KR101152902B1 - Evaluation of metabolic differences between hair samples by quantitative steroid signature - Google Patents
Evaluation of metabolic differences between hair samples by quantitative steroid signature Download PDFInfo
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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 concentration of steroid hormones in the hair in a statistical manner.
The present invention can grasp the overall metabolic distribution tendency by effectively extracting and analyzing steroid hormones from a small amount of hair, as well as indicating 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, and in particular, can be extended to the application of hair loss-related diseases.
Description
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 concentration of steroid hormones in the hair in a statistical manner.
Analytical steroid hormones are composed of male hormone (androgen), female hormone (estrogen), progesterone (corgestoid), corticoid, And sterols. 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 of the analysis, it is interpreted in connection with the physiological function, and studies to identify the cause of disease development are actively conducted.
There are various enzymes and receptors related to steroid metabolism in the hair root cells of hair and are involved in hair growth and hair loss. Because hair grows over long periods of time, it is an important biological sample with significant accuracy when retrospectively proven or measured. Therefore, the analysis of metabolites in hair has recently been used as a useful tool to include physiological information such as health status and early diagnosis of disease [ J. Invest. Dermatol. , 2001, 116: 57-61; Clin. Chem., 2001, 47: 143-144], unlike other in vivo samples such as urine and blood, has the advantage of being easy to collect and store. In addition, the analysis of hair has attracted attention as a complementary method of blood and urine tests that do not reflect past health conditions.
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.
In performing steroid analysis in hair, extraction with base hydrolysis is well known as a classical method but has limitations in the analysis of some steroids. Therefore, there is a need for an optimized assay to quantitatively evaluate the total steroid hormones present in trace amounts in hair samples and to measure the activity of enzymes associated therewith.
Therefore, the present inventors simultaneously extract 27 kinds of steroid hormones by ultrasonication using a small amount of hair, and re-extract the extracts through a solid phase extraction process, followed by gas chromatography through trimethylsilyl derivatization. The present invention was completed by simultaneously measuring the amount of steroid hormones and the activities of enzymes involved in their metabolism using a mass spectrometer, and comparing them to identify differentiation between sample groups.
Accordingly, an object of the present invention is to provide a method for determining and evaluating the cause of disease by measuring the relative concentrations and ratios of steroid hormones in hair and analyzing the differences between sample groups in connection with physiological functions.
The present invention,
Ultrasonically dissolving the hair to simultaneously extract steroid hormone biometabolites including male hormones, progesterone, sterols and corticosteroids;
Re-extracting steroid hormones from the biological metabolite with a lipophilic and hydrophilic copolymer adsorbent; And
Trimethylsilyl derivatizing the reextract and measuring the concentration of steroid hormone biometabolites in the hair using a gas chromatography-mass spectrometer; And
Characterized in that the method for detecting steroid hormones in the hair comprising.
In another aspect, the present invention, a method for analyzing the difference of the steroid hormone biometabolites between the two sample groups including the step of comparing the concentration of the steroid hormone biometabolites between the two sample groups obtained by the detection method with a statistical method It is done.
The present invention shows the overall metabolic distribution tendency by effectively extracting and analyzing steroid hormones, including corticosteroids, which have been difficult to analyze from a small amount of hair, using the conventional hydrolysis method. I can figure it out. Therefore, it can be used as a method for evaluating endocrine diseases and biological changes related to steroid hormone metabolism, and in particular, can be extended to the application of hair loss-related diseases.
1 is a schematic diagram illustrating a method of treating a sample for measuring the concentration of steroid hormones in hair.
FIG. 2 shows the concentration of 27 steroid hormones present in the hair of 36 adult males and plots them in the form of steroids. Since the horizontal axis represents 36 people and the vertical axis represents each compound corresponding to 27 steroid hormones, the data in the longitudinal direction represents hormonal information for each person.
Figure 3 is a result of the concentration of steroid hormones that are differentiated from the hair loss of male hair loss patients and normal people.
Figure 4 is a result of comparing the activity of enzymes that are differentiated in the course of steroid metabolism from male hair loss patients and normal human hair by the ratio of the production / precursor ratio.
FIG. 5 is a graph illustrating changes in the concentration distribution of steroid hormones according to hairs by measuring the concentrations of four steroid hormones present in the hair of nine normal adult women.
The present invention will be described in detail as follows.
The present invention extracts a small amount of steroid hormones by ultrasonic digestion to measure the concentration of steroid hormones in the hair, and graphically maps the distribution of the steroid hormones to detect endocrine diseases and biological changes related to steroid hormone metabolism. It's about how you can evaluate it.
The amount of hair used as a biological sample is preferably 1 to 100 mg, and when the amount is less than 1 mg, there is a problem in the detection limit for the analysis of steroid hormone biometabolites. Not only is the concentration too high, but there is a problem with a large amount of sampling.
First, steroid hormones in the hair are extracted by ultrasonic digestion. In the extraction using ultrasonic decomposition, the organic solvent is preferably methanol or a mixed solvent of methanol and dichloromethane, and it is preferable to proceed at a temperature of 35 ° C. or higher for efficient extraction. In order to extract enough steroid hormones in the hair, the ultrasonic decomposition time is suitable for 30 to 120 minutes. Such ultrasonic decomposition method is known in the conventional hydrolysis method [Analyst, 1999, 124: 1297-1300; Analyst, 2000, 125: 711-714] also allows the analysis of corticosteroids that are not detectable.
The extracts thus obtained are re-extracted steroid hormones 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 : 2221-2228], an Oasis HLB cartridge which is a copolymer adsorbent having lipophilic and hydrophilic properties.
Samples prepared through the above process are subjected to trimethylsilyl derivatization in order to facilitate detection through a gas chromatography-mass spectrometer, and then analyzed by a gas chromatography-mass spectrometer and the concentration thereof is measured.
After comparing the height ratio of the steroid hormones in the hair obtained by the gas chromatograph-mass spectrometer with the concentration of the standard substance, the concentration of each steroid was measured, and then the concentrations of the sample groups were compared to confirm the differentiation of the steroid hormone biological metabolites. do.
In particular, by hierarchical clustering analysis (hierarchical clustering analysis) it is possible to analyze the differentiation of the steroid hormone biometabolites between the two sample groups by plotting the distribution of the relative concentration of the steroid hormones present in the individual hair. The hierarchical cluster analysis is an analysis method in which clustering is performed by grouping close groups or separating long distance clusters one by one using the distance between the clusters. linkage). The dendogram represented by the hierarchical cluster analysis can provide information on the association of steroid hormones between clusters. The concentration of steroid hormones between sample groups can be expressed in the form of a heat-map. The difference can be plotted.
In addition, the precursor and the product of the steroid hormone metabolites are distinguished, the concentration ratio of the product / precursor is calculated and compared, and the ratio is plotted using a hierarchical cluster analysis. Analyze product differences.
Comparing the steroid concentration and the concentration of the product / precursor in the sample of the alopecia disease patients and normal persons by the analysis of the differentiation, it is possible to confirm the relationship between the activity involved in steroid metabolism and masculine hair loss, It can be applied to provide information necessary for diagnosing alopecia disease.
In particular, Dihydrotestosterone, 5β-Dihydroprogesterone, Isopregnanolone, Epipitosterone, Lanosterol, 20α-hydroxycholesterol , Concentration of steroid hormones selected from 24S-hydroxycholesterol and α-Cortolone or Dihydrotestosterone / Testosterone, Dihydrotestosterone / 5α-andro 5α-Androstanedione, Cortisol / Cortisone, Dihydroepiandrosterone / 17α-hydroxypregnenolone, Pregnenolone / 5β-di 5β-dihydroprogesterone and 17α-hydroxypregnenolone / pregne The generator / precursor concentration ratio selected from pregnenolone may be used as a factor having significance with the alopecia disease.
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) Ultrasonic Decomposition
In order to remove contaminants on the surface of the collected hair, wash the hair thoroughly with distilled water and isopropyl alcohol, and then cut the dried hair into a size of 1 to 2 mm. In vitro standard ( d 3 -testosterone, 40 ng / mL; methyltestosterone, 400 ng / mL; d 6 -cholesterol, 200 ng / mL; d 9 -progesterone, 400 ng / mL; d 8 -17a-OH- 15 μl of a mixed solution of progesterone (200 ng / mL) was added to 30 mg of hair, followed by sonication at 50 ° C. for 0.5 hour with 0.5 mL of methanol.
2) solid phase extraction
5.5 mL of 0.2 M sodium acetate buffer was added to the extract of 1), followed by solid phase extraction using an Oasis HLB cartridge [Oasis HLB, 60 mg, Waters, Co., Milford, MA]. , USA]. 2 mL each of methanol and distilled water was flowed twice, and the sample 1) containing 0.2 M sodium acetate buffer solution was flowed, followed by 5 mL of distilled water 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.
3) Gas Chromatography-Mass Spectrometry
After the process of 2), MSTFA ( N -methyl- N -trifluorotrimethylsilyl acetamide) / NH 4 I (ammonium iodide) / DTE (dithioerythritol) ( 500: 4: 2, v / w / w) 30 μl of the mixed solution was added and reacted at 60 ° C. for 20 minutes.
Selected ion monitoring (SIM) to select and detect only characteristic ions using an Agilent 7890 series Gas Chromatograph connected to a mass spectrometer (Agilent's 5975C Mass Selective Detector) for the analysis of steroid hormones The mass spectra were analyzed using a scan mode with a mass range of 50 m / m . 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 injecting unit was 280 ° C., and the sample injection was performed using a split mode of 8: 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.
Dihydrotestosterone
Dehydroepiandrosterone
Testosterone
Epitestosterone
Androstenedione
Androsterone
Etiocholanolone
5α-Androstanedione
DHT
DHEA
T
Epi-t
A-dione
An
Etio
5α-dione
434
432
432
432
430
419
419
432
18.56
17.07
19.74
17.96
19.00
14.54
14.70
17.81
Pregnenolone
5β-dihydroprogesterone
Pregnanolone
Isopregnanolone
Pregnanetriol
17α-hydroxypregnenolone
Preg
5β-DHP
P-one
Iso-p-one
P-triol
17α-OH-Preg
445
445
447
447
435
548
26.65
19.21
22.85
27.00
25.78
31.92
Cortisol
Cortisone
Tetrahydrocortisol
Allotetrahydrocortisol
Tetrahydrocortisone
20a-Dihydrocortisone
β-Cortol
α-Cortolone
F
E
THF
Allo-THF
THE
20α-DHE
632
615
636
636
634
617
343
431
47.88
45.77
40.93
41.93
38.04
47.34
38.90
38.94
Cholesterol
Desmosteroll
Lanosterol
24S-hydroxycholesterol
20α-hydroxycholesterol
Chol
24S-OH-Chol
20α-OH-Chol
443
343
393
413
461
40.83
42.14
47.62
49.06
48.03
Example 2 Schematic of Steroid Hormone Concentration
The quantitative value of each steroid hormone obtained through the process of 1) to 3) was standardized by using DecisionSite (version 9.1, Spotfire Inc., Palo Alto, CA, USA), which is a statistical program. Afterwards, the distribution tendency was expressed in color using a hierarchical clustering analysis method, with red representing a relatively high concentration, blue representing a low concentration, and white representing an average value. The results of plotting the concentrations of 27 steroid hormones for 36 adult males are shown in FIG. 2.
Example 3 Analysis of Male Hair Loss Patient Sample Using Steroid Recognition Method
Through the analysis and schematization method of Examples 1 and 2, the concentrations of 27 steroid hormone metabolites in the hair were measured from 18 male pattern baldness (MPB) patients and 18 normal persons, and their distribution patterns were detected by steroid recognition. Comparison was made using the method. Among the 27 steroids, those whose male and female alopecia patients had statistically significant differences ( P <0.05) were selected using the Student's T-test two-test method, and these were diagrammatically shown in FIG. 3. Of the eight statistically different steroids, three steroids, Dihydrotestosterone, 5β-Dihydroprogesterone, and Isopregnanolone, were distributed in relatively high concentrations in male hair loss patients. 5 types of steroids: Epitestosterone, Lanosterol, 20α-hydroxycholesterol, 24S-hydroxycholesterol and α-Cortolone Was relatively high in normal hair.
Example 4: Schematic representation of the activity of steroid metabolism related enzymes
The steroid concentrations present in male hair loss patients and normal human hairs were evaluated by quantitative ratios of the precursors and precursors converted by the following enzymes to evaluate the degree of activity of the enzymes involved in metabolism. 4 is shown. The types of enzymes involved in metabolism between each steroid are shown in Table 2 below.
In FIG. 4, DHT / T (5α-reductase), DHT / 5α-dione (17β-HSD), F / E (11β-HSD), and DHEA / 17α-OH-Preg among 15 kinds of product / precursor concentration ratios. The ratio of (17,20-lyase) was higher in male hair loss patients, and the ratio of P-one / 5β-DHP (3α-HSD) and 17α-OH-Preg / Preg (17α-hydroxylase) was higher in normal subjects. appear.
Example 5: measurement concentration change by hair part
Through the detection method of Example 1, the concentration of four representative steroid hormones (cholesterol, lanosterol, desosterol, pregnenolone) in the hair according to the site of hair from nine adult females was measured, and their distribution pattern Were compared, and the results are shown in FIG.
As shown in FIG. 5, the concentration of steroid hormones was detected highest in the adjacent hair part (scalp ~ 3 cm) in the scalp, and the concentration of steroid hormone was decreased in the hair at the end of the head away from the scalp.
Claims (8)
Re-extracting steroid hormones from the biological metabolite with a lipophilic and hydrophilic copolymer adsorbent; And
Trimethylsilyl derivatizing the reextract and measuring the concentration of steroid hormone biometabolites in the hair using a gas chromatography-mass spectrometer;
Method for detecting steroid hormones in hair, characterized in that consisting of.
Ultrasonically dissolving the hair to simultaneously extract steroid hormone biometabolites including male hormones, progesterone, sterols and corticosteroids;
Re-extracting steroid hormones from the biological metabolite with a lipophilic and hydrophilic copolymer adsorbent;
Trimethylsilyl derivatizing the reextract and measuring the concentration of steroid hormone biometabolites in the hair using a gas chromatography-mass spectrometer; And
Comparing and confirming the 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 comparison of the concentrations of steroid hormone biometabolites between the two sample groups is characterized by distinguishing the precursor and the product from the metabolites, and calculating and comparing the concentration ratio of the product / precursor. Method for Differentiating Metabolites.
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