces this hormone are actually clinically applied as ameliorating agents for androgenetic alopecia (non-patent literature). 9). Cortisol, a glucocorticoid, has been used as an indicator of stress. In addition, it has been reported that the recovery rate of the skin barrier function decreases in stressed animals, but that the recovery rate recovers when a glucocorticoid receptor antagonist is administered (Non-Patent Document 10). It is speculated that glucocorticoids are involved in the effects on human and animal skin functions.

Conventionally, when analyzing steroid hormones in skin, a method of measuring steroid hormones in a biopsy sample of skin is generally used (Non-Patent Document 11). However, this method is highly invasive and imposes a heavy burden on the subject. Patent Documents 1 and 2 disclose a less invasive method for quantifying estrogen by solvent-extracting stratum corneum samples collected by tape stripping and performing LC-MS analysis.

JP 2009-085946 A JP 2011-163944 A

J. Am. Acad. Dermatol., 2005, 53:555-568 Exp. Dermatol., 2006, 15:83-94 Maturitas, 2001, 39:43-55 Maturitas, 1995, 22:151-154 Maturitas, 1994, 19:211-223 Br. J. Dermatol., 2005, 153:626-634 Dermatol. Ther., 2008, 21:314-328 Mol. Cell Endocrinol., 2002, 198:89-95 Eur. J. Dermatol., 2002, 12:38-49 Am. J. Physiol. Regul. Integr. Comp. Physiol., 2006, 291: R1657-R1662 Mol. Cell Endocrinol., 2012, 362: 19-28

The present invention relates to a method for preparing or quantifying steroid hormones, and a method for analyzing skin characteristics or blood steroid hormones using the method.

The present inventors have found that steroid hormones can be separated from surface skin lipids collected from subjects and used for analysis. The present inventors also found that the amount of steroid hormone derived from lipids on the skin surface serves as an index representing the skin characteristics of a subject. Furthermore, since the amount of steroid hormones derived from lipids on the skin surface correlates with the amount of steroid hormones in the blood, the present inventors determined that the amount of steroid hormones in blood is based on the amount of steroid hormones derived from lipids on the skin surface. I found that it can be analyzed.

The present invention provides a method for preparing a steroid hormone, comprising isolating the steroid hormone in superficial skin lipids collected from a subject.
The present invention also provides a method for quantifying steroid hormones in skin surface lipids, which comprises quantifying the steroid hormones prepared by the preparation method described above.
Furthermore, the present invention provides a method for analyzing skin characteristics of a subject, which comprises quantifying steroid hormones in lipids on the skin surface collected from the subject by the above quantification method.
Furthermore, the present invention provides a method for analyzing the amount of steroid hormone in the blood of a subject, which comprises quantifying steroid hormone in lipids on the skin surface collected from the subject by the quantification method described above.

INDUSTRIAL APPLICABILITY According to the present invention, a subject's steroid hormone can be prepared and quantified simply and non-invasively. The amount of steroid hormone in the lipids on the skin surface prepared by the present invention correlates with the skin characteristics and blood steroid hormone amount. Therefore, according to the present invention, it is possible to analyze the skin characteristics and blood steroid hormone level of a subject based on the amount of steroid hormone in the lipids on the skin surface of the subject. Furthermore, according to the present invention, steroid hormones in lipids on the surface of the skin can be used instead of blood steroid hormones to evaluate biological functions or disease states, for which the amount of steroid hormones in the blood has conventionally been used as an index. It can be performed more easily and non-invasively.

Correlation between SSL steroid hormone concentration and blood steroid hormone concentration. R is the correlation coefficient.

All patents, non-patents, and other publications cited herein are hereby incorporated by reference in their entirety.

As used herein, the term “skin” is a general term for regions including epidermis, dermis, hair follicles, and tissues such as sweat glands, sebaceous glands, and other glands, unless otherwise specified.

As used herein, "skin surface lipids (SSL)" refers to a fat-soluble fraction present on the surface of the skin, and is sometimes called sebum. In general, SSL mainly contains secretions secreted from exocrine glands such as sebaceous glands in the skin, and exists on the skin surface in the form of a thin layer covering the skin surface.

In one embodiment, the present invention provides a method for preparing a steroid hormone comprising isolating the steroid hormone in superficial skin lipids collected from a subject.

Examples of steroid hormones prepared by the method of the invention include androgens, progesterone, estrogens, and glucocorticoids. Examples of androgens include dehydroepiandrosterone (DHEA), testosterone (T), dihydrotestosterone (DHT), and androstenedione (A-dione). Examples of estrogens include estrone (E1) and estradiol (E2). Examples of glucocorticoids include cortisol (F). In the method of the present invention, any one or more selected from the group consisting of various steroid hormones listed above may be prepared. In a preferred embodiment, the steroid hormone prepared by the method of the present invention contains androgen, more preferably any one or more selected from the group consisting of DHEA, T, DHT, and A-dione. and more preferably DHEA, T, DHT, and A-dione. In another preferred embodiment, the steroid hormone prepared by the method of the invention comprises progesterone (P4). In another preferred embodiment, the steroid hormone prepared by the method of the invention comprises estrogen, more preferably E1 or E2, more preferably E1 and E2. In another preferred embodiment, the steroid hormone prepared by the method of the invention comprises a glucocorticoid, more preferably cortisol (F).

The subject in the method of the present invention may be any organism that has SSL on its skin. Examples of subjects include mammals, including humans and non-human mammals, preferably humans. Preferably, the subject is a human or non-human mammal that requires or desires its own steroid hormone analysis.

In one embodiment, the method of the invention may further comprise harvesting the subject's SSL. The site of the skin from which SSL is collected includes any site of the body such as the head, face, neck, trunk, limbs, skin with diseases such as atopy, acne, inflammation, tumor, etc., skin with wounds, etc., preferably facial skin, but not particularly limited.

The collection of SSL from the subject's skin can employ any means used to collect or remove SSL from the skin. Preferably, an SSL absorbent material, an SSL adhesive material, or an instrument that scrapes the SSL off the skin, as described below, can be used. The SSL absorbent material or SSL adhesive material is not particularly limited as long as it has affinity for SSL, and examples thereof include polypropylene and pulp. More detailed examples of procedures for collecting SSL from the skin include a method of absorbing SSL into sheet-like materials such as blotting paper and blotting film, a method of adhering SSL to a glass plate, tape, etc., a spatula, a scraper, etc. and a method of scraping off and recovering SSL. In order to improve the adsorptivity of SSL, an SSL absorbent material previously impregnated with a solvent having high fat solubility may be used. On the other hand, if the SSL absorbent material contains a highly water-soluble solvent or water, it is not preferable because the adsorption of SSL is inhibited. Also, the SSL absorbent material preferably does not contain fat-soluble substances that interfere with the measurement of steroid hormones. Alternatively, the SSL absorbent material may be pretreated to remove the fat-soluble substances. Examples of the pretreatment include immersion in an organic solvent such as a chloroform/methanol solution. The SSL absorbent material is preferably used dry.

SSL collected from a subject may be immediately used for the separation of steroid hormones, which will be described later, or may be stored until the separation of steroid hormones. The collected SSL may be refrigerated or stored at a low temperature below freezing, but may also be stored at room temperature. For example, SSL absorbent material containing SSL or SSL adhesive material can be stored at room temperature, refrigerated, or cold stored.

Steroid hormones in SSL can be separated from harvested SSL by solvent extraction. Preferably, the steroid hormones in the SSL can be solvent extracted from the SSL absorbent material or SSL adhesive material containing the SSL described above. For example, after cutting the SSL absorbent material containing the SSL or the SSL adhesive material (e.g., oil blotting film or tape) into an appropriate size as necessary, ethers, alcohols, hexane, acetone, ethyl acetate or the like to extract the steroid hormone. The organic solvent used for the extraction is preferably ethers, more preferably methyl butyl ether. The extract obtained is preferably further purified. Purification means include column purification, washing with an organic solvent, and the like. For example, after removing the solvent from the resulting extract, it can be washed with hexane, acetonitrile, or the like.

The SSL-derived steroid hormone prepared by the above procedure can be used for various analyses. Analysis of SSL-derived steroid hormones can be performed according to a general procedure for analyzing steroid hormones. In a preferred embodiment, SSL-derived steroid hormones prepared by the above procedure are quantified.

Preferably, quantification of SSL-derived steroid hormones is performed by LC-MS measurements. As the LC-MS, for example, LC-MS/MS, LC-ESI-MS/MS, LC-APCI-MS/MS, etc. can be used, and LC-MS/MS is preferably used.

When steroid hormones are measured by LC-MS, it is preferable to appropriately introduce a substituent into the steroid hormone to obtain a steroid hormone derivative in order to improve the detection sensitivity. Derivatization of steroid hormones can be carried out according to the methods described in Japanese Patent No. 4634913 (“Method for measuring steroids”) and Patent Documents 1 and 2.

If androgens (eg DHEA, T, DHT or A-dione), progesterone (P4), or glucocorticoids (eg F) are to be detected more sensitively, they are preferably converted to their ester derivatives. For example, bases (e.g. sodium hydride, potassium carbonate, triethylamine, pyridine, etc.) and inert organic solvents (e.g., ethers such as dioxane, tetrahydrofuran, dimethoxyethane; aromatic hydrocarbons such as benzene, toluene, xylene; dimethyl An ester derivative can be obtained by reacting a steroid hormone with a pyridylcarbonyl compound such as picolinic acid in the presence of amides such as formamide and dimethylacetamide; acetonitrile; dimethylsulfoxide, etc.).

Alternatively, androgen can be converted to an acyl derivative, preferably a picolinoyl derivative, with an acylating reagent if the detection of the androgen is to be enhanced. Alternatively, 2-fluoro-1-methylpyridine may give the pyridinium derivative of androgen.

Alternatively, in the case of improving the detection sensitivity of progesterone, carbonyls at positions 3 and 20 of progesterone are reacted with a ketone derivatizing reagent O-ethylhydroxylammonium chloride, Girard reagent T or Girard reagent P to obtain an imino derivative of progesterone. Obtainable.

If the detection sensitivity of estrogens (eg E1 or E2) is to be improved, these are preferably converted to fluoropyridine ether derivatives. For example, reacting an estrogen with a pentahalogenated benzyl group or a pentahalogenated benzoyl group in the presence of a base and an inert organic solvent (such as those described above) to give a pentahalogenated benzyl or pentahalogenated benzoyl derivative. can be done. 2-(1-lower alkylhydrazino)pyridine may be further introduced into the resulting pentahalogenated benzyl or benzoyl derivative. Alternatively, an ester derivative may be obtained by reacting the obtained pentahalogenated benzyl or benzoyl derivative with a pyridylcarbonyl compound such as fusaric acid in the same procedure as the androgen ester derivatization reaction described above.

By extracting the obtained reaction solution containing the derivative with an organic solvent, a crude product of the target steroid hormone derivative can be obtained. Furthermore, by column-purifying the obtained crude product, the background in LC-MS measurement can be greatly reduced, and the detection accuracy of steroid hormones can be improved. For column purification, a normal phase column such as InertSep SI (GL Sciences) can be used.

SSL-derived steroid hormones can be measured by LC-MS according to a general LC-MS measurement procedure. From the measured values obtained, the amount of detected steroid hormone can be calculated based on an internal standard, a calibration curve, or the like, or the amount of steroid hormone in the sampled SSL can be quantified. When quantifying the amount of steroid hormones in SSL, for example, the amount of steroid hormones per mass of SSL collected may be quantified, or the amounts of triglycerides and free fatty acids, which are sebum components in the SSL, are measured, The amount of steroid hormone per triglyceride + free fatty acid can also be quantified.

As shown in Examples below, the present inventors have found that the amount of steroid hormone in SSL has a correlation with skin characteristics. This indicates that the subject's skin characteristics can be analyzed based on the steroid hormone content in the subject's SSL. Accordingly, the present invention also provides a method of analyzing skin characteristics of a subject comprising quantifying steroid hormones in SSL collected from the subject.

Skin characteristics analyzed in the present invention include sebum content, stratum corneum water content, transepidermal water loss (TEWL), melanin content (or melanin index, MI), erythema content (or erythema index, EI), Skin pH etc. are mentioned. The site of the skin whose skin characteristics are analyzed is not particularly limited, but is preferably the skin of the face, more preferably the skin of the forehead or cheeks. The amount of sebum includes the amount of casual sebum, the amount of recovered sebum (the amount of sebum excreted within a predetermined period of time after removing sebum, for example, the amount of sebum after a predetermined period of time from washing the face), and the like. A commercially available sebum measuring device (eg, Sebumeter SM815; Courage + Khazaka electronic) can be used to measure the amount of sebum. Sebumeter SM815 measures the amount of sebum by allowing sebum to adhere to a translucent tape attached to the tip of the measurement probe and measuring the increase in light transmittance of the tape due to sebum adsorption. The stratum corneum moisture content can be measured, for example, based on the capacitance of the skin. A commercially available measurement device (for example, orneometer CM825; Courage + Khazaka electronic) can be used to measure the moisture content of the stratum corneum. TEWL can be calculated based on the concentration gradient of water evaporating from the skin surface according to Fick's law. For example, the temperature difference and humidity difference of moisture passing through two sensors are measured, and TEWL is calculated from those values. TEWL measuring devices according to this principle are commercially available (eg TEWA meter TM300; Courage + Khazaka electronic). Alternatively, air or nitrogen gas can be circulated in a closed probe applied to the skin surface, and TEWL can be calculated from the moisture content of the collected gas. The amount of melanin in the skin can be determined by irradiating the skin with light of wavelengths different in melanin absorption and measuring the amount of light absorbed by the skin. Erythema mass in the skin can be measured based on light absorption by hemoglobin in the skin. The amount of erythema can be measured by irradiating the skin with light of wavelengths different in hemoglobin absorption and measuring the amount of light absorbed by the skin. The amount of melanin and the amount of erythema can be measured using a commercially available measuring device (eg, Mexameter MX18; Courage + Khazaka electronic). Skin pH can be measured by a commercially available pH meter (eg Skin-pH-Meter PH905; Courage + Khazaka electronic).

In one embodiment of the method for analyzing skin characteristics of the present invention, the steroid hormone to be quantified is androgen, and the skin characteristics to be analyzed are sebum content, stratum corneum water content, TEWL, melanin content (MI), and erythema content. (EI) and at least one selected from the group consisting of skin pH. The androgen is preferably at least one selected from the group consisting of DHEA, T, DHT and A-dione, more preferably DHEA, T, DHT and A-dione.

In one embodiment of the method for analyzing skin characteristics of the present invention, the steroid hormone to be quantified is progesterone (P4), and the skin characteristics to be analyzed are sebum content, stratum corneum water content, TEWL, melanin content (MI), It is at least one selected from the group consisting of erythema level (EI) and skin pH.

In one embodiment of the method for analyzing skin characteristics of the present invention, the steroid hormone to be quantified is estrogen, and the skin characteristics to be analyzed are sebum content, stratum corneum moisture content, TEWL, melanin content (MI), and erythema content. (EI) and at least one selected from the group consisting of skin pH. The estrogen is preferably at least one selected from the group consisting of E1 and E2, more preferably E1 and E2.

In one embodiment of the skin characteristic analysis method of the present invention, the steroid hormone to be quantified is a glucocorticoid, and the skin characteristic to be analyzed is at least one selected from the group consisting of stratum corneum water content and skin pH. is. The glucocorticoid is preferably cortisol (F).

In one embodiment, in the method for analyzing skin characteristics of the present invention, skin characteristics of a subject are evaluated based on the quantified amount of steroid hormone in SSL.
In a preferred embodiment, when the androgen content in the SSL taken from a subject is high, the subject's sebum content, TEWL, melanin content (MI) and erythema content (EI) are determined to be high, whereas the The subject's stratum corneum water content and skin pH are judged to be low. In addition, when the androgen content in the SSL is low, the subject's sebum content, TEWL, melanin content (MI), and erythema content (EI) are judged to be low, while the subject's stratum corneum water content and skin pH is judged to be high.
In a preferred embodiment, when the amount of P4 in the SSL sampled from a subject is high, it is determined that the stratum corneum water content and skin pH of the subject are high, while the sebum content, TEWL, and melanin content of the subject are determined to be high. (MI) and erythema (EI) are judged to be low. In addition, when the amount of P4 in the SSL is low, the subject's stratum corneum water content and skin pH are judged to be low, while the subject's sebum content, TEWL, melanin content (MI) and erythema content ( EI) is judged to be high.
In a preferred embodiment, when the amount of estrogen in the SSL sampled from a subject is high, it is determined that the stratum corneum water content and skin pH of the subject are high, while the sebum content, TEWL, and melanin content of the subject are determined to be high. (MI) and erythema (EI) are judged to be low. In addition, when the amount of estrogen in the SSL is low, the stratum corneum water content and skin pH of the subject are judged to be low. EI) is judged to be high.
In a preferred embodiment, when the amount of glucocorticoid in SSL collected from a subject is high, the stratum corneum water content and skin pH of the subject are judged to be low, while the amount of glucocorticoid in SSL is determined to be low. If low, the subject's stratum corneum water content and skin pH are judged to be high.

The determination of whether the steroid hormone level in SSL of the subject is high or low can be made based on, for example, a predetermined threshold or the like. For example, thresholds can be set based on statistical values of steroid hormone levels in SSL measured from a population. Alternatively, thresholds can be set based on previously measured steroid hormone levels in SSL from the same subject. Alternatively, as will be described later, the SSL steroid hormone level correlates with the blood steroid hormone level, so the threshold can be calculated based on a standard blood steroid hormone level. However, the method of setting the threshold in the method of the present invention and the method of judging the SSL hormone level are not limited to these.

In another embodiment, in the method for analyzing skin characteristics of the present invention, changes in SSL steroid hormone levels are measured, and skin characteristics of a subject are evaluated based on the changes in the hormone levels.
In a preferred embodiment, an increase in the amount of sebum, TEWL, amount of melanin (MI), and amount of erythema (EI) of the subject is determined or predicted when the amount of androgen in the SSL collected from the subject increases, On the other hand, a decrease in stratum corneum water content and skin pH of the subject is determined or predicted. In addition, when the androgen amount in the SSL decreases, a decrease in sebum content, TEWL, melanin content (MI), and erythema content (EI) in the subject is judged or predicted. An increase in volume and skin pH is determined or predicted.
In a preferred embodiment, when the amount of P4 in SSL collected from a subject increases, an increase in the stratum corneum water content and skin pH of the subject is determined or predicted, while the sebum content of the subject, TEWL , a decrease in melanin content (MI) and erythema content (EI) is determined or predicted. In addition, when the amount of P4 in the SSL decreases, it is judged or predicted that the stratum corneum water content and skin pH of the subject decrease, while the sebum content, TEWL, melanin content (MI) and redness of the subject An increase in crew quantity (EI) is determined or predicted.
In a preferred embodiment, when the amount of estrogen in SSL collected from a subject increases, an increase in the stratum corneum water content and skin pH of the subject is determined or predicted, while the amount of sebum, TEWL , a decrease in melanin content (MI) and erythema content (EI) is determined or predicted. In addition, when the amount of estrogen in the SSL decreases, a decrease in the moisture content of the stratum corneum and skin pH of the subject is judged or predicted, An increase in crew quantity (EI) is determined or predicted.
In a preferred embodiment, when the amount of glucocorticoid in SSL collected from a subject increases, a decrease in the stratum corneum water content and skin pH of the subject is determined or predicted, while the amount of glucocorticoid in the SSL If the amount of corticoid is decreased, an increase in stratum corneum water content and skin pH of the subject is determined or predicted.

A determination of an increase or decrease in SSL steroid hormone levels in a subject can be made based on previously measured SSL steroid hormone levels from the same subject. However, the technique for evaluating changes in SSL hormone levels in the method of the present invention is not limited to this.

In addition, as shown in Examples below, the present inventors have found that the amount of steroid hormone in SSL has a correlation with the amount of blood steroid hormone. This indicates that the blood steroid hormone level of the subject can be estimated based on the subject's SSL steroid hormone level. Therefore, the present invention also provides a method for analyzing the amount of steroid hormone in the blood of a subject, including quantifying the steroid hormone in SSL collected from the subject. In a preferred embodiment, the steroid hormone analyzed by this method is at least one selected from the group consisting of androgens (DHEA, T, DHT, and A-dione) and progesterone (P4).

In one embodiment, in the method for analyzing a blood steroid hormone level of the present invention, the blood steroid hormone level of a subject is analyzed based on the steroid hormone level in SSL collected from the subject. Preferably, in the method for analyzing the blood steroid hormone level of the present invention, the test is calculated from the SSL steroid hormone level of the subject based on a prediction formula for predicting the blood steroid hormone level from the SSL steroid hormone level. Estimate the amount of steroid hormones in the body's blood. The prediction calculation formula can be prepared in advance based on the steroid hormone levels in SSL and blood steroid hormone levels collected from the population. Furthermore, based on the obtained blood steroid hormone level of the subject, analyzing the direction (increase or decrease) or the extent (increase rate, decrease rate, etc.) of the change in the blood steroid hormone level of the subject. can be done. The blood steroid hormone level is used as an indicator of various physiological functions and diseases of the living body, and the blood steroid hormone level is a useful indicator for evaluating the physiological function and disease states. According to this method, the amount of steroid hormone in the blood of a subject can be analyzed noninvasively without the need for blood sampling, so that physiological functions and disease states can be examined without imposing a burden on the subject.

For example, the amount of testosterone in the blood is affected by male menopause, Addison's disease, hypopituitarism, liver cirrhosis, tension muscular dystrophy, prostate cancer, Cushing's syndrome, Turner's syndrome, hyperthyroidism, congenital adrenocortical hyperplasia, hyperplasia, and hyperplasia. It is used as a diagnostic index for cystic ovary syndrome, hypertrichosis, ovarian tumor, testicular tumor, and the like. In addition, testosterone and DHT control the growth of body hair and head hair (see Non-Patent Document 8), and DHT is closely involved in male alopecia (see Non-Patent Document 9). Addison's disease, body dysfunction, hypopituitarism, pituitary tumor, placental dysfunction, ovulation disorder, amenorrhea, ovarian hypofunction, miscarriage, Cushing's syndrome, congenital adrenocortical hyperplasia, It is used as a diagnostic index for adrenal cancer, adrenal genital syndrome, testicular stromal cell tumor, etc., or as a grasp of the menstrual cycle and pregnancy. Therefore, the amount of steroid hormone in SSL measured by the present invention may serve as a useful index for evaluating the above-described biological functions and disease states.

Further disclosed herein are the following materials, methods of manufacture, uses, methods, etc., as exemplary embodiments of the present invention. However, the invention is not limited to these embodiments.

[1] A method for preparing a steroid hormone, comprising separating the steroid hormone from superficial skin lipids (SSL) collected from a subject.
[2] Preferably, the method of [1], wherein the separation comprises solvent extraction of the steroid hormone in the SSL.
[3] Preferably, the steroid hormone is at least one selected from the group consisting of androgen, progesterone, estrogen, and glucocorticoid,
Preferably, the androgen is at least one selected from the group consisting of dehydroepiandrosterone (DHEA), testosterone (T), dihydrotestosterone (DHT), and androstenedione (A-dione),
Preferably, the estrogen is at least one selected from the group consisting of estrone (E1) and estradiol (E2),
Preferably, said glucocorticoid is cortisol (F)
The method according to [1] or [2].
[4] The method according to [2] or [3], wherein the solvent is preferably an organic solvent, more preferably an ether.
[5] Preferably, the method according to any one of [1] to [4], further comprising collecting the SSL of the subject.

[6] A method for quantifying steroid hormones in SSL, which comprises quantifying the steroid hormones prepared by the method according to any one of [1] to [5].
[7] Preferably, the method of [6], wherein the quantification is performed by LC-MS measurement.
[8] Preferably, the method of [7] further comprises preparing the steroid hormone derivative prior to the LC-MS measurement.
[9] Preferably, the method of [8], wherein the steroid hormone is androgen, progesterone, or glucocorticoid, and the derivative is an ester derivative.
[10] Preferably, the method of [8], wherein the steroid hormone is estrogen and the derivative is a pentahalogenated benzyl derivative or a pentahalogenated benzoyl derivative.
[11] The method according to [10], which preferably further comprises preparing a fluoropyridine ether derivative or an ester derivative of the pentahalogenated benzyl derivative or pentahalogenated benzoyl derivative.

[12] A method for analyzing skin characteristics of a subject, comprising quantifying steroid hormones in SSL collected from the subject by the method according to any one of [6] to [11].
[13] Preferably, the method of [12], further comprising evaluating skin characteristics of the subject based on the amount of steroid hormone in SSL.
[14] Preferably, the method of [12] further comprises evaluating skin characteristics of the subject based on changes in the SSL steroid hormone level.
[15] Preferably, the steroid hormone is an androgen, and the subject's skin characteristics are selected from the group consisting of sebum content, stratum corneum water content, transepidermal water loss, melanin content, erythema content, and skin pH. The method according to any one of [12] to [14].
[16] The method of [15], preferably further comprising:
judging that when the androgen level is high, the sebum level, TEWL, melanin level and erythema level of the subject are high, while the stratum corneum water level and skin pH of the subject are low;
judging that when the androgen level is low, the sebum level, TEWL, melanin level and erythema level of the subject are low, while the stratum corneum water level and skin pH of the subject are high;
When the amount of androgen increases, determining or predicting an increase in sebum content, TEWL, melanin content, and erythema content of the subject, while determining or predicting a decrease in stratum corneum water content and skin pH in the subject. to do; or
When the androgen level decreases, determining or predicting a decrease in the subject's sebum content, TEWL, melanin content, and erythema content, while determining or predicting an increase in the stratum corneum water content and skin pH of the subject. to do.
[17] Preferably, the steroid hormone is progesterone, and the subject's skin characteristics are selected from the group consisting of sebum content, stratum corneum water content, transepidermal water loss, melanin content, erythema content, and skin pH. The method according to any one of [12] to [14].
[18] The method of [17], preferably further comprising:
judging that when the progesterone level is high, the subject's stratum corneum water content and skin pH are high, while the subject's sebum content, TEWL, melanin content, and erythema content are low;
judging that when the progesterone level is low, the stratum corneum water content and skin pH of the subject are low, while the sebum content, TEWL, melanin content and erythema content of the subject are high;
When the amount of progesterone increases, determining or predicting an increase in the stratum corneum water content and skin pH of the subject, while determining or predicting a decrease in the sebum content, TEWL, melanin content, and erythema content of the subject. to do; or
When the progesterone level decreases, determining or predicting a decrease in the stratum corneum water content and skin pH of the subject, while determining or predicting an increase in sebum content, TEWL, melanin content, and erythema content in the subject. to do.
[19] Preferably, the steroid hormone is estrogen, and the subject's skin characteristics are selected from the group consisting of sebum content, stratum corneum water content, transepidermal water loss, melanin content, erythema content, and skin pH. The method according to any one of [12] to [14].
[20] The method of [19], preferably further comprising:
judging that when the estrogen level is high, the subject's stratum corneum water content and skin pH are high, while the subject's sebum content, TEWL, melanin content and erythema content are low;
judging that when the estrogen level is low, the subject's stratum corneum water content and skin pH are low, while the subject's sebum content, TEWL, melanin content and erythema content are high;
When the estrogen level increases, determining or predicting an increase in the stratum corneum water content and skin pH of the subject, while determining or predicting a decrease in the sebum content, TEWL, melanin content and erythema content of the subject. to do; or
If the estrogen level decreases, determining or predicting a decrease in the stratum corneum water content and skin pH of the subject, while determining or predicting an increase in the sebum content, TEWL, melanin content, and erythema content of the subject. to do.
[21] Preferably, the steroid hormone is a glucocorticoid, and the subject's skin characteristics are at least one selected from the group consisting of stratum corneum water content and skin pH, [12] to [14] ] any 1 item|term method.
[22] The method of [21], preferably further comprising:
judging that when the glucocorticoid amount is high, the stratum corneum water content and skin pH of the subject are low;
judging that when the glucocorticoid level is low, the stratum corneum water content and skin pH of the subject are high;
Determining or predicting a decrease in the stratum corneum water content and skin pH of the subject when the amount of glucocorticoid increases; or
Determining or predicting an increase in the stratum corneum water content and skin pH of the subject when the glucocorticoid content decreases.

[23] A method for analyzing a blood steroid hormone level in a subject, which comprises quantifying the steroid hormone in SSL collected from the subject by the method according to any one of [6] to [11].
[24] Preferably, the method of [23] further comprises analyzing changes in blood steroid hormone levels of the subject based on changes in the steroid hormone levels in the SSL.
[25] The method of [23] or [24], wherein the steroid hormone is preferably at least one selected from the group consisting of androgen and progesterone.

EXAMPLES The present invention will be described in more detail below based on examples, but the present invention is not limited to these.

In the following examples, steroid hormones are represented by the following abbreviations.
DHEA; dehydroepiandrosterone T; testosterone DHT; dihydrotestosterone A-dione; androstenedione P4; progesterone F;

Test Example 1 Determination of Steroid Hormone in SSL (1) Subjects Eighty healthy males and healthy females (20 to 59 years old, BMI: 18.5 or more and less than 5.0) were used as subjects. The subject was confirmed in advance by a dermatologist that there was no skin abnormality.

(2) Collection of SSL In order to previously remove fat-soluble substances that may interfere with steroid hormone measurement, a blotting film (3M) was immersed in a chloroform/methanol solution. After immersion, the oil removing film was sufficiently dried. Using the oil blotting film, SSL was collected from the entire face of each subject, the mass was measured with a precision balance, and the mass of SSL (mass of oil blotting film after SSL collection - mass of oil blotting film before SSL collection) ). The blotting films were transferred to glass vials and stored at -80°C.

(3) Extraction of Steroid Hormone from SSL This procedure was entrusted to Asuka Pharmaceutical Medical Co., Ltd. and implemented. The oil blotting film was finely cut with scissors and put into a test tube, and internal standards (DHEA- ¹³ C ₃ (ISOSciences) 400 pg, T- ¹³ C ₃ (Sigma-Aldrich) 600 pg, DHT- ¹³ C ₃ (ISO Sciences) 200 pg, A -dione- ¹³ C ₃ (ISO Sciences) 400 pg, P4- ¹³ C ₃ (Hayashi Pure Chemical Industries Co., Ltd.) 400 pg, F-d4 (CDN Isotopes) 2000 ng, E1- ¹³ C ₄ (Hayashi Pure Chemical Industries Co., Ltd.) 100 pg, and E2- ¹³ C ₄ (Hayashi Pure Chemical Industries Co., Ltd.)) were added, and steroid hormones were extracted with 4 mL of methyl-t-butyl ether (MTBE, Sigma-Aldrich). After extraction, MTBE was distilled off, 2 mL of hexane and 0.5 mL of acetonitrile were added to the resulting residue, the mixture was shaken, and the acetonitrile layer was separated and distilled off. The residue was dissolved in 1 mL of methanol to obtain a sample solution containing SSL-derived steroid hormones. The obtained sample solution was divided into two equal parts for the measurement of DHEA, T, DHT, A-dione, P4 and F, and E1 and E2 respectively.

(4) Quantification of DHEA, T, DHT, A-dione, P4 and F This measurement was performed according to the method described in Japanese Patent No. 4634913 (“Method for measuring steroid”). The sample solution for measurement of DHEA, T, DHT, A-dione, P4 and F obtained in (3) was diluted with 1 mL of purified water and loaded onto an Oasis MAX cartridge (Waters). The cartridge was washed sequentially with 1 mL of purified water, 1 mL of purified water/methanol/acetic acid aqueous solution (55:45:1, v/v/v), and 1 mL of purified water/pyridine aqueous solution (100:1, v/v). After that, the sample was eluted with 1 mL of methanol/pyridine solution (100:1, v/v), and the eluate was distilled off from the obtained eluate. To the residue was added 0.05 mL of picolinic acid ester derivatization reagent (40 mg of picolinic acid (Tokyo Chemical Industry), 80 mg of 2-methyl-6-nitrobenzoic anhydride (MNBA, Tokyo Chemical Industry) and 20 mg of 4-dimethyl 1 mL of an acetonitrile solution containing aminopyridine (DMAP)) and 0.01 mL of triethylamine (TEA, Fujifilm Wako Pure Chemical Industries, Ltd.) were added, and the steroid picolinate ester derivatization reaction was carried out at room temperature for 30 minutes. The reaction was diluted with 0.5 mL of ethyl acetate/hexane/acetic acid solution (15:35:1, v/v/v) and loaded onto an InertSep SI cartridge (GL Sciences). After sequentially washing the cartridge with 1 mL of hexane and 2 mL of ethyl acetate/hexane solution (3:7, v/v), the sample was eluted with 2.5 mL of acetone/hexane solution (7:3, v/v).
The eluent was distilled off from the resulting eluate, and the residue was dissolved in 0.1 mL of purified water/acetonitrile aqueous solution (3:2, v/v). Using the resulting solution, LC-MS / MS [mass spectrometer: API4000, UHPLC: Nexera UHPLC system, analytical column: Kinetex C18 (1.7 μm, 2.1 × 150 mm), column temperature: 50 ° C., flow rate : 0.5 mL/min, mobile phase: 0.1% formic acid aqueous solution-acetonitrile gradient analysis], DHEA, T, DHT, A-dione, P4 and F were measured. From the measured values of LC-MS/MS, the amounts of DHEA, T, DHT, A-dione, P4, and F were quantified according to calibration curves based on respective internal standards. The value obtained by dividing the obtained value by the mass of SSL measured in (2) was obtained as the amount of DHEA, T, DHT, A-dione, P4 and F in SSL.

(5) Quantification of E1 and E2 This measurement was carried out according to the method described in Japanese Patent No. 6041125 (“Method for ultra-sensitive determination of estrogen”). The sample solution for measurement of E1 and E2 obtained in (3) was diluted with 1 mL of purified water and loaded onto an Oasis MAX cartridge (Waters). 1 mL of purified water/acetic acid aqueous solution (100:1, v/v), 1 mL of purified water/acetonitrile aqueous solution (7:3, v/v), 1 mL of 1 mol/L sodium hydroxide solution, 3 mL of methanol, After washing sequentially with 1 mL of purified water/aqueous acetic acid (100:1, v/v) and 1 mL of methanol/purified water/aqueous pyridine (60:40:1, v/v/v), 1 mL of methanol/purified The sample was eluted with water/pyridine aqueous solution (90:10:1, v/v/v) and the eluate was distilled off from the resulting eluate. 0.05 mL of pentafluoropyridine (Alfa Aesar), 0.1 mL of acetonitrile and 0.02 mL of TEA were added to the residue, and the steroid tetrafluoropyridine ether derivatization reaction was carried out at room temperature for 20 minutes.
The liquid was distilled off from the reaction, and the resulting residue (including E1-Tfpy and E2-Tfpy) was added to 2-hydrazino-1-methylpyridine (HMP 0.05 mg, Asuka Pharmaceutical Medical), trifluoroacetic acid (Fuji Film Wako Pure Chemical Industries, Ltd.) (0.01 mL) and acetonitrile (0.14 mL) were added and reacted at room temperature for 1 hour to derivatize E1-Tfpy in the residue with HMP. The resulting reaction was evaporated and the residue dissolved in 0.5 mL of methanol, further diluted with 0.5 mL of purified water and loaded onto an Oasis WCX cartridge (Waters). After sequentially washing the cartridge with 1 mL of 0.1 mol/L potassium dihydrogen phosphate aqueous solution, 2 mL of purified water, and 1 mL of purified water/acetonitrile aqueous solution (3:2, v/v), 1 mL of methanol was added to the E2-Tfpy fraction. minutes were eluted. After washing the E2-Tfpy eluted cartridge with 2 ml of acetonitrile, 0.5 mL of purified water and 1 mL of methanol/purified water/formic acid aqueous solution (65:35:2, v/v/v), 1 mL of methanol/ The E1 derivative (E1-TfpyHMP) fraction was eluted with a formic acid solution (50:1, v/v). The eluent was distilled off from each fraction.
To the residue of the E2-Tfpy fraction was added 0.05 mL of fusaric ester derivatization reagent (40 mg of fusaric acid (Chem-Impex International, Inc.), 80 mg of MNBA and 20 mg of DMAP dissolved in 1 mL of acetonitrile solution) and 0. .01 mL of TEA was added and the fusarate derivatization reaction of E2-Tfpy was carried out for 30 minutes at room temperature. The reaction was diluted with 0.5 mL of hexane/acetic acid solution (50:1, v/v) and loaded onto an InertSep SI cartridge. After sequentially washing the cartridge with 1 mL of hexane and 2 mL of ethyl acetate/hexane solution (3:17, v/v), E2 derivative (E2 -TfpyFU) was eluted and the eluent was evaporated from the eluate.
The residue containing E1-TfpyHMP was dissolved in 0.1 mL of acetonitrile/purified water aqueous solution (7:3, v/v). The residue containing E2-TfpyFU was dissolved in 0.1 mL of acetonitrile/purified water aqueous solution (4:1, v/v). Using the obtained solution, E1 and E2 were measured by LC-MS/MS. LC-MS/MS measurement of E1 [mass spectrometer: API5000, UHPLC: Agilent 1290 Infinity LC system, first-dimensional analytical column: Cortex C18 (1.6 μm, 2.1 × 50 mm), second-dimensional analytical column: Luna Omega PS C18 (1.6 μm, 2.1×50 mm), column temperature: 60° C., flow rate: 0.5 mL/min, first dimension mobile phase: 0.1% formic acid aqueous solution-methanol gradient analysis, second dimension Mobile phase: 20 mM ammonium formate aqueous solution-acetonitrile gradient analysis]. LC-MS/MS measurement of E2 [mass spectrometer: API5000, UHPLC: Agilent 1290 Infinity LC system, first-dimensional analysis column: Capcellcore PFP (2.7 μm, 2.1 × 50 mm), second-dimensional analysis column: Cortex C18 (1.6 μm, 2.1 × 150 mm), column temperature: 60 ° C., flow rate: 0.5 mL / min, first-dimensional mobile phase: 0.1% formic acid aqueous solution-acetonitrile gradient analysis, second-dimensional mobile phase : 0.1% formic acid aqueous solution-acetonitrile gradient analysis]. From the obtained LC-MS/MS measurement values, the amounts of E1 and E2 were each quantified according to a calibration curve based on each internal standard. The value obtained by dividing the obtained value by the mass of SSL measured in (2) was obtained as the amount of E1 and E2 in SSL.

(6) Results All eight steroid hormones (DHEA, T, DHT, A-dione, P4, F, E1 and E2) could be detected and quantified in all subjects from whom SSL was taken. Table 1 shows the average value and standard deviation of the amount of each steroid hormone among all subjects. From the results in Table 1, it was clarified that DHEA, T, DHT, A-dione, P4, and F were present abundantly in SSL, although the amounts of E1 and E2 were small.

Test Example 2 Correlation Between Steroid Hormone Level in SSL and Skin Characteristics In the subject population of Test Example 1, various measurements of skin characteristics were performed on the same day as the SSL was taken. In the measurement of skin characteristics, first, the amount of casual sebum on the forehead and cheeks after washing the face for 4 hours or longer was measured using Sebumeter SM815 (Courage + Khazaka electronic). After that, the face was washed and acclimatized for 20 minutes, and then the transepidermal water loss was measured using Tewameter TM300 (Courage + Khazaka electronic), and the stratum corneum water content was measured using Corneometer CM825 (Courage + Khazaka electronic). Skin pH was measured with a Skin-pH-Meter PH905 (Courage + Khazaka electronic), melanin index and erythma index with a Mexameter MX18 (Courage + Khazaka electronic), respectively. Furthermore, after washing the face for 60 minutes, the amount of recovered sebum on the forehead and cheeks was measured using Sebumeter SM815. Correlations between the various skin characteristic values obtained and the amounts of various steroid hormones in SSL measured in Test Example 1 were analyzed by Pearson's correlation analysis. Table 2 shows the results.

From the results in Table 2, multiple statistically significant correlations were confirmed between DHEA, T, DHT, A-dione, P4, and F and various skin properties. Androgens (DHEA, T, DHT, and A-dione) mainly show a positive correlation with sebum content, transepidermal water loss, melanin index and erythma index, while showing a negative correlation with stratum corneum water content and pH. rice field. Progesterone (P4) showed a positive correlation with stratum corneum water content and pH, and a negative correlation with sebum content, transepidermal water loss, melanin index and erythma index. Cortisol (F) showed a negative correlation with stratum corneum water content and pH. Estrogen (E1 and E2) showed a positive correlation with stratum corneum water content and pH, and showed a negative correlation with sebum content, transepidermal water loss, melanin index and erythma index.

Test Example 3 Correlation Between SSL Steroid Hormone Level and Blood Steroid Hormone Level From the test subject group in Test Example 1, 3 mL of blood was collected from the arm on the same day as the SSL collection. Serum was separated from collected blood using a centrifuge and stored at -80°C until analysis. From the obtained serum samples, blood concentrations of T and P4 were quantified using an ELISA measurement kit (Cayman) according to the attached protocol. For T and P4, the correlation between the blood concentration and the SSL concentration measured in Test Example 1 was analyzed. The results of plotting the blood and SSL concentrations of T and P4 are shown in FIG. For T and P4, a statistically significant positive correlation was observed between SSL concentration and blood concentration.

Claims (7)

extracting at least one steroid hormone selected from the group consisting of testosterone and progesterone in lipids on the skin surface collected from a subject using a sheet-like lipid-absorbing material on the skin surface with methyl-t-butyl ether ; A method for preparing the steroid hormone, comprising: A method for quantifying steroid hormones in lipids on the skin surface, comprising quantifying at least one steroid hormone selected from the group consisting of testosterone and progesterone prepared by the method according to claim 1 . Skin of a subject, including extracting androgen in lipids on the skin surface collected from a subject using a sheet-like lipid-absorbing material on the skin surface with methyl-t-butyl ether, and quantifying the androgen at least one analysis method selected from the group consisting of sebum content, stratum corneum water content, transepidermal water loss, melanin content, erythema content and skin pH. The skin of a subject , including extracting progesterone in lipids on the skin surface collected from a subject using a sheet-like lipid-absorbing material on the skin surface with methyl-t-butyl ether, and quantifying the progesterone at least one analysis method selected from the group consisting of sebum content, stratum corneum water content, transepidermal water loss, melanin content, erythema content and skin pH. The skin of a subject, including extracting estrogen in lipids on the skin surface collected from a subject using a sheet-like lipid-absorbing material on the skin surface with methyl-t-butyl ether, and quantifying the estrogen at least one analysis method selected from the group consisting of sebum content, stratum corneum water content, transepidermal water loss, melanin content, erythema content and skin pH. extracting with methyl-t-butyl ether glucocorticoids in skin surface lipids collected from a subject using a sheet-shaped skin surface lipid-absorbing material, and quantifying the glucocorticoids , At least one analysis method selected from the group consisting of stratum corneum water content and skin pH of the subject's skin. The steroid in the blood of the subject, which comprises quantifying at least one steroid hormone selected from the group consisting of testosterone and progesterone in lipids on the skin surface collected from the subject by the method according to claim 2 . A method for analyzing hormone levels.

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