CN1100993C - Testing method of body steroid hormone and derivative content detected according to the keratin growth-gen of body surface - Google Patents
Testing method of body steroid hormone and derivative content detected according to the keratin growth-gen of body surface Download PDFInfo
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- CN1100993C CN1100993C CN97107498A CN97107498A CN1100993C CN 1100993 C CN1100993 C CN 1100993C CN 97107498 A CN97107498 A CN 97107498A CN 97107498 A CN97107498 A CN 97107498A CN 1100993 C CN1100993 C CN 1100993C
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Abstract
The present invention relates to a detection method for measuring the content of in-vivo steroid hormones and derivatives thereof by cuticular growers on the surface of a human body, which comprises the steps that cuticular growers on the surface of a human body are soaked by an organic solvent which can dissolve steroid hormones or derivatives thereof to be detected after being pulverized, and the solution is fully extracted for at least once; all the extraction solution is completely evaporated, and an extraction product is obtained; the obtained extraction product is arranged in a phosphate buffer solution with the pH value of 7.2 to 7.4; the phosphate buffer solution with the extraction product is kept for 25 to 35 minutes at the temperature of 35 to 39DEG C and is stirred for at least 1 minute at room temperature; the content of the steroid hormones or the derivatives thereof in the stirred solution is measured in a conventional radio-immunity method or an enzyme immune method.
Description
Technical field
What the present invention relates to is the detection method that cutin growth-gens such as a kind of hair by body surface, nail or toenail are measured steroid hormone in the bodies and derivative content thereof.
Background technology
Steroid hormone is one of chief component in the human endocrine hormone, not only have important and tremendous influence to normal metabolic and physiological function, and the variation of different types of steroid hormone contents level also often can indicate and the different physiological situations of reflection health in the body, thus to its in vivo the detection of contents level be usually used in the analysis and judgement in medical diagnosis and other relevant field.For example, to the detection of reproductive hormones such as estradiol, progesterone, in women's physiological change or medical diagnosis on disease, be an important test item and basis for estimation; In the drug-testing of sports tournament, also be the important evidence that is used to judge to the detection of the steroid hormone or derivatives thereof content such as male sex hormone in sportsman's body.In addition, the detection to the steroid hormone or derivatives thereof content of multiple adrenal cortex class in the body also often has crucial meaning and value in medical diagnosis and treatment.
At present clinically in the human body method of steroid hormone content all be by just detecting after the samplings such as blood, saliva, urine, seminal fluid or tissue to human body, wherein great majority again all dependence take a blood sample and carry out.These methods, the method for particularly taking a blood sample, many shortcomings are fairly obvious.At first, the person of being sampled must arrive hospital or have the place of corresponding conditions just can get blood, and the extraction amount of blood can not be too big, and also therefore propagates easily or infected upward disease, thereby many people are difficult for accepting.Secondly, the processing procedure of blood sample trouble, easily contaminated, and must in refrigerator, preserve, should not transport for long-distance again.And, use the method that the urine sample of gathering is detected for a long time to the time based on the drug-testing of male sex hormone or derivatives thereof always.And the collection of urine also has many difficulties, and also trouble very of the processing of urine, and detecting instrument is very expensive again, can take certain methods to avoid drug-testing to its true urine but maximum problem still is anti-depressant user.Therefore, also existing at present more and more appealing the in anti-depressant detection with blood testing replacement urine detection, but come inevitably above-mentioned many shortcomings of institute with blood testing, must can become the problem that can't avoid and will solve again.
Summary of the invention
The objective of the invention is the detection method that provides steroid hormone in a kind of new human body and derivative content level thereof for addressing the above problem, it is to be that sample detects by the body surface cutin growth-gen to human body, thereby overcoming and avoided at present with body fluid, particularly is many shortcomings and the problem that sample detects with blood.
The detection method of steroid hormone and derivative content thereof in the body of the present invention, the cutin growth-gen that is the body surfaces such as hair, beard, nail or toenail with human body is the sample of sampling, and wherein special hair with the people is the sample most convenient and actual application value is arranged.Assay method is operated by following step:
1) the cutin growth-gen of getting body surface is pulverized, for example, can cut into segment in small, broken bits or broken last, organic solvent with the detected steroid hormone or derivatives thereof composition of solubilized soaks, fully extraction at least once, generally can be twice, then whole extracts are waved most solvent, obtain extracting extract;
2) will go up the phosphate buffer that the extraction extract that obtains of step places pH7.2-7.4,, promptly place after 25-35 minute for 35-39 ℃, again in room temperature DL 1 minute at least in the temperature that is equivalent to body temperature;
3) measure the content of going up steroid hormone or derivatives thereof tested in the step mixed rotary liquid with conventional radioimmunoassay or enzyme immunization method.
The organic solvent of said extraction usefulness is preferably the low boiling kind solvent that boiling point is lower than 100 ℃ in the said method, so that wave the operation that removes or reclaim extraction solvent after the extraction.For example can use lower boiling oxo solvent such as conventional methyl alcohol, ethanol, ether, acetone, also can use boiling range to be 30-60 ℃ non-polar solvents such as sherwood oil.Be good to use ether as extraction solvent especially wherein, effect of extracting is good, and boiling point is low, be easy to wave remove and reclaim, and convenient post-treatment, and cheap and easy to get.
Embodiment
The invention of present patent application is an example to gather human hair's sample, handles as follows, extracts different steroid hormones, has carried out detecting and relevant analysis and research.
After people's hair scissors is cut into secondary segment in small, broken bits, make extraction solvent with ether (or boiling range is 30-60 ℃ a sherwood oil), in every 25-50 milligram hair sample with the about 2 milliliters ratio DL of solvent after at least 1 minute, place more than 1 hour, after isolating extract, add homogeneous solvent in above-mentioned half ratio again and repeat above-mentioned extracting operation 1-2 time.Merge that each time extract is waved to the greatest extent naturally or after the water-bath heating waves most solvent, obtain extracting extract.The phosphate buffer 0.2-0.3 milliliter of the pH7.2-7.4 that sodium dihydrogen phosphate-sodium hydrogen phosphate is formed joins in this extraction extract DL and places 35-39 ℃, usually preferably can in 37 ± 0.5 ℃ water-bath, take out after about 30 minutes, at room temperature about 1 minute of DL more promptly becomes for the standby that detects usefulness.
Adopt with to identical radioimmunoassay (RIA) or the enzyme immunization method conventional sense methods such as (EIA) of human serum (or blood plasma) steroid hormone detection, perhaps the radioimmunoassay or the enzyme immunization method of The World Health Organization's regulation detect the relevant steroid hormone in the above-mentioned detection standby.
To adopt
125The RIA detection method of iodine labeling is an example, above-mentioned people is sent out the content situation and the influence factor of steroidal class reproductive hormone contained in the sample and has carried out detecting and further furtheing investigate.
After women's hair sample that sample number n=17 example was not washed with any shampoo in 24 hours is taken a sample respectively; three kinds of detected specimen sample that " the flower trump shampoo " and " poem sweet smell board shampoo " of be divided into direct detection, producing respectively with commercially available Sino-Japan joint Shanghai KAO. Corp. SA washed; and all it is detected respectively by sending out tip, a stage casing and root of hair portion three parts again, to investigate the estradiol (E in the hair different parts
2) and the distributional difference of two kinds of steroid hormones of progesterone (P), and shampoo is to the influence of its content in hair.Experimental result respectively as shown in Table 1 and Table 2.The experimental data of table 1 and table 2 is clear to be demonstrated, between the different parts of hair, and E
2With the content of two kinds of steroid hormones of P, and shampoo serves as big to the influence that these two kinds of hormone-contents in the hair different parts change with the difference between listed each data of table 1." three kinds of hair condition are divided the E at position in the his-and-hers watches 1
2Amount mean value " data in the processing learned by statistics of maximum different value (42.8 and 47.1), t=0.90,0.677＜t＜1.291 are so 0.50＞p＞0.20 shows between its maximum difference group there was no significant difference.Hence one can see that, and other has respectively organized between data obviously also more there was no significant difference.This had both illustrated that the steroid hormone in the human body was ubiquitous in people's hair, and its existence in hair and the root can not be subjected to hair of distributing, in, the influence of sampling position difference such as the tip, shown also its content can not change because of whether in the influence with external treatment factors such as shampoo cleanings, have relative stability, but thereby make method of the present invention have the basis and the value of practical application.Simultaneously, the indicated important meaning on the other hand of this experiment and being worth also is: if a certain shampoo or makeup use the steroid hormone of the body surface cutin growth-gen at position to detect to produce big or seriously influence hair or other, then can be used as at least and show that this shampoo or makeup are likely and be not suitable for the important evidence that human body uses.
Respectively the hair sample of the masculinity and femininity of different sample numbers and the content of the hormone of the same race in the blood sample are detected, and carry out statistical procedures, make its correlation research.The result shows, this two have tangible correlativity.About the experiment of correlativity and statistics data referring to table 3.
With the women of sample number n=37 example in its normal menstrual cycle follicular phase (menstruation 7-10 days), the onset of ovulation (menstruation the 14th day) and get hair sample respectively in three physiological stages luteal phase (menstruation 20-24 days) and detect, concern to investigate steroid hormone content in the hair rule between changing with body physiological state.Detect wherein the average content of estradiol be respectively 22.8,21.8 and 25.6 (pg/g).The blood plasma level of this content rule and L. Te Leige women's estradiol hormone of record in its " steroid hormone " of showing (Science Press, 1980) and the Changing Pattern that concerns in the result of study sexual cycle to be explained fit like a glove.
Outside experimentizing divided by above-mentioned hair sample, be test sample, carried out the check experiment of steroid hormone content equally with people's nail.After the nail samples that will take from the women is pulverized, obtain mixed rotary liquid to be measured with above-mentioned identical disposal route after, detect wherein that the content of estradiol is 102pg/g, progesterone levels is 5.9ng/g.Show that except that hair other cutin growth-gen of body surface equally also contains can utilize and have the steroid hormone that detects meaning and value.
Except that above-mentioned hormone, be feasible too to the detection of other steroid hormone in the human body.For example, to the male sex of n=7 example and n=7 example women's the discovery that detects, the average content of cortisol hormone is 9ng in its per 50 milligrams of hairs respectively.
These experimental results fully show, the no matter male sex or women, and steroid hormone is equal stable existence in hair not only, and its content and change and its situation that exists in blood has significant correlativity.That is be to have equal meaning and value, to the detection of its content in hair and to its detection of content in blood.
With test experience result and the conclusion thereof of radioimmunoassay to above-mentioned relevant steroid hormone, adopting " the immuno-enzymatic connection determination method (IEMA) that magnetic particle separates " to detect is feasible equally.
Obviously, above-mentioned detection principle and method of operating equally also are to be suitable for and feasible for the detection of relevant steroid hormone derivant undoubtedly.
Because up to now, people do not recognize in the people sends out, certainly more impossiblely recognize beard, refer to contain steroid hormone in these cutin growth-gens of (toe) first-class body surface the people, and have and the variation of Human Physiology, pathological condition adapts self Changing Pattern and important use thereof are worth, therefore method of the present invention is conspicuous in scientific significance.Practical application meaning that above-mentioned experimental result is indicated and value equally also are conspicuous, it can thoroughly change the traditional approach that must arrive hospital or have the place of corresponding conditions to take a blood sample the detection of human body steroid hormone and derivant thereof, cutin growth-gen with body surface, be test sample particularly to measure that the people who is easy to get greatly and is regarded as at present discarded object usually sends out, can have equal meaning and effect, simple and easy to do, without any misery, can not infect and spread disease, thereby be easy to accept extensively for people, and avoided the many drawbacks and the inconvenience that bring with sampling and detection to blood, greatly reduce the expense cost of detection again, not only the medical test in disease has great use meaning and value with diagnosing, also significant in scientific research, it can become the important means of endocrine research.Simplifying and conveniently also having important and huge realistic meaning and value as application to other relevant fields such as athletic drug-testings.
Estradiol (E in table 1 hair
2) assay result (n=17, E
2Content unit is pg/g)
| Hair condition | Root of hair E 2Content | Send out middle part E 2Content | Send out E sharp 2Content | Average E 2Content |
| Do not wash | 49.1±22.7 | 42.2±18.2 | 45.6±25.5 | 45.6±22.1 |
| Wash with " flower king " shampoo | 43.2±15.2 | 42.6±21.7 | 45.8±15.1 | 43.7±17.3 |
| Wash with " poem sweet smell " shampoo | 45.7±26.8 | 43.7±27.0 | 49.8±28.3 | 46.4±27.4 |
| Three kinds of hair condition are divided the E at position 2Amount mean value | 46.0±21.6 | 42.8±22.3 | 47.1±25.2 | / |
Annotate: the pg in the content unit is 10
-12Gram
The assay result of progesterone in table 2 hair (P) (n=17, P content unit are ng/g)
| Hair condition | Root of hair P content | Send out middle part P content | Send out P content sharp | Mean P content |
| Do not wash | 6.93±1.69 | 6.83±2.51 | 6.90±2.56 | 6.89±2.25 |
| Wash with " flower king " shampoo | 6.67±2.51 | 6.74±3.10 | 6.93±3.98 | 6.78±3.20 |
| Wash with " poem sweet smell " shampoo | 7.15±3.62 | 7.19±3.08 | 6.78±3.70 | 7.04±3.47 |
| Three kinds of hair condition are divided the P content mean value at position | 6.92±2.61 | 6.92±2.90 | 6.87±3.41 | / |
Annotate: the ng in the content unit is 10
-9Gram
Table 3 people send out and blood in correlativity between steroid hormone of the same race
| Sex and sample number | Hormone kind | Content in the hair | Content in the serum | Content in content/blood in sending out | Correlation (r) | Correlativity |
| Woman n=35 | Estradiol | 25.6 pg/g | 59.8 pg/ml | 42.8％ | 0.396 | 0.05＞p＞0.025 conspicuousness is relevant |
| Woman n=35 | Progesterone | 8.25 ng/g | 13.1 ng/ml | 63.5％ | 0.440 | 0.025＞p＞0.01 conspicuousness is relevant |
| Man n=5 | Testosterone | 1.30 ng/g | 4.86 ng/ml | 26.7％ | 0.920 | 0.025＞p＞0.01 conspicuousness is relevant |
Claims (9)
1. measure the detection method of steroid hormone in the body and derivative content thereof by body surface cutin growth-gen for one kind, it is characterized in that by following step operation:
1) after the cutin growth-gen of getting body surface is pulverized, the low boiling point organic solvent that is lower than 100 ℃ with the boiling point of the detected steroid hormone or derivatives thereof composition of solubilized soaks, fully after the extraction at least once, whole extracts are waved most solvent, obtain extracting extract;
2) will go up the phosphate buffer that the extraction extract that obtains of step places pH7.2-7.4, in 35-39 ℃ place 25-35 minute after, again in room temperature DL 1 minute at least;
3) measure the content of going up tested steroid hormone or derivatives thereof in the step mixed rotary liquid with conventional radioimmunology or enzyme immunoassay.
2. detection method as claimed in claim 1 is characterized in that said organic solvent is oxygen containing low boiling point organic solvent.
3. detection method as claimed in claim 2 is characterized in that said organic solvent is an ether.
4. detection method as claimed in claim 1 is characterized in that said organic solvent is that boiling spread is 30-60 ℃ a sherwood oil.
5. detection method as claimed in claim 1, it is characterized in that said during with solvent extraction the consumption of organic solvent be the ratio of horn that be extracted of 25-50 milligram with 2 milliliters of solvents, extraction times is at least twice.
6. detection method as claimed in claim 3, it is characterized in that said during with solvent extraction the consumption of organic solvent be the ratio of horn that be extracted of 25-50 milligram with 2 milliliters of solvents, extraction times is at least twice, each extraction and being at least standing time 60 minutes merges each time extract and waves naturally and use up or water-bath obtains extracting extract after waving most extraction solvent.
7. detection method as claimed in claim 1, it is characterized in that said extraction extract is placed the damping fluid of being made up of sodium dihydrogen phosphate-sodium hydrogen phosphate, after placing about 30 minutes down, 37 ± 0.5 ℃ of water-baths take out, after 1 minute, use conventional method of immunity mensuration steroid hormone or derivatives thereof content wherein in DL under the room temperature again.
8. as the described detection method of one of claim 1 to 7, it is characterized in that said body surface cutin growth-gen is the hair of human body.
9. as the described detection method of one of claim 1 to 7, it is characterized in that said body surface cutin growth-gen is the nail or the toenail of human body.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN97107498A CN1100993C (en) | 1997-05-19 | 1997-05-19 | Testing method of body steroid hormone and derivative content detected according to the keratin growth-gen of body surface |
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|---|---|---|---|
| CN97107498A CN1100993C (en) | 1997-05-19 | 1997-05-19 | Testing method of body steroid hormone and derivative content detected according to the keratin growth-gen of body surface |
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| CN1173641A CN1173641A (en) | 1998-02-18 |
| CN1100993C true CN1100993C (en) | 2003-02-05 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4433056A (en) * | 1981-09-24 | 1984-02-21 | Baranczuk Richard J | Process for preparation of control for use in estrogen receptor test |
| DE4130915A1 (en) * | 1990-09-19 | 1992-03-26 | Pola Kasei Kogyo Kk | Topical compsn. contg. steroid or tri:terpenoid glycoside - and sphingolipid, opt. also steroid hormone, as moisture retaining agent for scalp and for treating rough skin |
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1997
- 1997-05-19 CN CN97107498A patent/CN1100993C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4433056A (en) * | 1981-09-24 | 1984-02-21 | Baranczuk Richard J | Process for preparation of control for use in estrogen receptor test |
| DE4130915A1 (en) * | 1990-09-19 | 1992-03-26 | Pola Kasei Kogyo Kk | Topical compsn. contg. steroid or tri:terpenoid glycoside - and sphingolipid, opt. also steroid hormone, as moisture retaining agent for scalp and for treating rough skin |
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| CN1173641A (en) | 1998-02-18 |
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