KR20120066597A - A method of diagnosing a menstrual activation using saliva - Google Patents

Abstract

PURPOSE: A method for measuring sex hormone in saliva is provided to detect trace amounts of estradiol and to quickly and objectively determine menstrual regulation. CONSTITUTION: A method for measuring estradiol concentration comprises: a step of treating strong base of pH 12-14 to collected saliva; a step of heating at 70-90°C and cooling; a step of removing digestive enzymes and bacteria from saliva by centrifuging; a step of neutralizing; and a step of removing mucosaccharide-proteins. The saliva is collected one hour after getting up. The strong base includes NaOH, LiOH, KOH or Ca(OH)_2.

Description

A method of diagnosing a menstrual activation using saliva

The present invention relates to a method for measuring sex hormones in saliva in order to provide information necessary for diagnosing physiological (menstrual) activity, and more specifically, by effectively purifying saliva through specific pH control and temperature control, the spheroid in saliva It relates to a method of measuring the concentration of hormones such as estradiol.

Women's ovaries are composed of follicles at various stages of development. The follicles are composed of germ cells, somatic cells, and follicular cells. Oocytes and capsular cells surrounding the egg produce and secrete various steroid hormones, which are known to regulate the growth, maturation and ovulation of the egg and the functions of the appendages necessary for reproductive activities such as the uterus and mammary glands. have.

The circadian rhythms of hormone production and secretion are known to be controlled by the cranial nerves, hypothalamus, and pituitary, and are fundamentally determined by the individual's sleep cycle. After a normal night's sleep, waking up in the morning, the brain's nerves wake up, and this signal reaches the suprachiasmatic nucleus (SCN) in the hypothalamus, where the bioclock gene is activated. The signal is again transmitted to the paraventricular nucleus (PVN) and GnRH neurone. In PVN, corticotropin releasing hormone (CRH) is released in response to this stimulus, and GnRH is released in GnRH neurone. Secreted CRH and GnRH stimulate the pituitary gland to produce and secrete adrenocorticotrophic hormone (ACTH), follicle stimulating hormone (FSH) and luteinizing hormone (LH), respectively.

ACTH, FSH, and LH produced and secreted by the pituitary gland act on the adrenal glands and gonads (ovaries), respectively, to produce and regulate cortisol and estradiol or progesterone.

Cortisol production in the adrenal cortex is characterized by a very pronounced circadian rhythm. The circadian rhythm of cortisol produced and secreted in the adrenal cortex shows a sharp increase immediately after waking up to the highest concentration of the day 30 minutes after waking up. This phenomenon is called 'rhythm right after the weather'. Cortisol concentration then gradually decreases to the lowest concentration before bedtime. Cortisol, a hormone produced in the adrenal cortex, is known to have the highest concentration of the day 30 minutes after wake up. However, it is not known how female hormones change after the weather.

In particular, women's menstrual cycles are controlled by various hormones and nerves in the hypothalamus-pituitary-gonad's (HPG) axis. You can see that it is. Until now, however, ovarian function was assessed by evaluating hormonal function by circulating hormonal rhythms of hormone production and secretion. Therefore, functional parts such as changes in physiological activity were relatively difficult to diagnose and estradiol contained in saliva. There is no information to objectively determine the change in physiological activity by measuring.

Therefore, the inventors found that the concentration of estradiol during the first hour after the gas phase was increased, and the estradiol concentration contained in the saliva collected every 30 minutes after the vapor phase and at 1 hour after the vapor phase By measuring the change, it is confirmed that the normal physiological activity and normal ovarian function and hormone secretion function can be discriminated, and the saliva purification process for detecting the trace amount of estradiol present in the saliva completed the present invention. It was.

It is an object of the present invention to provide a method for measuring the concentration of estradiol in saliva in order to provide information necessary for diagnosing physiological activity.

Another object of the present invention is to provide a method for purifying saliva through specific pH and temperature control in order to measure the concentration of estradiol in saliva.

Still another object of the present invention is to provide a method for determining whether the physiological activity is normal by changing the concentration of estradiol.

In order to solve the above problems, the present invention provides a method for measuring the concentration of estradiol hormone comprising the following steps, in order to provide information necessary for diagnosing the menstrual cycle:

(a) treating the collected saliva with a strong base of pH 12-14;

(b) cooling after heating;

(c) neutralization step; And

(d) mucus glycoprotein (mucosugar) removal step.

At this time, the saliva collection in step (a) is preferably made within 1 hour after the gaseous phase, for example, it is more preferably collected immediately after the vapor phase, 30 minutes after the gas phase and 1 hour after the gas phase.

In addition, the strong base of pH 12-14 in step (a) may be one selected from the group consisting of NaOH, LiOH, KOH, Ca (OH) ₂ . In one embodiment of the present invention 1N NaOH was used.

In addition, the heating in step (b) is preferably made of a temperature of 70 ~ 90 ℃, more preferably about 80 ℃. In addition, the cooling may be performed by quenching using, for example, an ice bath.

And, after the step (b), it is preferable to further include the step of removing the denatured digestive enzymes and bacteria by pH and heat from saliva by centrifugation.

In addition, the (c) neutralization step is most typically carried out by treating a strong acid of pH 1-2, in particular, the strong acid of pH 1-2 is in the group consisting of HCl, H ₂ SO ₄ , HNO ₃ It may be one selected. In one embodiment of the present invention 1N HCl was used.

In addition, in step (d), the removal of mucus (mucosaccharides) is achieved by treating the mucus glycoprotein (mucosaccharide) degrading enzyme. Since the mucoglycoprotein (mucosaccharide) is not denatured by heat, a method of removing by using an enzyme is used.

At this time, the mucolytic enzyme can be any enzyme capable of degrading β-Nacetylhexosamine- [1 → 4] glycosidic bond, and in one embodiment of the present invention, hyaluronidase type 1 (H 3506, Sigmaaldrich). chemical co.) 500 IU was used.

Further, after removing the mucoglycoprotein (mucosugar), it is more preferable to perform the step of removing the mucoglycoprotein (mucosaccharide) degrading enzyme by centrifugation.

Finally, by further performing the step of mixing the buffer, it is possible to finish the saliva purification process. At this time, the buffer solution preferably contains the same amount of gelatin as saliva. This gelatin leads to a homogeneous distribution of trace estradiol in saliva and maintains optimum acidity even when saliva is exposed to outside air during the analysis.

In addition, the concentration of estradiol is most preferably measured using radioimmunoassay.

On the other hand, the present invention provides a method for analyzing the concentration of estradiol, including:

(a) measuring the concentration of estradiol in saliva of a woman having a normal menstrual cycle by the method of claim 1;

(b) setting a reference indicator to the measured concentration of estradiol;

(c) measuring the concentration of estradiol in the saliva of the test subject woman by the method of claim 1;

(d) comparing the obtained estradiol concentration with the reference indicator value set in step (b).

As mentioned above, the concentration of estradiol is preferably measured from saliva collected within 1 hour after gas phase.

In particular, the concentration analysis of estradiol is more preferably performed for the change in the concentration of estradiol measured from saliva collected every time immediately after the gas phase, 30 minutes after the gas phase and 1 hour after the gas phase.

At this time, it is compared whether the concentration change of estradiol in the saliva of the test subject is out of the range of the reference indicator set as the concentration change value of the woman with a normal menstrual cycle. Can be determined as "physiologically abnormal".

The present invention first identified the phenomenon of increasing the concentration of estradiol, a sex hormone after morning weather, and to accurately measure the concentration of estradiol in saliva, using a rapid temperature change and a pH change to denature the protein in the saliva It is characterized by using a process to make. By accurate analysis of estradiol concentration it is possible to collect the information necessary for diagnosing the physiological activity of women from various viewpoints on the function of the ovary, physiological impurities.

The present invention enables the detection of trace amounts of estradiol in saliva by denaturing proteins contained in saliva through rapid temperature changes and pH changes.

Therefore, the present invention measures the estradiol concentration contained in the saliva collected within 1 hour after the weather, it is possible to quickly and objectively determine whether the normal physiological activity through the change in the concentration of estradiol after the indicated weather In addition, the evaluation of abnormal increase or abnormal decrease of estradiol in the ovary based on the change in post-weather concentration of estradiol may provide information necessary for diagnosing abnormality of the ovary, early diagnosis of menopause, and unknown physiological impurity. Can be.

1 shows the changes in cortisol concentrations in saliva collected immediately after waking up, 30 minutes after waking up and 60 minutes after waking up in women (a), menopausal women (b) and menstrual disorders (c) with normal menstrual cycles, respectively. Is a graph.
Figure 2 shows the change of estradiol concentration in saliva collected immediately after waking up, 30 minutes after waking up and 60 minutes after waking up in women (a), menopausal women (b) and menstrual disorders (c) with menstrual cycles, respectively. Is a graph.
3 is a histogram showing the distribution of saliva estradiol concentrations in women with normal menstrual cycles immediately after waking (a), 30 minutes after waking (b) and 60 minutes after waking (c).
4 is a diagram illustrating a change (a) of Peak / A0% and a change (b) of MinInc% according to a menstrual cycle of a woman having a normal menstrual cycle.
FIG. 5 shows the difference between Peak / A0% and MinInc% in women with normal menstrual cycles, postmenopausal women, and women with menstrual irregularities.

Definitions of terms used in the present invention are as follows.

"Diagnostic" means identifying the presence or activity of a particular pathology or physiological condition, and the like. For the purposes of the present invention, the diagnosis is to identify the physiological activity, menstrual cycle and related conditions of a woman. Diagnosis of such physiological activity is useful for providing information on the incidence of infertility, contraception and female disease.

"Subject" or "subject" refers to an animal, in some embodiments a mammal, and in other embodiments a human being to be treated, observed or tested. In particular, in the present invention, a human female was targeted.

"Sample" or "sample" means a collection of similar cells obtained from a tissue of a subject or patient. Sources of tissue or cell samples may include solid tissue from fresh, frozen and / or preserved organ or tissue samples or biopsies or aspirates; Blood or any liquid tissue. In the present invention, saliva is used.

Sex hormones are hormones secreted from the gonads of females and males of vertebrates, which promote the development of the genital organs and maintain their function. The gonads that secrete hormones are hepatocytes in the testes in males and the females are mainly follicles or corpus luteum in the ovaries. Not only the gonads but also the adrenal cortex secrete substances with the action of sex hormones. During pregnancy, sex hormones are also released from the placenta, contributing to the maintenance of pregnancy. All sex hormones are steroidal substances

"Physiological or menstrual" refers to a physiological phenomenon in which an adult female periodically releases an egg from the ovary in the body and then fertilizes the vagina with bleeding after the uterine mucosa to implant the fertilized egg into the uterus. It usually has a 28-day cycle and lasts 3 to 5 days. However, cycles can vary between individuals and depending on their physical condition. Most men grow up to about 12 to 15 years of age and begin menstruation for the first time. Women have about 400 menstruation cycles in their lifetimes, and when they are 45 or 55 years old, they become menopause and stop menstruation. Blood flow to menstruation is about 35 ml, within 10 to 80 ml is normal.

The "menstrual cycle or menstrual cycle" is a regular change in the reproductive organs under the influence of endocrine hormones.It is not constant between 21 and 35 days, but usually repeats about 28 days. The period of time until the appearance of menstruation. The endometrium and ovary represent periodicity by the level of female hormone secretion. When the endometrium collapses, bleeding is called menstruation, and when the follicle matures in the ovary to release the egg, it is called ovulation day. Ovulation occurs about 13 to 15 days from the beginning of menstruation, which is the best time to conceive.

The present invention includes determining and diagnosing menstrual activation by diagnosing the menstrual cycle or menstrual cycle. Therefore, in this specification, the terms "physiological cycle diagnosis" and "physiological activity diagnosis" are used interchangeably.

"Biophysiological impurity" is a period of menstrual cycle and abnormalities, menstrual irregularities in which menstrual cycle is more than a week ahead of the normal date (frequent menstruation) and more than one week later (heavy menstruation) There is a large amount of blood coming out every time (overmenstrual), a small amount (overmenstrual), and irregular cases (negative uterine bleeding).

"Menopause" (閉經 期) is the period when women's menstruation stops completely, also known as menopause (更年期). It usually appears between the ages of 40 and 55, and some women may be early menopause due to surgery or premature ovarian insufficiency. If you haven't had menstruation for the last 12 months, you're in menopause. During menopause, the production of hormones called estrogen and progesterone slowly decreases.

As used herein, the terms "about", "substantially", and the like, are used at, or in close proximity to, numerical values when manufacturing and material tolerances inherent in the meanings indicated are intended to aid the understanding of the invention. Accurate or absolute figures are used to assist in the prevention of unfair use of unscrupulous infringers.

All technical terms used in the present invention, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention. The contents of all publications described herein by reference are incorporated into the present invention.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention is directed to a method of measuring the concentration of estradiol in saliva to provide information necessary for diagnosing a menstrual cycle.

(1) In the present invention, the concentration change of "estradiol" is used to provide information necessary for diagnosing physiological activity of a woman.

Women's menstrual cycles begin as part of puberty until menopause and are part of women's lives, except during pregnancy and lactation. The vitality of women is largely divided into follicular, ovulatory, luteal and menstrual periods.

The follicle is the growth phase of the follicle, which is the time when 6-12 follicles are grown at the same time. Estradiol concentrations are known to peak before ovulation and decrease gradually. Progesterone is produced and secreted mainly in the ovulation follicles. The concentration of progesterone is highest during the luteal phase and decreases during menstruation. This type of cycle is called the ovarian cycle. Interestingly, the production of steroid hormones in the body occurs in both endocrine organs, the adrenal glands and the gonads, where cortisol is produced mainly in the adrenal cortex and estradiol in the ovary.

The estradiol and progesterone production of follicles is controlled by hormones produced and secreted by the hypothalamus and pituitary gland. The biaxial axis is called the hypothalamus-pituitary-gondola (HPG axis). Hormone production and secretion by endocrine organs (adrenal glands, gonads, etc.), which are controlled by hormones produced by the pituitary gland, have a regular cycle of one day. The hormonal cycle is called 'daily rhythm' and the rhythm that continues after waking up to bedtime is called 'daily rhythm'. These hormone production and secretion of the follicles are, after all, essential for the regulation of menstrual cycles and maintenance of pregnancy.

Changes in female hormones are determined by the follicles that make up the ovary, but women who are part of the reproductive age are affected by misuse factors such as hormone intake (contraceptives), stress, and nutritional status. There may be changes (shortening and delaying), and abnormal hormone concentrations and hormonal imbalances of estradiol and progesterone are known to be highly associated with polycystic ovary syndrome (ovary), endometriosis, breast cancer, and cardiovascular diseases.

In particular, estradiol is the final product of ovarian steroid production and acts as a regulator of egg growth and uterine thickening. The delay of menstrual cycles is characterized by a longer follicle during the ovarian cycle. As a result, estradiol concentrations in postmenopausal women are much lower than those in menstrual cycles.

In the present invention, by measuring the concentration of such "estradiol" to obtain information on the biological activity for the menstrual cycle and the like.

Estradiol shows a distinct hormonal cycle according to the menstrual cycle, the abnormality of the hormonal cycle is directly connected to the ovarian cycle.

Menopause no longer results in follicle growth, resulting in the ovarian female hormone production reaching its lowest state and not having a constant rhythm. In addition, physiological changes in the woman's uterus and other reproductive organs are not present periodically. In other words, the function of all the organs involved in reproduction is stopped. However, menstrual irregularities (shortening and delaying) are usually determined by whether the follicle shortens or slows down.

In particular, it is preferable that beta estradiol (β-estradiol) as estradiol in the present invention.

Beta estradiol is a type of estradiol, a steroid hormone secreted from the ovaries of females of vertebrates, and is one of the follicle hormones extracted and crystallized from the ovaries and the placenta. Female gonads develop secondary to tertiary features, particularly in mammals, causing estrus. In addition, there is an action to enter the cell nucleus to promote the synthesis of new proteins, causing cell proliferation. In particular, beta estradiol is a highly variable reproductive hormone in a woman's body that counteracts the caffeine-like action, adenosine's original inhibitory effect, that women experience during the menstrual cycle, just before ovulation, shortly before ovulation. It is known to cause a mild excitement. In addition, it is also used as a prolactin releasing factor (PRF) to promote prolactin secretion.

In the meantime, early diagnosis of menopause and the diagnosis of unknown menstrual disorders have been made by measuring the pituitary hormones (FSH and LH) contained in the blood. However, as confirmed in the embodiment of the present invention, when the indicator of the present invention is used, the reliability of the concentration change after the estradiol gas phase is not only highly reliable, objective and rapid, but also after the vapor phase of estradiol. It is possible to evaluate abnormal increase or abnormal reduction of estradiol in ovary based on the change in concentration, which is helpful for diagnosing abnormality of ovary, early diagnosis of menopause, and unknown physiological impurity.

(2) The present invention measures the concentration of estradiol present in "saliva".

In the meantime, hormone concentrations were measured mainly in blood. From an endocrinology point of view, blood is not a hormone's target organ, but a simple hormone carrier. Most blood protein hormones (growth hormones, insulin, etc.) are well soluble in water and present in an activated form in the blood, but steroids produced in the adrenal cortex and gonads are insoluble in water and therefore combined with other media (carrier proteins). Is present in the blood. When steroids are combined with the carrier, they do not act on the hormone receptors in the target organs, so they are called 'inactive' and cannot induce physiological mechanisms. Most of the steroids in the blood (95-99%) are present in this inactive state and only a fraction (1-4%) are known to be in an unbound state (active state). Therefore, the concentration of steroids in the blood can be said to be the concentration of most inactive rather than the concentration of the hormone in the active state.

On the contrary, unlike the blood, the steroid contained in the saliva is not in an active state in which all of the steroids are bound to the carrier medium, and the active steroid contained in the extracelluar fluid is the salivary gland by passive diffusion. Will be moved to. Therefore, the steroid concentration contained in saliva is recognized as an active steroid contained in the tissue.

In addition, unlike blood, saliva has the advantage of being able to easily study the daily rhythm of the hormone of the individual because it can be easily collected by the general public in accordance with the sleep cycle of the individual without the help of the medical staff. In other words, using saliva has the advantage of easy access to the concentration of active steroids and the hormonal circadian rhythms, which were difficult to access to blood.

The present invention measures the concentration of estradiol present in saliva, the above advantages can be exerted effectively.

However, since the concentration of steroid contained in saliva is very low, such as 1 / 100-1 / 1000 of blood, there was a problem that cannot be analyzed with a commercially available hormone analysis kit.

(3) Therefore, the present invention is characterized by including the following saliva purification method to measure the trace estradiol concentration contained in saliva:

(a) treating the collected saliva with a strong base of pH 12-14;

(b) cooling after heating; And

(c) neutralization step; And

(d) mucus glycoprotein (mucosugar) removal step.

At this time, after the step (b), it is preferable to further include the step of removing the digestive enzymes and bacteria modified by pH and heat from the saliva by centrifugation, and after the step (d), mixing the buffer solution It is more preferable to carry out further.

First, saliva is collected from the subject.

Preferably, the females can be collected by spitting the saliva sample directly into the provided sampling tube at the time of waking up in the morning (just after waking up, within 1 minute), 30 minutes after waking up and 60 minutes after waking up. At this time, it is preferable not to gargle, smoke, drink a drink containing caffeine, and not eat or eat for one hour after waking up.

The saliva is then treated with a strong base solution having a pH of 12-14.

The strong base of pH 12-14 may be one strong base solution selected from the group consisting of NaOH, LiOH, KOH, Ca (OH) ₂ , preferably about 1N concentration.

The saliva treated with the strong base is heated to a temperature of 70 ° C. to 90 ° C. for 20 to 30 minutes, and then the temperature is rapidly lowered. In one embodiment of the present invention was heated for about 20 minutes at 80 ℃ and then quenched using an ice bath.

Thereafter, centrifugation is more preferably performed to remove digestive enzymes, bacteria, and the like contained in saliva.

As such, the present invention enables the detection of trace amounts of estradiol in saliva by purifying saliva using the "protein denaturation principle".

Protein denaturation means that proteins are affected by physical factors (heating, drying, stirring, pressure, X-rays, ultrasound, vibration, freezing), chemical factors (acids, bases, urea, organic solvents, heavy metals, surfactants) and enzymatic reactions. It refers to a phenomenon in which the unique higher-order structure (secondary, third-order, fourth-order) changes without changing the peptide structure. Among them, the temperature of the thermal denaturation is 60 ~ 70 ℃ in most proteins, the higher the temperature, the faster the denaturation rate.

In the present invention, particularly, proteins contained in saliva are denatured through rapid temperature changes and pH changes. And, through this, it has the effect of increasing the accuracy and shortening the analysis time.

In a typical saliva purification process, heat treatment is performed at about 60 ° C. for 2 hours, followed by cooling, followed by centrifugation and direct analysis (Howard et al., 1989; Tamate et al., 1997). When the temperature is gradually lowered, there is a property to return to the original state and there is a problem that the separation is difficult by centrifugation.

On the contrary, the present invention does not have this problem because the protein is denatured by exposure to high heat for a short time of 20-30 minutes after strong base treatment and quenching using an ice bath or the like.

Next, in the present invention, a neutralization step of adjusting the pH to the neutral pH is performed. The most representative method can be used to treat strong acid solution of pH 1-2. The strong acid having a pH of 1 to 2 may be used, for example, one selected from the group consisting of HCl, H ₂ SO ₄ , HNO ₃ , and the like, and may be used at a concentration of about 1N.

Preferably, the supernatant obtained after centrifugation is treated with a strong acid solution having a pH of 1 to 2 at a 1N concentration to adjust the neutral acidity (pH).

The obtained neutral sample is treated with a mucin glycoproteinase to remove mucus glycoprotein (mucosaccharide) in saliva.

Mucus is a biological liquid that can form a gel, which is a mixture of components that contain water and secretions of various cells. Mucins are also called mucus glycoproteins or epithelial glycoproteins, and a number of disaccharide side chains are linked by N- and O-links to the peptide core. It is a major component of the mucus and glycoconjugates that are characterized

Since the mucoglycoprotein (mucosaccharide) is not denatured by heat, various mucolytic agents or mucoglycoproteinases that can directly degrade it are used.

The mucin glycoprotein (mucosaccharide) degrading enzyme of the present invention may be any enzyme capable of degrading β-Nacetylhexosamine- [1 → 4] glycosidic bond, and in one embodiment of the present invention, hyaluronidase type 1 (H 3506, Sigmaaldrich chemical co.) 500 IU was used.

In addition, based on the mechanisms of action of mucin glycoproteinases, mucolytic agents or mucin glycoproteinases can often be classified into the following groups: proteases that break down the protein core of mucin glycoproteins (Eg, pronase, papain); Sulfhydryl compounds that cleave mucoprotein disulfide linkages; And detergents that destroy non-covalent bonds in mucus (eg, Triton X-100, Tween 20). Additional compounds may be used in this context, but not limited to, bile salts and surfactants such as sodium deoxycholate, sodium taurodeoxycholate, sodium glycolate, and Lysophosphatidylcholine.

In the present invention, the mucus glycoproteinase can be treated and reacted at about 37 ° C. for 10 to 15 minutes to break down the mucus glycoprotein in saliva.

Then, after heating the sample to a temperature of 70 ℃ ~ 90 ℃, the temperature is rapidly lowered to induce denaturation of the mucoglycoproteinase. In one embodiment of the present invention was heated for about 10 minutes at 80 ℃ and then quenched using an ice bath. Thereafter, the denatured mucin glycoproteinase is removed by centrifugation.

Finally, the obtained sample is mixed with a buffer to complete the saliva purification process.

In particular, the buffer preferably contains the same amount of gelatin as saliva. The gelatin induces a homogeneous distribution of trace steroids, estradiol and maintains optimal acidity even when saliva is exposed to outside air during the analysis.

The saliva purification process for measuring the estradiol concentration of the present invention takes about 25 minutes to 30 minutes in the strong base treatment, heating and quenching step, about 20 minutes to remove the mucosugar protein (muco sugar) and the enzyme used therein It takes ~ 25 minutes, so it corresponds to a total of 50 to 60 minutes. This has the effect of being much shorter in time compared to the conventional saliva purification process, which required heating for 2 hours or more.

On the other hand, the estradiol concentration measurement in the present invention, although there is no limitation, it is preferable to use a radioimmunoassay.

For example, enzyme immunoassay (EIA) using enzymes as markers, chemiluminescence immunoassay (CIA) with increased sensitivity with luminol or derivatives thereof or aminobutyl-ethylisoluminol (ABEI), or fluorescent label (fluorescent label) Fluorescence immunoassay (FIA), etc. may be used, but in particular, it is preferable to use radioimmunoassay.

Radioimmunoassays have many advantages, such as high sensitivity, precision, and accuracy, and allow for clear and clear detection after the reaction. Most preferably, radioimmunoassays can be used. For this purpose, a liquid-phased-double antibody method can be used. More detailed measurement procedures are available in the literature on hormone measurement using the liquid-dual antibody method (Salivary cortisol and DHEA levels in the Korean population; age-related differences, diurnal, and correlations with serum levels.Yonsei Med J. 2007; 48: 379 -88).

Most of the hormone analyzes mainly use immunoassay using an antigen-antibody reaction. Since various foreign substances act as a factor of disturbance in the antigen-antibody reaction process and often show different results from actual hormone levels, Immunoassays allow more accurate concentration measurements.

In the present invention, the measurement of the concentration of estradiol is preferably carried out after the gas phase, preferably from the saliva within 1 hour after the gas phase. More preferably, it may be made immediately after the weather, 30 minutes after the weather, at intervals of 1 hour after the weather.

That is, it is possible to provide information necessary for diagnosing the menstrual cycle and the biological activity through the change in the concentration of saliva estradiol during the first hour after the weather.

At this time, it is preferable to index the concentration change of estradiol within the first hour after wake-up of a woman having a normal menstrual cycle. That is, the indicators can be used to quickly and objectively determine whether the physiological activity of the target women is normal, and further determine whether the ovarian function and hormone secretion function are normal.

Accordingly, in another aspect, the present invention relates to a method for determining whether a menstrual cycle is normal, in order to provide information necessary for diagnosing a menstrual cycle, including:

(a) measuring the concentration of estradiol in saliva of a woman having a normal menstrual cycle by the method described above;

(b) setting a reference indicator with the measured concentration of estradiol;

(c) measuring the concentration of estradiol in the saliva of the woman to be tested by the method described above;

(d) comparing the obtained estradiol concentration with the reference indicator value set in step (b).

As described above, the present invention can quickly and objectively determine whether the menstrual cycle is normal by indexing the concentration change of estradiol after wake-up of a woman having a normal menstrual cycle.

The indicator is based on the change in estradiol concentration in women with a normal menstrual cycle for 3 months. Normally, 'normal menstruation' is given in an amount of 33 to 35ml (15 to 20 pads) every 24 to 32 days.

Immediately after waking up, saliva samples are collected at 30 minutes after waking up and 1 hour after waking up to determine the concentration (establishment) of estradiol in saliva, and by measuring and adjusting the individual's other habits and exercises, The best yields are well known to those skilled in the art. The sample may then be sent to a test facility and the results may be obtained, for example, via the internet. In addition, measurements can be made in situ and supplied to a Palm Pilot.

These samples analyze the estradiol concentration values at each time point using appropriate analytical techniques. Although estradiol in saliva is very low, saliva purified by the method of the present invention significantly increases the detection efficiency of the estradiol, and can be accurately measured using radioimmunoassay.

Since the sample does not need to be handled in any particular way and is stable at room temperature for a long time, there is a way to chart and place estradiol levels over time within 1 hour after vapor phase. In addition, the objective measurement is provided to the individual, allowing the individual to make judgments about changes in estradiol concentrations without having to consult with a healthcare professional to manage the trial and interpret the results. The time course data obtained is used to calculate three to six different values for the estradiol concentration of the individual. In one embodiment of the present invention, three values were calculated immediately after waking up, 30 minutes after waking up, and 1 hour after waking up. This data set is used as a reference value for an individual.

Then, it is determined by comparing whether the set of data values falls within the range of the indicators created by women with normal menstrual cycles. As a result of comparison, if the range does not fall within the range of the indicator, it may be determined that the normal menstrual cycle diagnosis result is not good.

In other words, it is possible to evaluate abnormal increase or abnormal decrease of estradiol in the ovary based on the change in the concentration of estradiol after the weather, which can provide information for diagnosing ovarian abnormality, early diagnosis of menopause, and unknown physiological impurity. Can be.

The present invention first identified (i) an increase in the concentration of estradiol, a sex hormone after morning weather, and (ii) a follicle capable of producing ovarian functions, such as sex hormones, by the concentration of estradiol. The presence or absence of perimenopausal and menopausal follicles that can produce sex hormones can be easily checked, and (iii) whether or not menstrual irregularities are caused by factors such as excessive stress, deficiency of influence, and excessive exercise. From a variety of perspectives, women may be able to collect information needed to diagnose women's biological activity.

[Example]

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

It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1 Screening of Screening Subjects

The women who collected saliva were checked for their therapeutic purpose, menstrual cycle, menopause and timing, and their weight, height and body mass index were measured. The women were screened for women who had a normal menstrual cycle for the last three months and menopausal women who did not have a normal menstrual cycle for last year.

However, ① women who take hormone replacement therapy, oral contraceptives, drugs containing tamoxifen or estrogen, and progesterone cortisol, ② women who are diagnosed with pregnancy, lactation, infertility, sexually transmitted diseases or ovarian function, ③ Women with severe disease such as cancer, diabetes, high / low blood pressure or abnormal body mass index (BMI ≤18 or ≥25) were excluded.

Women included in the screening were asked to spit 1 ml of saliva samples directly into the provided collection tubes each time they woke up in the morning (less than 1 minute after waking up), 30 minutes after waking up and 60 minutes after waking up.

Here, women included in the screening were not allowed to brush their teeth, smoke, drink intake of caffeine and eat snacks or meals for 1 hour after waking up, and the saliva collection time was marked on the surface of the collecting tube using an oil pen. .

Cortisol produced and secreted by the adrenal gland has a very pronounced circadian rhythm, especially at 30 minutes immediately after waking up. However, if saliva is not collected immediately after the weather, there is no difference from the cortisol concentration measured in the saliva 30 minutes after the weather. These results should be at least 2.8 nmol / L, which means that saliva was collected immediately after the weather. Therefore, after cortisol was quantitatively analyzed in the saliva of women included in the screening, only samples with a difference of more than 2.8 nmol / L were selected from samples taken immediately after the weather and 30 minutes after the weather. In addition, since sexual function is affected by internal and external stress factors, samples that are considered to have a shift in adrenal function due to extremely low or high cortisol concentration were excluded from screening.

Among the women who met the above criteria, 80 women with normal menstrual cycles, 57 postmenopausal women, and 13 menstrual irregularities whose cause was not identified for more than 60 days were included in the screening. Women with normal menstrual cycles were subdivided into three groups based on the menstrual start date:

phase Ⅰ: small group 2-3 days after menstrual period (menstrual women)

phase II: Small group 12-14 days after menstruation (late follicle-early ovulation women)

phase III: Small group 18-23 days after menstruation (early luteal female)

phase Ⅳ: Small group 25-28 days after menstruation (late luteal female)

Example 2: Saliva Tablets

The collected saliva sample was purified.

First, saliva each treated with 1N NaOH was heated at 80 ° C. for about 20 minutes and then quenched using an ice bath. Then, centrifugation was performed to remove digestive enzymes, bacteria, and the like contained in saliva. The supernatant was then collected and treated with 1N HCl to adjust the pH to neutral acidity.

Then, the mucosaccharide degrading enzyme hyaluronidase type 1 (H 3506, Sigmaaldrich chemical co.) 500 IU was treated and reacted at about 37 ° C. for 10 minutes to decompose the mucus glycoprotein (mucosaccharide) in saliva.

The sample was heated at 80 ° C. for about 10 minutes and then quenched using an ice bath. Thereafter, the denatured mucin glycoproteinase was removed by centrifugation.

Finally, the saliva purification process was completed by mixing saliva with a buffer containing 0.2% gelatin in the same amount.

Comparative Example 1 Comparison of Accuracy of Estradiol Detection in Pretreatment by Heat Treatment and Pretreatment by pH Control

Method 1: Pretreated saliva sample by heating at 56 ° C for 2 hours and centrifugation

Based on the existing saliva pretreatment method (Howard et al., 1989; Tamate et al., 1997), the sputum and foreign substances contained in the saliva were removed by centrifugation (3000g, 10 minutes), and the saliva sample was removed at 56 ° C. After heating for 2 hours, the mixture was left at room temperature (25 ° C.) for 10 minutes, and then centrifuged (3000 g, 20 minutes) to obtain a supernatant. (Approximately three hours)

 Method 2: Saliva Sample Pretreated by Acidity (pH) Change (Invention)

The saliva sample was first centrifuged (3000 g, 10 minutes) to remove sputum components first, and the supernatant was taken. 50 ul of a 1N NaOH solution was added to a supernatant of 200 ul and heated at 80 ° C. for 20 minutes. The test tube containing the heated saliva was quenched by standing in an ice bath (0 ° C.) for 5 minutes, centrifuged (12000 g, 10 minutes), and the supernatant was taken. The supernatant was treated with 50ul of 1N HCl and treated with hyaluronidase type 1 (H 3506, Sigmaaldrich chemical co.) 500 IU to remove mucosaccharides and left at 37 ° C. for 10 minutes. In order to remove the activity of the enzyme and remove it, it was heated for 10 minutes at 80 ℃ and centrifuged (12000g, 10 minutes). (Time required: about 1 hour 30 minutes)

The effects of estradiol quantitative analysis by radioimmunoassay on the treated saliva samples were investigated.

For this purpose, the saliva sample was first centrifuged (3000 g, 10 minutes) to remove the sputum component first, and the supernatant was taken. Dextrane-coated activated carbon (dextran coated 0.1% activated charcoal (w / v)) in the supernatant was stirred for 12 hours in a low temperature chamber maintained at 4 ° C to remove small molecular weight substances contained in saliva samples (charcoal stripped). saliva, CSS). The RIA method confirmed that the CSS contained residual steroid hormones. The CSS did not contain cortisol and sex hormones (below the detection range). After adding the estradiol (50 pg / 100 ul) of known concentration to CSS, the pretreatment of saliva samples according to method 1) and method 2) was carried out, and then the estradiol was quantitatively analyzed in the saliva samples. It was.

The results are shown in Table 1.

No pretreatment
(pg / 100 ul) Method 1
(pg / 100 ul) Method 2
(pg / 100 ul) Estradiol addition
(50 pg / 100ul) 56.2 ± 3.2 44.6 ± 2.2 47.9 ± 2.3

As shown in Table 1, steroid hormones were detected slightly higher than expected in saliva samples that were not pretreated. This is interpreted as the effect that the high-molecular weight substances (proteins, mucosaccharides, etc.) that could not be removed by activated carbon contained in the saliva sample interfered with the antigen-antibody reaction in hormonal quantitative analysis.

In the saliva sample pretreated by method 1, the sex hormone except cortisol was detected slightly lower than expected concentration, but it was significantly higher than the detection concentration of steroid hormones detected in the saliva sample pretreated by method 2). Detected.

In addition, in the case of method 2), steroid hormones are detected without significant difference from expected concentration, and it is believed that the decomposition of polysaccharides and the decomposition of polysaccharides improve the accuracy of the detection concentration with the removal of the large molecular weight material of method 2).

Example 3 Hormone Analysis

Saliva samples purified by the method of Example 2 were measured by radioimmunoassay, in particular liquid-phased-double antibody method.

Pure cortisol and estradiol used as standard samples were purchased from Sigma-Aldrich Co., Ltd., and dissolved in an organic solvent in which ethyl alcohol and propylene glycol were mixed in a 1: 1 ratio. Thus, a standard sample was prepared by further diluting with 0.1% gelatin-dissolved phosphate buffer solution (GPBS, pH = 7.2, GPBS). Cortisol-3-labeled cortisol-3- (O-carboxymethyloximino) -2-125Iiodohistamine and estradiol-3-estradiol-3- (O-carboxymethyloximino) -2-125Iiodohistamine were purchased from PerkinElmer Life and Analytical Sciences. Was used.

Antibodies of cortisol and estradiol were purchased from BioGenesis. Cortisol antibodies include aldosterone and 1.001% 11-deoxycorticosterone and 4.1%, deoxycortisol and 5.7%, 21-deoxycortisol and 0.5% corti. Correosterone (corticosterone) and other steroid hormones other than 2.1% cross-reacted less than 0.001%. Estradiol antibody cross-reacted 0.4% with estrone, 0.1% with estriol, 0.05% with dehydrotestosterone and less than 0.001% with other steroids. The detection range of cortisol is 1-250 nmol / L and the detection range of estradiol is 0.1-100 pmol / L. The within assay variation of the cortisol assay was 5.4% (n = 20) and the betweenwen assay variation was 8.8% (n = 16). The intra-analysis variation of estradiol was 9.7% (n = 20) and the inter-analysis variation was 13.4%.

In describing the quantitative analysis of hormones in saliva, a more detailed measurement is available in the literature on hormone measurement using the liquid-dual antibody method (Salivary cortisol and DHEA levels in the Korean population; age-related differences, diurnal, and correlations with serum levels). Yonsei Med J. 2007; 48: 379-88.

0.1 ml of standard and saliva samples were added to the test tube for measurement, and in order to measure the nonspecific response, the test tube without steroid hormone was replaced with GPBS instead of the antibody, and the standard sample contained steroid hormone. The test tube was added with charcoal-stripped saliva purified using activated carbon.

The total radiation dose of 125I-labeled cortisol or estradiol was approximately 20,000 cpm per tube and the antibody was diluted to bind 30-35%. The first reaction was 0.4ml per test tube, and the standard and saliva samples were reacted with antibody and radiolabeled cortisol or estradiol at 4 ° C. for 24 hours. Then, the reaction was further reacted at room temperature for 30 minutes for the second reaction, and then 0.1 ml of the secondary antibody was added to each test tube for 30 minutes. Then, 0.5 ml of 10% PEG was added, followed by reaction at room temperature for 15 minutes. After the reaction was completed, all the tubes were centrifuged at 1500 g at 4 ° C. for 20 minutes, the supernatant was removed using a vacuum pump, and the radiation dose of the precipitate remaining in each test tube was measured using a gamma ray meter (Cobra 5005, PerkinElmer Life and Analytic Sciences). ) Was measured. Each measured cortisol or estradiol concentration was calculated from a standard curve using RIASMART software (PerkinElmer Life and Analytic Sciences).

Example 4 Data Analysis

The distribution test of the concentrations of each of the coatsol and estradiol measured in saliva samples taken immediately after the gas phase (0 min), 30 minutes after the gas phase, and 60 minutes after the gas phase was performed. In addition, from the concentration of hormones obtained from each individual, ① the relative percentage of the highest concentration among 30 60 minutes after waking up, Peak / A0% [(highest hormone concentration / 0 minute hormone concentration) * 100], ② Relative percentage of estradiol concentration changed during 30 and 60 minutes after waking, based on estradiol concentration, MinInc% [(30 minutes hormone concentration + 60 minutes hormone concentration / 2) / concentration immediately after the weather]] * 100, two Statistical test between groups was performed by Mann Whitney test U-test, and statistical significance test of more than three groups was performed by Dunn's Multiple Comparison test of Kruskal-Wallis test. The concentrations of each hormone and the indicators were sequenced, and percentile coefficients were determined using SAS statistical programs.

The change in cortisol concentration immediately after waking in a woman with normal menstrual cycle,

As shown in FIG. 1A, the concentration of cortisol reached a normal value at 30 minutes after the weather, and an increase of 11.2 ± 5.8 nmol / L occurred immediately after the weather and 30 minutes of gas phase, and the increased range was 3.0-22.7 nmol / L. It was. MinInc% was 209.3 ± 107.0%.

These results indicate that saliva was collected at the correct time in women with normal menstrual cycles in the present invention and they had normal adrenal function.

Changes in estradiol levels immediately after wake-up in women with normal menstrual cycles,

Immediately after the weather, the concentration of estradiol contained in saliva collected 30 minutes after the weather and 60 minutes after the weather was measured. As shown in Fig. 2a, the estradiol concentrations of immediately after the vapor phase, 30 minutes after the gas phase, and 60 minutes after the gas phase were 1.4 ± 0.2, 2.8 ± 0.5, and 2.7 ± 0.3 nmol / L, respectively. The concentration was shown to increase significantly (p <0.05).

As shown in FIGS. 3A to 3C, the concentrations of estradiol immediately after vapor phase, after 30 minutes, and after 60 minutes ranged from 0.1 to 12.70, 0.1 to 25.90, and 0.1 to 16.20 pmol / L, and did not follow a normal distribution (p <0.001). ).

The estradiol peak / A0% and estradiol Minlnc% indicators of women who have a normal menstrual cycle are

 As shown in Figures 3a to 3c the concentration of estradiol over time existed in a wide range, which is due to the large difference in individual hormone concentrations, the difference is the menstrual cycle (follicular, ovulatory, luteal, menstrual) of each woman ) Is interpreted as different. In general, however, the estradiol concentration gradually increases after the weather regardless of the menstrual cycle. Therefore, the above indicators were calculated for each individual regardless of the menstrual cycle, and the indicators were sequenced and expressed as a percentage.

The peak / A0% and estradiol MinInc% indices of estradiol in women with normal menstrual cycles ranged from 135.4 to 427.1% and 115.6 to 309.8%, respectively. The mean (standard deviation) of these indicators was 239.1 ± 99.3% and 187.2 ± 64.1%.

This means that the peak concentration increased by 239.1% on the basis of estradiol concentration immediately after the weather, regardless of the menstrual cycle, and that the mean concentration during the first hour immediately after the weather increased by 187.2% over the ground. . The percentile coefficients of these indicators are shown in Table 2, which shows the main percentiles of the Peak / A0% and Estradiol MinInc% indicators of estradiol in women with normal menstrual cycles below.

5% 10% 25% 50% 75% 90% 95% Peak / A0% 135.4 143.0 166.2 215.1 284.4 372.1 437.1 MinInc% 115.6 118.4 142.5 171.3 214.8 287.4 309.8

In addition, the changes in the peak / A0% index and the estradiol MinInc% index of estradiol were investigated according to the menstrual cycle.

As a result, the peak / A0% index and the estradiol MinInc% index of estradiol were highest in the ovulation phase and relatively low in menstruation, as shown in FIGS. 4A and 4B.

Therefore, 12-14 days from the start of menstruation is most effective for determining normal menstrual cycle using Peak / A0% index of estradiol and MinInc% index of estradiol.

Example 5 blind test 1 using extraction indicators

Estimates of estradiol concentrations in postmenopausal women were performed in the same manner as the screening of the indicators described above, and blind tests were performed using the extracted indicators.

First, as shown in FIG. 1B, in the postmenopausal women (n = 58, age; 30 minutes after 61.7 ± 8.7 centuries, the concentration of cortisol reached a normal value and an increase of 8.3 ± 5.7 nmol / L was observed between 30 minutes after wake up and 30 minutes after wake up). And increased range was 2.8-24.4 nmol / L MinInc% was 173.8 ± 78.6%.

These results indicate that saliva was collected at the correct time in postmenopausal women and that they had normal adrenal function.

As shown in FIG. 2 b, estradiol concentrations in postmenopausal women were 1.7 ± 1.0, 1.3 ± 0.9, and 1.2 ± 0.9 pmol / L, respectively, immediately after waking, 30 minutes after waking, and 60 minutes after waking. However, the decrease tended to decrease gradually, but the decrease was not statistically significant. (p〉 0.05)

As a result of evaluating the change in estradiol concentration after menopause using peak / A0% and MinInc% indices, individual indicators of postmenopausal women were obtained and compared with women with normal menstrual cycles. None of the subjects were within the normal range of the above indicators.

Menopause women
Normal range (5 ~ 95% range)
Synthesis Peak / A0% Minlnc% Participants Peak / A0% Minlnc% 135.4 ~ 437.1 115.6 ~ 309.8 One 92.7 97.6 abnormal abnormal abnormal 2 78.1 81.3 abnormal abnormal abnormal 3 128.2 118.7 abnormal abnormal abnormal 4 73.0 55.9 abnormal abnormal abnormal 5 18.9 50.9 abnormal abnormal abnormal 6 134.9 117.3 abnormal abnormal abnormal 7 79.2 70.9 abnormal abnormal abnormal 8 42.7 43.0 abnormal abnormal abnormal 9 15.4 37.5 abnormal abnormal abnormal 10 111.8 115.5 abnormal normal abnormal 11 61.3 51.8 abnormal abnormal abnormal 12 108.3 113.6 abnormal abnormal abnormal 13 129.4 86.1 abnormal abnormal abnormal 14 67.6 83.3 abnormal abnormal abnormal 15 61.3 51.8 abnormal abnormal abnormal 16 32.6 72.2 abnormal abnormal abnormal 17 115.9 104.6 abnormal abnormal abnormal 18 61.6 89.8 abnormal abnormal abnormal 19 49.2 50.0 abnormal abnormal abnormal 20 106.2 83.9 abnormal abnormal abnormal 21 83.1 65.9 abnormal abnormal abnormal 22 94.9 81.9 abnormal abnormal abnormal 23 42.4 63.0 abnormal abnormal abnormal 24 106.2 108.2 abnormal abnormal abnormal 25 119.0 86.9 abnormal abnormal abnormal 26 101.8 98.2 abnormal abnormal abnormal 27 58.0 60.9 abnormal abnormal abnormal 28 3.7 13.2 abnormal abnormal abnormal 29 58.8 102.6 abnormal abnormal abnormal 30 26.6 63.6 abnormal abnormal abnormal 31 45.6 60.6 abnormal abnormal abnormal 32 42.3 75.3 abnormal abnormal abnormal 33 105.5 101.6 abnormal abnormal abnormal 34 26.7 55.7 abnormal abnormal abnormal 35 58.1 48.0 abnormal abnormal abnormal 36 66.4 71.7 abnormal abnormal abnormal 37 24.0 41.3 abnormal abnormal abnormal 38 75.0 53.7 abnormal abnormal abnormal 39 55.7 80.5 abnormal abnormal abnormal 40 34.6 62.4 abnormal abnormal abnormal 41 63.1 75.2 abnormal abnormal abnormal 42 97.7 83.5 abnormal abnormal abnormal 43 127.7 115.2 abnormal abnormal abnormal 44 105.1 92.7 abnormal abnormal abnormal 45 36.3 45.8 abnormal abnormal abnormal 46 49.6 99.3 abnormal abnormal abnormal 47 144.4 99.3 normal abnormal abnormal 48 54.0 86.2 abnormal abnormal abnormal 49 72.0 60.7 abnormal abnormal abnormal 50 34.2 51.0 abnormal abnormal abnormal 51 52.4 51.1 abnormal abnormal abnormal 52 106.2 83.9 abnormal abnormal abnormal 53 65.3 70.3 abnormal abnormal abnormal 54 111.8 115.5 abnormal abnormal abnormal 55 69.4 76.9 abnormal abnormal abnormal 56 118.0 119.6 abnormal abnormal abnormal

This is because the Peak / A0% index and the MinInc% index are significantly different from those in the normal menstrual cycle as shown in FIG. (p <0.05)

Example 6 blind test 2 using extraction indicator

In the same way as the screening of the above-described indicators, estradiol concentrations were measured in women with dysmenorrhea (women who had delayed menstruation for more than 60 days, n = 13, age; 31.5 ± 12.9 years), Blind test was performed. First, as shown in FIG. 1C, the concentration of cortisol reached a normal value at 30 minutes after the weather, and the concentration increased from 12.5 ± 9.1 nmol / L immediately after the weather to 30 minutes, and the range was 2.9-28.6 nmol / L. MinInc% was 176.1 ± 75.5%.

These results indicate that saliva was collected at the correct time in women with menstrual disorders and they had normal adrenal function.

In addition, as shown in FIG. 2C, estradiol concentrations in women were 1.1 ± 0.5, 0.8 ± 0.6, and 0.8 ± 0.6 pmol / L, respectively, immediately after waking, 30 minutes, and 60 minutes, respectively. Was not statistically significant (p> 0.05).

As shown in Table 4 below, which shows the evaluation of estradiol concentration change after wakeup using peak / A0% and MinInc% indices, the individual indices of dysmenorrhea were obtained and compared with women with normal menstrual cycles. All 13 impure women were not within the normal range of the above indicators.

Menstrual disorder Normal range (5% -95% range) Peak / A0% MinInc% Synthesis Participant ID Peak / A0% MinInc% 135.4-437.1 115.6-309.8 One 114.9 99.3 abnormal abnormal abnormal 2 117.4 100.6 abnormal abnormal abnormal 3 128.8 114.1 abnormal abnormal abnormal 4 18.8 56.8 abnormal abnormal abnormal 5 78.1 89.1 abnormal abnormal abnormal 6 81.4 85.1 abnormal abnormal abnormal 7 50.3 53.7 abnormal abnormal abnormal 8 36.9 39.7 abnormal abnormal abnormal 9 101.5 121.1 abnormal abnormal abnormal 10 44.8 36.5 abnormal abnormal abnormal 11 100.8 92.1 abnormal abnormal abnormal 12 39.8 33.1 abnormal abnormal abnormal 13 89.1 90.6 abnormal abnormal abnormal

This is because the Peak / A0% and MinInc% indices are significantly different from women with normal menstrual cycles, as shown in FIG. 5. (p <0.05)

As can be seen from the test results of the present invention, when the indicator of the present invention is used, it is possible to determine whether the concentration change after estradiol concentration is normal, objective and rapid, and also after the estradiol after meteorology. It is possible to evaluate abnormal increase or abnormal reduction of estradiol in ovary based on the change in concentration, which is helpful for diagnosing abnormality of ovary, early diagnosis of menopause, and unknown physiological impurity.

Claims (20)

To provide the information necessary to diagnose the menstrual cycle, a method of measuring the concentration of estradiol comprising the following steps:
(a) treating the collected saliva with a strong base of pH 12-14;
(b) cooling after heating;
(c) neutralization step; And
(d) mucus glycoprotein (mucosugar) removal step.
The method according to claim 1, wherein the collection of saliva in step (a) is performed within one hour after the gaseous phase.
The method of claim 2, wherein in the step (a), the saliva is collected immediately after the weather, 30 minutes after the weather, and 1 hour after the weather.
The method of claim 1, wherein the strong base having a pH of 12 to 14 in step (a) is one selected from the group consisting of NaOH, LiOH, KOH, Ca (OH) ₂ .
The method of claim 1, wherein the heating in step (b) is characterized in that at a temperature of 70 ~ 90 ℃.
The method of claim 1, wherein the cooling in step (b) is performed by quenching using an ice bath.
The method of claim 1, further comprising, after step (b), removing digestive enzymes and bacteria from saliva by centrifugation.
The method of claim 1, wherein the step (c) neutralization is carried out by treating a strong acid of pH 1-2.
The method of claim 8, wherein the strong acid having a pH of 1 to 2 is one selected from the group consisting of HCl, H ₂ SO ₄ , and HNO ₃ .
The method of claim 1, wherein in step (d), the mucus glycoprotein (mucosugar) removal is performed by treating the mucoglycoprotein (mucosaccharide) degrading enzyme.
11. The method of claim 10, wherein the mucoglycoprotein (mucosaccharide) degrading enzyme is characterized in that to decompose Nacetylhexosamine- [1 → 4] glycosidic bond.
The method of claim 1, further comprising removing mucus glycoprotein (mucosaccharide) degrading enzyme by centrifugation after removing mucus glycoprotein (mucosaccharide) in step (d).
The method of claim 12, further comprising the step of mixing the buffer after removing the mucoglycoprotein (mucosugar) degrading enzyme.
The method of claim 13, wherein the buffer comprises the same amount of gelatin as saliva.
The method of claim 1, wherein the concentration of estradiol is measured using radioimmunoassay.
To provide information necessary for diagnosing physiological activity, a method for analyzing the concentration of estradiol comprising:
(a) measuring the concentration of estradiol in saliva of a woman having a normal menstrual cycle by the method of claim 1;
(b) setting a reference indicator to the measured concentration of estradiol;
(c) measuring the concentration of estradiol in the saliva of the test subject woman by the method of claim 1;
(d) comparing the obtained estradiol concentration with the reference indicator value set in step (b).
The method of claim 16, wherein the concentration of estradiol is measured from saliva collected within 1 hour after the gas phase.
The method of claim 17, wherein the concentration of estradiol is measured from saliva collected immediately after the gas phase, 30 minutes after the gas phase, and 1 hour after the gas phase.
17. The method of claim 16, wherein the concentration analysis of estradiol is performed on the change in the concentration of estradiol measured from saliva collected immediately after the vapor phase, 30 minutes after the vapor phase, and 1 hour after the vapor phase.
20. The method of claim 19, wherein the change in the concentration of estradiol in the saliva of the test subject is out of the range of the reference indicator set as the change in concentration of the female having a normal menstrual cycle.

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