KR101068993B1 - Evaluation of metabolic differences between urine samples by quantitative steroid signature - Google Patents

Abstract

The present invention relates to an analytical method capable of simultaneously measuring and comparing the activity of enzymes involved in their metabolism as well as the concentration of each steroid hormone by treating the measured concentrations of steroid hormones in urine in a statistical manner.

The present invention can determine the overall metabolic distribution tendency by measuring the concentration of steroid hormones in the urine as well as the ratio between steroids involved in metabolism. Therefore, it can be used as a method for evaluating endocrine diseases and biological changes related to steroid hormone metabolism, such as enlarged prostate, and can extend its application to various diseases of hormone dependency.

Urine, Steroids, Enzyme Activity, Prostatic Hyperplasia

Description

Evaluation of metabolic differences between urine samples by quantitative steroid signature}

The present invention relates to an analytical method capable of simultaneously measuring and comparing the activity of enzymes involved in their metabolism as well as the concentration of each steroid hormone by treating the measured concentrations of steroid hormones in urine in a statistical manner.

Analytical steroid hormones are composed of male hormone (androgen), female hormone (estrogen), progestin, corticosteroid (corticoid), which are synthesized by the action of various enzymes using cholesterol (cholesterol) as a precursor Sterols and the like. Steroid hormones that control the endocrine system cause diseases when biological changes occur due to enzyme deficiency or excess of enzymes involved in their metabolism. Recently, changes in related steroid hormones in certain physiological and pathological conditions After analyzing the results and interpreting them in relation to physiological functions, studies are being actively conducted to investigate the causes of disease development.

In general, the analysis of steroid hormones is limited to analyzing specific steroid hormones related to the target disease rather than simultaneously extracting and analyzing a wide range of steroid hormones in one sample pretreatment. To explain, it is necessary to understand the overall metabolic trend of steroid hormones. This method has the advantage of being able to simultaneously quantify and identify a number of compounds produced and secreted from each organ in the body, as well as to measure the activity of related enzymes by changing the relative concentration ratios between compounds [ Endo. J. , 2003, 50: 783-792; Endocrinology , 2007, 148: 2505-2517. The overall metabolic trend of steroid hormones is shown in FIG. 1.

Therefore, a wide range of assays have been developed for the simultaneous and quantitative evaluation of various steroid hormones present in trace amounts in urine samples [ Endo. J. , 2003, 50: 783-792], whereby 1) measuring relative changes between highly active steroids, 2) identifying changes in metabolic balance between precursors and products on metabolic pathways, and 3) appearing on metabolic pathways. By measuring the activity of enzymes related thereto from the imbalance, it is possible to comprehensively evaluate the metabolic processes of endogenous substances according to disease.

However, using a large amount of data obtained by analyzing various steroid hormones simultaneously, it is a lot of time and effort to compare the activities of various types of enzymes involved in their metabolism as well as the changes of all steroid hormones in patients with normal diseases and normal subjects. And a high degree of expertise in the interpretation of the quantitative results obtained. In addition, it enzymes generally used for measuring the activity of the relevant enzyme immunoaffinity methods are also impossible to measure the various kinds of related enzymes at the same time, it has a low reproducibility disadvantages of the results by a number of various experimental conditions [Cancer Epidemiol. Biomarkers Prev ., 2007, 16: 1004-1008; Ann. Clin. Biochem ., 2008, 45: 380-388.

In recent years, a large amount of data the statistical methods which way by multivariate analysis to compare easily the normal subjects and patients showing disease and illustrates the quantitative changes of the body metabolites are being used to complement the above-mentioned problems, but [Cancer Res ., 2006, 66: 10795-10804; Annu. Rev. Pharmacol. Toxicol ., 2008, 48: 653-683], there is no method to simultaneously measure the overall metabolic trends of steroid hormone metabolism and related enzyme activity associated with endocrine diseases such as prostate hyperplasia.

In view of the above, the present inventors simultaneously extract about 70 kinds of steroid hormones from urine, enzymatic hydrolysis of the extracts, and re-extraction using an organic solvent, followed by gas chromatography through trimethylsilyl derivatization. Complete the present invention by measuring the concentration of steroid hormones in urine using a graph-mass spectrometer and plotting their distribution patterns in an array form for evaluating the relative concentrations of each steroid hormone using multivariate analysis, a statistical method. It became.

Accordingly, an object of the present invention is to provide information necessary for diagnosing prostate disease by diagramming the relative concentrations and ratios of the measured urine steroid hormones.

The present invention,

As a method of analyzing the differentiation of steroid hormone metabolites in two sample groups,

Simultaneously extracting steroid hormone biological metabolites including male hormones, female hormones, progesterone, sterols, and corticosteroids in urine by solid phase extraction;

Re-extracting the extract with an organic solvent after enzymatic hydrolysis;

Trimethylsilyl derivatization of the reextract and measuring the concentration of steroid hormone biometabolites in urine using gas chromatography-mass spectrometry; And

Comparing and confirming concentrations of steroid hormone biometabolites between two sample groups;

Characterized by the differential analysis method of the steroid hormone biological metabolite between two sample groups comprising a.

In addition, the present invention,

As a method of analyzing the differentiation of steroid hormone metabolites in two sample groups,

Simultaneously extracting steroid hormone biological metabolites including male hormones, female hormones, progesterone, sterols, and corticosteroids in urine by solid phase extraction;

Re-extracting the extract with an organic solvent after enzymatic hydrolysis;

Trimethylsilyl derivatization of the reextract and measuring the concentration of steroid hormone biometabolites in urine using gas chromatography-mass spectrometry;

Distinguishing the precursor from the product in the steroid hormone metabolites, calculating the concentration ratio of the product / precursor, and comparing the ratio between the two sample groups; And

Schematizing the product / precursor concentration ratio using a partial least-squares discriminant analysis (PLS-DA) method

Another feature of the method for analyzing the difference between the steroid hormone biometabolites between the two sample groups comprising.

The present invention can determine the overall metabolic distribution tendency by measuring the concentration of steroid hormones in the urine as well as the ratio between steroids involved in metabolism. Therefore, it can be used as a method for evaluating endocrine diseases and biological changes related to steroid hormone metabolism, such as enlarged prostate gland, and can be extended to a variety of hormone-dependent diseases.

The present invention will be described in detail as follows.

The present invention relates to a method of determining the concentration of steroid hormones in urine and the distribution of the steroids in a statistical manner to identify biologic changes caused by hormone-dependent diseases including prostatic hyperplasia.

The amount of urine which is a biological sample to be used is preferably 0.2 to 5 mL, more preferably 0.2 to 2 mL. If it is less than 0.2 mL, there is a problem in the detection limit for the analysis of some steroid metabolites. If it is larger than 5 mL, the detection concentration of some compounds, such as cholesterol, is too high, and a lot of biological samples are collected. .

First, steroid hormones are extracted by solid phase extraction. In the present invention, a high recovery rate and reproducible assay results for many compounds present in vivo [Rapid Commun. Mass Spectrom., 2002, 16:22 1-2228] was used.

Steroid hormones adsorbed on the adsorbent were eluted with methanol and the eluate was evaporated to dryness.

The extract thus obtained is enzymatic hydrolyzed at 55 ° C. for 3 hours under pH 5.2 and re-extracted with an organic solvent. The enzyme used for hydrolysis may be glucuronidase or a mixture of arylsulfatase / glucuronidase, which is used to simultaneously decompose glucuronic acid and sulfuric acid polymers in urine samples. It is preferable to use a mixed solution of kurondase, and glucuronidase is preferred to more accurately evaluate steroid hormones having a specific form (3β-hydroxy-5-ene). It is preferable to use a mixed solvent of ethyl acetate (EA) and normal hexane ( n- hexane) as the organic solvent. More preferably, a mixed solvent in which ethyl acetate and normal hexane are mixed in a volume ratio of 1: 1 to 1: 2 is used. The reextract is evaporated to dryness, and then subjected to a TMS derivatization reaction to be analyzed by a gas chromatograph-mass spectrometer.

The height ratio of the steroid hormones in urine obtained by the gas chromatograph-mass spectrometer was measured by comparing the concentrations of the steroids with the concentrations of the standard substance, and then the concentrations of the two sample groups were compared to confirm the differentiation of the steroid hormone biological metabolites. Analyze

In particular, the multivariate analysis can be used to plot the distribution of relative concentrations of steroid hormones in individual urine, thereby analyzing the differences in steroid hormone biometabolites between two sample groups.

In addition, the precursor and the progenitor in the steroid hormone biometabolites are distinguished, the concentration ratio of the progenitor / precursor is calculated, the proportion of the prostate hypertrophy patients and the normal person is compared, and the ratio is plotted using a multivariate analysis method. Differentiation of steroid hormone metabolites between sample groups can be analyzed and the relationship between enzyme activity involved in steroid metabolism and prostate disease can be identified.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited to the following examples.

Example 1 Analysis of Steroid Hormones

1) Solid phase extraction

In vitro, 8 internal standards ( d ₃ -testosterone, 1 μg / mL; d ₄ -androsterone, 10 μg / mL; methyltestosterone, 10 μg / mL; d ₄ -17β-estradiol, 1 μg / mL; d ₄ 20 μl of a mixed solution of -cortisol, 5 μg / mL; d ₆ -cholesterol, 10 μg / mL; d ₉ -progesterone, 10 μg / mL; d ₈ -17a-OH-progesterone, 5 μg / mL) After adding 2 mL, a solid phase extraction using an Oasis HLB cartridge was used [Oasis HLB, 60 mg, Waters, Co., Milford, MA, USA]. Methanol and distilled water were flowed twice, 2 mL each, and the urine sample was flowed. Then, 2 mL of distilled water was flowed to remove impurities. Thereafter, the mixture was poured twice into 2 mL of methanol to elute the steroid hormones adsorbed on the cartridge, and the eluate was evaporated using nitrogen gas at 40 ° C.

2) hydrolysis

1 mL of 0.2 M sodium acetate buffer is added to the extract of 1) to prevent degradation by oxidation of catechol-type female hormones (catechol estrogens) metabolites. To this, 100 μl of 0.2% ascorbic acid and 50 μl of arylsulfatase / glucuronidase were further added, followed by hydrolysis at 55 ° C. for 3 hours.

3) Liquid phase extraction

The hydrolyzate of 2) was extracted using a solvent in which ethyl acetate and normal hexane were mixed in a volume ratio of 2: 3. Although ether and tetrahydrofuran (THF) can be used as a solvent, it is preferable to use a mixed solvent of ethyl acetate and normal hexane from the viewpoint of extraction efficiency. The eluate was evaporated using nitrogen gas at 40 ° C., and then sufficiently dried in a vacuum desiccator for at least 30 minutes.

4) Gas Chromatography-Mass Spectrometry

After the process of 3), to apply the derivatization method in order to use gas chromatography-mass spectrometry, MSTFA ( N- methyl- N- trifluorotrimethylsilyl acetamide) / NH ₄ I (ammonium iodide) / DTE (dithioerythritol) ( 40 μl of the mixture for 500: 4: 2, v / w / w) was added and reacted at 60 ° C. for 20 minutes.

Selected ion monitoring (SIM) to select and detect only characteristic ions using an Agilent 6890 series Gas Chromatograph connected to a mass spectrometer (Agilent's 5975 Mass Selective Detector) for the analysis of steroid hormones The mass spectrometry was analyzed using a scan mode with a mass range of 50 m / z. The separation of steroid hormones was performed using Agilent's Ultra-1 as a fused-silica capillary column. The length of the column was 25 m, the inner diameter was 0.2 mm, and the film thickness was 0.33 μm. The flow rate of the carrier gas helium was 1.0 mL / min. The temperature of the injector was 280 ° C., and the sample injection was performed using a split mode of 10: 1. The temperature conditions for the analysis were as follows. The starting temperature of the oven was 215 ° C. and the temperature was raised to 260 ° C. at a rate of 1 ° C./min and finally raised to 320 ° C. at a rate of 15 ° C./min and maintained for 1 minute. The ionization method used an electron impact method (EI), and the electron energy used was 70 eV.

In the selective ion detection method (SIM) used for the quantitative analysis of steroid hormones, the concentration was calculated by comparing the height ratio of the metabolites with the height ratio of the standard, and the symbol and mass of the analytes used to distinguish the metabolites. Quantitative ions on the spectrum and retention times in the analytical column are shown in Table 1 below.

Steroid hormones
sign Fixed ion Retention time
(minute) Male Hormone
Dihydrotestosterone
Epidihydrotestosterone
Dehydroepiandrosterone
Testosterone
Epitestosterone
5α-androstane-3α, 17β-diol
5α-androstane-3β, 17β-diol
5β-androstane-3α, 17β-diol
5β-androstane-3α, 17α-diol
5β-androstane-3β, 17α-diol
5α-androstane-3β, 17α-diol
Androstenedione
Androstenediol
Androsterone
Etiocholanolone
11-keto-androsterone
11-keto-etiocholanolone
11β-OH-androsterone
11β-OH-etiocholanolone
16α-OH-dehydroepiandrosterone
Epiandrosterone
5α-Androstanedione
DHT
Epi-DHT
DHEA
T
Epi-t
ααβ-diol
αββ-diol
βαβ-diol
βαα-diol
ββα-diol
αβα-diol
A-dione
A-diol
An
Etio
11-keto-an
11-keto-Etio
11β-OH-An
11β-OH-Etio
16α-OH-DHEA
Epi-an

434
434
432
432
432
241
241
256
256
256
241
430
434
434
434
520
520
522
522
505
419
432
19.43
17.54
17.83
20.68
18.87
16.08
18.95
16.14
12.25
12.94
17.13
19.90
18.69
15.34
15.50
17.65
17.70
20.83
21.15
28.62
18.19
18.71
Female hormone
Estrone
17β-estradiol
17α-estradiol
Estriol
2-hydroxyestrone
2-hydroxyestradiol
4-hydroxyestrone
4-hydroxyestradiol
16α-hydroxyestrone
16-keto-estradiol
2-methoxyestrone
2-methoxyestradiol
4-methoxyestradiol
17-Epiestriol
16-Epiestriol
2-hydroxyestriol
2-methoxyestradiol-3-methylether
2-hydroxyestradiol-3-methylether

E1
17β-E2
17α-E2
E3
2-OH-E1
2-OH-E2
4-OH-E1
4-OH-E2
16α-OH-E1
16-keto-E2
2-MeO-E1
2-MeO-E2
4-MeO-E2
17-epi-E3
16-epi-E3
2-OH-E3


414
416
416
504
502
504
502
504
487
487
444
446
446
504
504
592
388
446
19.23
20.08
18.71
29.96
26.00
26.84
27.46
28.55
30.28
30.28
24.67
25.65
23.83
29.29
31.29
37.36
23.48
25.18 Progesterone
Pregnenolone
Progesterone
5β-dihydroprogesterone
5α-dihydroprogesterone
20α-dihydroprogesterone
Epipregnanolone
Pregnanolone
Allopregnanolone
Pregnanediol
Pregnanetriol
17α-hydroxypregnenolone
17α-hydroxyprogesterone
11β-hydroxyprogesterone

Preg
Prog
5β-DHP
5α-DHP
20α-DHP
Epi-p-one
P-one
Allo-p-one
P-diol
P-triol
17α-OH-Preg
17β-OH-Prog
11β-OH-Prog
445
458
445
445
445
447
447
462
117
435
548
546
531

27.48
30.02
20.11
28.71
30.40
23.43
23.73
24.07
25.15
26.42
32.74
35.83
41.41 Sterol
Cholesterol
24S-hydroxycholesterol

Chol
24S-OH-Chol
458
413
40.90
49.00 Corticosteroids
Cortisol
Allodihydrocortisol
Corticosterone
Allodihydrocorticosterone
Dehydrodeoxycorticosterone
11-Deoxycorticosterone
11-Deoxycortisol
Cortisone
Allotetrahydrocortisol
21-Deoxycortisol
11-dehydrocorticosterone
Tetrahydrodeoxycortisol
Tetrahydrocortisone
Tetrahydrocortisol
Tetrahydrodeoxycorticosterone
F
Allo-DHF
B
Allo-DHB
DHDOC
11-DeoxyB
11-DeoxyF
E
Allo-THF
21-DeoxyF
11-DehydroB
THS
THE
THF
THDOC
632
634
634
636
548
546
544
615
636
634
617
548
634
636
550
47.92
47.04
47.82
46.92
42.14
43.61
42.89
46.07
42.51
42.65
46.83
35.06
38.72
41.41
36.35

Example  2: Schematic of Steroid Hormone Concentration

The quantitative value of each steroid hormone obtained from 60 adult men and women through the process of 1) to 4) using the statistical program DecisionSite (version 9.1, Spotfire Inc., Palo Alto, CA, USA) Standardized by individual, and then the distribution trend was analyzed using PLS-DA (partial least-squares discriminant analysis) [ Anal. Chem ., 2007, 79: 6102-6110], and the magnitude of the relative concentration is indicated in color, red means relatively high concentration, blue means low concentration, and white means average value. The results of plotting a total of 70 steroid hormones are shown in FIG. 2.

Example 3 Analysis of Patients with Prostatic Hyperplasia Using Steroid Recognition Method

Through the analysis and schematization method of Examples 1 and 2, the concentration of 70 steroid hormone metabolites in urine was measured from 59 patients with Benign prostatic hyperplasia (BPH) and 41 normal persons, and their distribution patterns were detected by steroid recognition. Comparison was made using the method. Of the total 70 kinds of staidoids, those whose prostate hyperplasia and normal humans were statistically different ( P <0.01) were selected using Student's T-test two-test, and their schematics are shown in FIG. 3. Among 24 steroids with statistical differences, Epidihydrotestosterone, 5α-androstan-3β, 17β-diol, Androstenediol, 11β -OH-androsterone (11β-OH-androsterone), 11β-OH-etiocholanolone, 2-hydroxyestriol, 17α-hydroxypregnenolone ( Nine steroids, 17α-hydroxypregnenolone, Allodihydrocortisol and 11-Deoxycortisol, were found to be relatively high in patients with prostatic hyperplasia. ), 5α-androstan-3α, 17β-diol (5α-androstane-3α, 17β-diol), 5β-androstan-3α, 17β-diol (5β-androstane-3α, 17β-diol), 5β- Androstane-3α, 17α-diol (5β-androstane-3α, 17α-diol), Ethioochola nolone, Epiandrosterone, Estrone, 17β-estradiol, Estriol, Pregnanediol, 11β-hydroxyprogesterone, 11β-hydroxyprogesterone, The 15 steroids of allodihydrocorticosterone, 11-dehydrocorticosterone and tetrahydrocortisone showed relatively high concentrations in normal urine.

Example 4: Schematic representation of the activity of steroid metabolism related enzymes

As shown in FIG. 1, the steroid concentrations present in the prostate hypertrophy patients and the normal human urine were evaluated for the degree of activity of the enzymes involved in metabolism through quantitative ratios of the precursors and precursors converted by each enzyme. Among them, the enzyme activity between prostatic hypertrophy patients and normal humans was selected and plotted only by the ratio of the production / progenitors showing statistically differentiation ( P <0.01) using Student's T-test bilateral assay, which is shown in FIG. 4.

In FIG. 4, 7 ratios of DHT / T to αββ-diol / ααβ-diol among 14 types of progenitor / progenitor concentrations were high in patients with prostatic hyperplasia, and T / A-diol to P-diol / The proportion of seven species to P-one was higher in normal subjects. The types of enzymes involved in metabolism between each steroid are shown in Table 2 below.

Construct / precursor Related enzymes DHT / T 5α-reductase Epi-DHT / Epi-T 5α-reductase 17α-OH-Prog / 17α-OH-Preg 3β-HSD 5a-dione / A-dione 5α-reductase αββ-diol / DHT 3β-HSD A-diol / DHEA 17β-HSD αββ-diol / ααβ-diol 3α / β-HSD T / A-diol 3β-HSD 17β-E2 / E1 17β-HSD βαβ-diol / Etio 17β-HSD ααβ-diol / An 17β-HSD ααβ-diol / DHT 3α-HSD F / 11-deoxyF 11β-Hydroxylase P-diol / P-one 20α-HSD

Figure 1 shows the overall metabolic trend of steroid hormones.

FIG. 2 shows the concentration of 70 steroid hormones present in urine in 60 adult men and women and plots them in the form of steroids. Since the horizontal axis represents 60 people and the vertical axis represents each compound corresponding to 70 steroid hormones, the data in the longitudinal direction represents hormonal information for each person.

Figure 3 shows the results of the concentration of steroids to be differentiated from the urine of the patients with prostatic hyperplasia according to the present invention.

Figure 4 is a result of comparing the activity of enzymes that are differentiated in the steroid metabolism process from the urine of the prostate hyperplasia patients and normal people according to the invention by the diagram of the production / progenitor ratio.

Claims (7)

As a method of analyzing the differentiation of steroid hormone metabolites in two sample groups, Simultaneously extracting steroid hormone biometabolites including male hormones, female hormones, progesterone, sterols and corticosteroids in urine by solid phase extraction; Enzymatic hydrolysis of the extract followed by re-extraction with an organic solvent to obtain a re-extract; Trimethylsilyl derivatization of the reextract and measuring the concentration of steroid hormone biometabolites in urine using gas chromatography-mass spectrometry; And Comparing and confirming concentrations of steroid hormone biometabolites between two sample groups; Differentiation analysis method of the steroid hormone biological metabolite between two sample groups, characterized in that consisting of. The method of claim 1, wherein the organic solvent is a mixed solvent in which ethyl acetate and normal hexane are mixed. According to claim 1, Epidihydrotestosterone (Epidihydrotestosterone), 5α- androstane-3β, 17β-diol, Androstenediol, 11β-OH-andros 11 beta-OH-androsterone, 11 beta-OH-etiocholanolone, 2-hydroxyestriol, 17α-hydroxypregnenolone Analytical method, characterized in comparing the concentration of one or two or more urine steroid hormones biometabolite selected from allodihydrocortisol (Allodihydrocortisol) and 11-deoxycortisol (11-Deoxycortisol). According to claim 1, Testosterone (Epiestosterone), 5α-androstan-3α, 17β-diol (5α-androstane-3α, 17β-diol), 5β-androstan-3α, 17β-die O- (5β-androstane-3α, 17β-diol), 5β-androstan-3α, 17α-diol (5β-androstane-3α, 17α-diol), Etiocholanolone, Epiandrosterone , Estrone, 17β-estradiol, Estriol, Pregnanediol, 11β-hydroxyprogesterone, Allodihydrocorticosterone , 11-dehydrocorticosterone (11-dehydrocorticosterone) and tetrahydrocortisone (Tetrahydrocortisone) The analysis method characterized in that the concentration of one or two or more urine steroid hormone biometabolites selected from. The analysis method according to claim 1, wherein the concentrations of steroid hormone biometabolites between the two sample groups are compared by a schematization by a partial least-squares discriminant analysis (PLS-DA) method. As a method of analyzing the differentiation of steroid hormone metabolites in two sample groups, Simultaneously extracting steroid hormone biometabolites including male hormones, female hormones, progesterone, sterols and corticosteroids in urine by solid phase extraction; Enzymatic hydrolysis of the extract followed by re-extraction with an organic solvent to obtain a re-extract; Trimethylsilyl derivatization of the reextract and measuring the concentration of steroid hormone biometabolites in urine using gas chromatography-mass spectrometry; Distinguishing the precursor from the product in the steroid hormone metabolites, calculating the concentration ratio of the product / precursor, and comparing the ratio between the two sample groups; And Schematizing the product / precursor concentration ratio using a partial least-squares discriminant analysis (PLS-DA) method Differentiation analysis method of the steroid hormone biological metabolite between two sample groups, characterized in that consisting of. The analysis method of any one of claims 1 to 6, wherein the two sample groups are sample groups of a patient and a normal person, respectively.

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