Method for aromatase activity test

FIELD: medicine.

SUBSTANCE: blood serum is examined for lutropin, follitropin, oestradiol, total testosterone and free testosterone levels before and 48 hours after the oral administration of 450 aromatase inhibitor, letrozole 10 mg, and the variations of lutropin and follitropin, as well as of the oestradiol/total testosterone and oestradiol/free testosterone relations are used to asses aromatase activity in points assigned by a certain way to consider low aromatase activity shown by total score 0 to 7 points, normal aromatase activity - 8 to 14 points, and high aromatase activity - 15 points or more.

EFFECT: high testing accuracy and simplicity of the implementation.

8 tbl, 3 ex

The invention relates to medicine, namely to the endocrinology of reproduction, and can be used to assess aromatase activity.

Aromatase P450 - enzyme complex that catalyzes the conversion of C19-androgens into estrogens, realizing the biosynthesis of estrone from Androstenedione and estradiol from testosterone. The human aromatase P450 are found in many tissues and organs such as the gonads, brain, adipose tissue, placenta, liver, skin, bones, blood vessels, endometrium, and in endometrioid heterotopia, tissue leiomyoma, endometrial cancer and breast cancer. Gene aromatase man is localized on the 15th chromosome. In the literature there are descriptions of patients with a defect of the gene aromatase P450 CYP19. The clinical picture in patients with a female depends on the activity of this enzyme and is expressed prenatal virilization of varying severity, hypergonadotropic hypogonadism, underdeveloped secondary sexual characteristics, primary amenorrhea, infertility. In addition, a partial defect of the gene aromatase P450 CYP19 may be responsible for insufficient production of estradiol dominant follicle and to play a role in the pathogenesis of anovulation and infertility.

There is a method of assessment aromatase activity by mapping the content of androgens and estrogens in the blood or follicular who Arnau fluid {Misharin E.V. The content of estrone, Androstenedione and β-endorphin in women with obesity and hormonal ovarian failure: author. Dis. ... candles. the honey. Sciences. - SPb., 1993]. However, this method is acceptable for indirect estimation aromatase activity in groups of patients, but not acceptable to determine a particular patient.

There is a method of assessment aromatase activity by protein Western blot turns [Lin L. Variable phenotypes associated with aromatase (CYP19) insufficiency in humans / L. Lin [et al.] // J. Clin. Endocrinol. Metab. - 2007. - V.92. P.982-990]. In the process of determining aromatase activity is used electrophoresis of proteins in polyacrylamide gel for separation denaturirovannykh polypeptides in length or three-dimensional structure of native proteins. Next, proteins are transferred to nitrocellulose membrane, where aromatase is determined using specific antibodies. This method is distinguished by the complexity and the need for special equipment.

There is a method of assessment aromatase activity by immunohistochemical studies in various tissues and organs [Suzuki T. Quantitation of P450 aromatase immunoreactivity in human ovary during the menstrual cycle: the relationship between the enzyme activity and immunointensity / T.Suzuki [et al.] // J.Histochem. Cytochem. - 1994. - Vol.42, N12. - P.1565-1573]. The method is based on the binding of unlabelled primary rabbit polyclonal antibodies to aromatase P450 with the formation of the immune complex. Further immune Sands is with visualized using a labeled secondary antibody, the primary antibodies used for secondary antigens. Immunohistochemistry performed on paraffin sections. The tissue section thickness of 5 μm, placed on glass slides, covered with a film of poly-L-lysine. Quantitative evaluation of the results of immunohistochemical reactions performed on micrographs obtained using a fixation system for microscopic images, consisting of a microscope, digital camera, personal computer, software, AST-1, version 2.12 and Videotest-Morphology 5.0". The photography is carried at 400x magnification (the eyepiece 10x, 40x lens), a complete closure of the aperture diaphragm, when raised contendere, in Photo mode, the exposure time 1/20 sec, the sensitivity of the maximum image size of 1280×1024 pixels, the graphic image format JPEG (normal). Determined aromataza activity in the granular cells of the dominant follicle, using a measure of the integral optical density and the number of granular cells in the object.

The disadvantage of this method is the need for invasive procedures, the definition aromatase activity only in the ovaries, the extraordinary complexity.

The technical result of the invention is to increase the efficiency of the method, obtaining accurate data about the level aromatase activity more simple among the properties.

This technical result is achieved in that in the method of estimating aromatase activity according to the invention determine the content of the hormones LH (lotensinbuy hormone), FSH (follicle stimulating hormone), estradiol, total testosterone and free testosterone before and 48 hours after oral administration of 10 mg aromatase inhibitor of letrozole and change of LH, FSH, and relations estradiol/total testosterone and estradiol/free testosterone appreciate aromatase activity points. Assign points in a certain way (table 1), this estimate was reduced aromatase activity, normal and high.

Aromatase activity assessed according to the amount of points:

0-7 points - reduced aromataza activity; 8-14 points - normal; 15 points or more - increased.

To determine the evaluation criteria were studied 13 women of reproductive age (mean age was 28±0,7 years) with saved ovulatory cycle confirmed by ultrasound examination of the pelvic organs and the level of progesterone in the blood on the 20-21st day of the menstrual cycle. Sample with letrozole held on the 2nd day of the menstrual cycle. The content of gonadotropins and sex steroids in the blood was determined to receive letrozole and 2-nd, 3-th and 4-th day after taking the drug. The most pronounced changes (decrease in the level of estradiol in the blood, increased levels of testosterone, FSH and LH) had on day 3 (48 hours) after administration of letrozole.

Table 2 presents data of the mountains of the national survey (M±m) and limits of confidence intervals (p=0.05) before and after administration of 10 mg of letrozole women of reproductive age with a full ovulatory cycle (n=13).

As follows from the data presented in table 2, under the influence of letrozole in healthy women, there is a decrease of estradiol in the blood by 44.2±9.8 pmol/l (26,6±5,4%). According to the mechanism of negative feedback on the 3-day samples, there was an increase in blood FSH 4.1±0,6 IU/l (74,9±11,8%) and an increase in LH 3.8±0,6 IU/l (146,9±26.8 per cent). However, changes in the level of testosterone in the blood under the influence of letrozole was not statistically significant. Given the breadth of changes in androgen and estrogen under the influence of letrozole, to assess the response to drug use attitudes estradiol/total testosterone and estradiol/free testosterone. In the group of healthy women on day 3 sample limit of confidence interval (p=0.05), the relationship estradiol/testosterone ranged from 88,2 to 53.6 and relationships estradiol/free testosterone from 47,8 to 11.7.

The method is as follows.

At 9 a.m. on the 2nd day of the menstrual cycle from the cubital vein of the patient collect 10 ml of blood for hormonal studies. Then the patient takes 10 mg aromatase inhibitor of letrozole per os. After 48 hours produce repeated blood sampling. In both portions determine blood levels of FSH, LH, estradiol, testosterone, and free testosterone by an enzyme immunoassay. About ar is Machesney activity is judged using a point system, taking into account the dynamics of the estimated parameters, the amount of points is reduced when aromatase activity from 0 to 7 points; under normal aromatase activity from 8 to 14 points; at high aromatase activity from 15 points or more.

The essence of the method is illustrated by the following clinical examples.

Example 1. Patient I., 31, chronic normogonadotropic anovulation and infertility.

Reaction to letrozole patient And. presented in table 3. As follows from table 3, in response to receiving letrozole in this patient occurred paradoxical increase in the level of estradiol in the blood with 91.7 pmol/l to 113,7 pmol/l (23,9%), an absolute increase of estradiol was equal to 22 pmol/L. the Level of testosterone increased from 0.1 nmol/l to 1.0 nmol/l, i.e. by 0.9 nmol/l, the content of free, metabolically active testosterone remained practically at the same level of 0.6 pmol/l, compared with baseline levels (0.7 pmol/l). The content of LH in the blood increased from 1.6 IU/l to 2.3 IU/l (37,5%), while the absolute increase was only 0.6 IU/L. Response of FSH was even less pronounced (4,2%), absolute growth rate was 0.2 IU/L. the Ratio of estradiol/testosterone on day 3 of the sample was equal to 113,7, estradiol/free-testosterone - 189,5 that significantly exceeded the corresponding figures in healthy women (table 3).