CN106483212B - Urine estrogen metabolism object marker relevant to idiopathic male infertility and its detection method and application - Google Patents
Urine estrogen metabolism object marker relevant to idiopathic male infertility and its detection method and application Download PDFInfo
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Abstract
The invention belongs to analytical chemistry and clinical medicine domain, urine estrogen metabolism object marker relevant to idiopathic male infertility and its detection method and application are disclosed.The marker is urine estrogen metabolism object methoxyestradiol and/or androstenedione, it is detected using UPLC-Q exactive MS method, the marker can be used for the auxiliary diagnosis and monitoring of idiopathic male infertility, sensitivity with higher and specificity, have clinical generalization value.
Description
Invention field
The invention belongs to analytical chemistry and clinical medicine domain, it is related to urine estrogen relevant to idiopathic male infertility
Metabolite markers and its detection method and application based on UPLC-Q exactive MS.
Background technique
Currently, there are about the couple at child-bearing age of 10-15% to suffer from growing barrier in the whole world.China is due to large population base, newly-married husband
Infertile patient numbers are in woman far more than million, and wherein infertility caused by male factor is higher than 50%.Mesh in male sterility
It is preceding still to there is about 40%~75% to can not find reason, referred to as idiopathic male infertility disease.Idiopathic male infertility causes extremely tight
The Disease Spectrum of weight.
In fact, there are great difficulties for idiopathic male infertility diagnosis.The diagnostic criteria of the WHO of male sterility is man and wife's wedding
After live together 1 year or more, do not take contraceptives, the infertile person in the wife's side caused due to the bridegroom's or husband's side.However, since there are many practical reasons
Man and wife do not ensure that it is stringent live together 1 year so that the judgement of last male sterility becomes extremely difficult；It lives together up to 1 year
Observing time has delayed the time that early treatment and intervention are carried out to male sterility significantly；In order to exclude wife's side reason, the wife's side is also
It needs to carry out detailed inspection, brings heavy economy and medical burden.What existing male sterility coherence check relied on is
Traditional Sperm recovery, it only focuses on the conventional parameters such as sperm quantity, degrees of motion, semen volume, pH and liquefying time.Because
It is influenced by factors such as abstinence times, Sperm parameters conventional analysis result shows as biggish fluctuation.Thus, it is clinical
Diagnosis generally requires to bring burden to doctor and patient with reference to multiple semen routine analysis.More importantly traditional essence
Liquid parameter testing cannot comprehensively react whole situations of sperm.Therefore, male sterility often show as Sperm parameters without
It is obvious abnormal, but also traditional seminal parameters inspection can not efficient diagnosis male sterility.Thus, it clinically needs for spy
The new diagnostic method of hair property male sterility.
Metabolism group is the omics technology emerging after the latter door of genomics, protein science, transcription group, it is to pass through
High throughput investigates a Men Ke of biosystem Changeement biosystem of metabolite after genetic change and/or environment change
It learns.Metabolism group research has the characteristics that " unbiased ", " overall situation ", " no to assume ", " being assumed by data-driven ", thus the knot studied
By often reliability and novelty with higher.Metabolism group can be with systematic overview, analysis and exploration cell, tissue and device
The small molecule biomarkers object and its situation of change that official generates, more intuitively understand the material base of function phenotypic alternation,
Object is related to the metabolism network of the organisms such as lipid metaboli, glycometabolism, amino acid metabolism, nucleic acid metabolism, coenzyme metabolism, is to compare gene
Group, protein science, the group of transcription group closer " phenotype ".Metabolism group due to its analysis sample can for urine this
A little noninvasive samples, can greatly reduce the wound of patient's sampling.Metabolism group shows high in the diagnosis of complex disease
Application potential and value have the characteristics that high sensitivity and stable.Metabolism group is carried out by the whole metabolic components to body
Analysis and research, therefrom find out specific biomarkers, and timely, accurate, highly sensitive and Gao Teyi can be carried out to clinical disease
The diagnosis of property.At present metabolism group be mainly used in terms of the diagnosis of disease coronary heart disease, liver diseases, diabetes, hypertension,
Fat and tumour.It is worth noting that, urine is the biological sample being clinically easy to get, have the advantages that noninvasive, bulky.
It is particularly suitable for the diagnosis and screening of disease.However, using metabonomic analysis urine metabolism small molecule in idiopathic male infertility
Diagnostic monitoring in application do not paid close attention to accordingly also.
Metabolism group detection platform common at present mainly includes nuclear magnetic resonance (NMR), gas-chromatography tandem mass spectrum (GC-
MS), Liquid Chromatography-Tandem Mass Spectrometry (LC-MS) and Capillary Electrophoresis tandem mass spectrum (CE-MS).Although nuclear magnetic resonance is that chemicals are fixed
Property goldstandard, but have the shortcomings that sensitivity is low, it is difficult to obtain more complete metabolism spectrum.Tandem mass spectrum has high sensitivity
With qualitative accurate advantage.But gas-chromatography tandem mass spectrum is only limited to detect volatile substance, detection range cannot cover
The requirement of metabolism spectrum.In order to make up the defect, the method that gas-chromatography tandem mass spectrum often uses derivatization detects chemicals, this
So that pre-treating method becomes complicated, more errors are introduced.Meanwhile the detection of gas-chromatography tandem mass spectrum is due to isolation technics
Limitation, general detection time is longer, extends experimental period, increases instrument occupancy, limits it and examines in large sample crowd
Application in survey.Capillary Electrophoresis tandem mass spectrum has an advantage to the separation detection of polar compound, but stability of instrument is asked
Topic is relatively difficult to resolve certainly.And Liquid Chromatography-Tandem Mass Spectrometry has sample process simple, high sensitivity, the strong feature of Clinical practicability can
To well solve the above problem.UPLC-Q exactive MS is the combination of high resolution mass spectrum of new generation Yu ultra high efficiency liquid phase,
With compared to the stronger sensitivity of traditional LC-MS, specificity and stability.So being urinated using UPLC-Q exactive MS
Liquid is metabolized the metabonomic analysis of small molecule, if stable special urine generation relevant to idiopathic male infertility morbidity can be found
It thanks to small molecule as biomarker, and researches and develops the detection side UPLC-Q exactive MS of corresponding metabolism small molecule mark
Method is not only in first place in the world in the field, can also create the economic benefit to attract people's attention, to raising China's male genetic
The general level of the health also will be primary strong promotion.
Summary of the invention
The object of the present invention is to provide a kind of urine estrogen metabolism object markers relevant to idiopathic male infertility.
Another object of the present invention is to provide the detection method of above-mentioned urine estrogen metabolism object marker.
A further object of the present invention is to provide the kit for detecting above-mentioned urine estrogen metabolism object marker.
The purpose of the present invention is what is realized by following technical measures:
Urine estrogen metabolism object marker relevant to idiopathic male infertility, the marker are urine estrogen metabolism
Object methoxyestradiol and/or androstenedione.
The urine estrogen metabolism object marker is in preparing idiopathic male infertility diagnosis or monitoring reagent box
Using.
A kind of kit diagnosed or monitor idiopathic male infertility, the kit contain detection urine estrogen metabolism object
The reagent of methoxyestradiol and/or androstenedione.
The kit, which, which contains, detects urine estrogen metabolism using UPLC-Q exactive MS method
The reagent of object methoxyestradiol and/or androstenedione.
The kit, the kit contain following reagent:
Methoxyestradiol standard items；
Androstenedione standard items；
Internal standard A: creatinine, valine, niacin, thymidine, glutaric acid, L- phenylpropylamine acid, N- acetaminophen, horse
The Isotopic Internal Standard (deuterium mark, aqueous solution) of the one or more substances of uric acid；
Internal standard B: the Isotopic Internal Standard (deuterium mark, methanol solution) of pentadecanoic acid；
Internal standard C: the Isotopic Internal Standard (deuterium mark, methanol solution) of lignoceric acid；
Further, which also contains:
Hypersil GOLD C18 chromatographic column；
Reagent A: 100% methanol (protein precipitation use)；
Reagent B: the water (mobile phase use) containing 0.1% formic acid；
Reagent C: the acetonitrile (mobile phase use) containing 0.1% formic acid；
Reagent D: 100% ultrapure water (is redissolved and is used).
A method of above-mentioned urine estrogen metabolism object marker relevant to idiopathic male infertility is detected, it is special
Sign is this method using UPLC-Q exactive MS method, detect urine estrogen metabolism object methoxyestradiol and/or
The content of androstenedione.
In this method:
One, liquid-phase condition:
Liquid-phase chromatographic column is Hypersil GOLD C18 chromatographic column (100mm × 2.1mm, 1.9 μm of Thermo of partial size
Scientific, Germany), column temperature is 40 DEG C.
Mobile phase A is the water containing 0.1% formic acid, and Mobile phase B is the acetonitrile containing 0.1% formic acid, and flow velocity is 400 μ L/min；
Instrument gradient are as follows: 0-3min 1%B, 3-10min 1% arrives 99%B, 10-13min 99%B, 13-13.1min
99% arrives 1%B, 13.1-17min 1%B；(B refers to Mobile phase B, the amount of the amount of mobile phase A and corresponding Mobile phase B in each gradient
Totally 100%, similarly hereinafter)
Input mode: 5 μ l of volume；
Two, Mass Spectrometry Conditions
Using heating electrospray ionisation mode (HESI), positive ion mode spray voltage: 3.5kV；Negative ion mode is spraying
Voltage: 2.5kV；Capillary temperature under both of which: 250 DEG C, heter temperature: 425 DEG C, sheath gas air-flow: 50AU assists gas gas
Stream: 13AU, blowback air air-flow: 0AU；Lens voltage: 60V.Using mode is swept entirely, scanning range: 70 arrive 1050m/z；Resolution ratio:
70000。
The present invention is described in detail as follows:
The present inventor acquires standard compliant urine specimen with S.O.P. (SOP), and system collects complete crowd
Basic information and clinical data, and use the metabolism group method based on UPLC-Q exactive MS and analyzed.
The experimental method specifically studied mainly includes following components:
One, research object selection and group basis
First stage: screening stage
It is included in 430 people of 607 people of idiopathic male infertility and normal healthy controls to clarify a diagnosis at random, totally 1037 people.
A group: healthy control group (430 people):
1. the age is between 19 to 39 years old；
2. constitutional index is between 17 to 31；
3. the male of fecundity health, and have healthy offspring after 5-8 months；
4. without whole body major disease.
B group: idiopathic male infertility disease group (607 people):
1. the age matches with control group；
2. constitutional index is matched with control group
3. pregnancy is attempted not succeed within 12 months, and spouse does not have the male of infertile disease；.
4. without the clear male sterility cause of disease；
5. Smoking And Drinking history is matched with control group；
6. nationality matches with control group；
7. without whole body major disease.
Second stage: Qualify Phase
It is included in 15 people of 15 people of idiopathic male infertility and normal healthy controls to clarify a diagnosis, totally 30 people.
A group: healthy control group (15 people):
1. the age is between 24 to 36 years old；
2. constitutional index is between 19 to 24；
3. the male of fecundity health, and have healthy offspring after 5-8 months；
4. without whole body major disease.
B group: idiopathic male infertility disease group (15 people):
1. the age matches with control group；
2. constitutional index is matched with control group；
3. pregnancy is attempted not succeed within 12 months, and spouse does not have the male of infertile disease；
4. without the clear male sterility cause of disease；
5. Smoking And Drinking history is matched with control group；
6. nationality matches with control group；
7. without whole body major disease.
Two, UPLC-Q exactive MS metabonomic analysis and idiopathic male infertility diagnosis are sieved with estrogen metabolism object
Choosing and verifying
1. Sample pretreatment
1. taking 300 μ L urines, 10 μ L internal standard A being added, 10 μ L internal standard B are added, 10 μ L internal standard C are added, 900 μ L of methanol is added
(reagent A), vortex 30s.
2. 4 DEG C of 16000g are centrifuged 15min in centrifuge, supernatant is transferred to 1.5mL import EP pipe, and supernatant is existed
It is concentrated to dryness in centrifugal concentrating drying instrument under room temperature.
3. it is redissolved with 10 μ L ultrapure waters (reagent D), it is to be analyzed.
2. instrument detects
1. analysis instrument: UPLC Ultimate 3000system (Dionex) high performance liquid chromatograph；Q-Exactive
High-resolution mass spectrometer.
2. liquid-phase condition:
2.1. liquid-phase chromatographic column be Hypersil GOLD C18 chromatographic column (100mm × 2.1mm, 1.9 μm of partial size, Thermo
Scientific, Germany), column temperature is 40 DEG C.
Water (reagent B) and (B) of 2.2 mobile phases used for (A) containing 0.1% formic acid contain the acetonitrile (reagent of 0.1% formic acid
C), flow velocity is 400 μ L/min.
2.3 instrument gradients are as follows: instrument gradient are as follows: 0-3min 1%B, 3-10min 1% arrives 99%B, 10-13min 99%
B, 13-13.1min 99% arrives 1%B, 13.1-17min 1%B.
2.4 input modes: 5 μ l of volume.
3. Mass Spectrometry Conditions
3.1 heating electrospray ionisation mode (HESI) are analyzed.
3.2 using heating electrospray ionisation mode (HESI), positive ion mode spray voltage: 3.5kV；Negative ion mode spray
Mist voltage: 2.5kV；Capillary temperature under both of which: 250 DEG C, heter temperature: 425 DEG C, sheath gas air-flow: 50AU assists gas
Air-flow: 13AU, blowback air air-flow: 0AU；Lens voltage: 60V.Using mode is swept entirely, scanning range: 70 arrive 1050m/z；It differentiates
Rate: 70000.
3. substance is qualitative
Estrogen metabolism object is qualitative to compare Chromatographic information (when reservation using with standard items methoxyestradiol and androstenedione
Between) and Information in Mass Spectra (accurate molecular weight), and compare the Chromatographic information of Isotopic Internal Standard standard items series in sample in real time with school
Positive retention time.
4. data are analyzed:
Biomarker screening confirms key metabolites using Multivariate Logistic Regression.
5. the difference and diagnostic significance of estrogen metabolism object in healthy control group, idiopathic male infertility group urine specimen.
The corrected age, constitutional index, smoking and history of drinking history information, logistic regression find urine
The content increase of methoxyestradiol and androstenedione significantly improves the generation of idiopathic male infertility.It is answered using random crowd
With above-mentioned estrogen metabolism object combined diagnosis idiopathic male infertility, sensitivity 93.33%, specificity 93.33%, ROC
Area under the curve is 0.9867, additive diagnostic value with higher.
Three, diagnostic reagent box preparation method
According to above-mentioned a series of experiments as a result, the present inventor is also prepared for a kind of diagnosis or monitoring idiopathic male infertility
Kit, the diagnostic kit include to be stabilized in measurement subject's urine and detectable methoxyestradiol and androstene
The standard items of diketone and the internal standard standard items series of assistant analysis.Diagnostic kit can also include a set of urine estrogen generation
Thank object extraction and used in chromatograph reagent and equipment.
Beneficial effects of the present invention:
The present inventor compares in normal control and idiopathic male infertility urine by using UPLC-Q exactive MS
Metabolism small molecule, it was found that exist in urine and can be used for assessing whether that there is auxiliary diagnosis valence with idiopathic male infertility
The urine estrogen metabolism object marker of value combines and the UPLC-Q exactive of the estrogen metabolism object marker detection
The application of MS, developing can be convenient for the idiopathic male infertility diagnosis of clinical application, monitoring reagent box.
The present invention is advantageous in that using the marker that urine metabolism small molecule is evaluated as idiopathic male infertility:
(1) urine metabolism small molecule is a kind of new biomarkers, is associated with disease outcome by force, not only stable, nothing
It creates, be easy to detect, and is quantitative accurate, will greatly improve the sensibility and specificity of idiopathic male infertility diagnosis, such small point
The successful exploitation of sub- biomarker will start completely new situation for the prevention and treatment of idiopathic male infertility, be other diseases biological marker
The development of object is offered reference.
(2) urine metabolism small molecule marker provided by the invention can be used as the diagnosis marker of idiopathic male infertility,
Can in Check-up crowd clear idiopathic male infertility disease condition, thus for the further testing in depth testing of clinician provide according to
According to quick and precisely to grasp the morbid state of patient and coincident with severity degree of condition, taking the control prece of more personalized to mention in time
For supporting, delay and prevent progression of disease.
(3) present invention is verified using the urine specimen of idiopathic male infertility and the random crowd of normal healthy controls, it was demonstrated that
Methoxyestradiol and Androstenedione levels have higher sensitivity and special in diagnosis idiopathic male infertility in urine
Degree can be used as marker use.
(4) present invention uses tight, multistage verifying and appraisement system, and initial stage screens a variety of urine generations by preliminary experiment
Thank to small molecule, carry out independent crowd's verifying using UPLC-Q exactive MS, ensure that the urine metabolism biomarker and
The reliability of diagnostic method.
(5) UPLC-Q exactive MS technology sample process is simple, and instrument is analyzed rapidly and accurately, clinic with higher
Diagnose practical value.
Detailed description of the invention
Fig. 1 screening stage, the corrected age, constitutional index, smoking and history of drinking history information, Multivariate Logistic Regression
Analysis finds that the content increase of urine methoxyestradiol and androstenedione significantly improves the generation of idiopathic male infertility.a
The single factor test Logistic regression result of Confounding Factor is not adjusted.bIt is more after adjusting age, constitutional index, smoking and history of drinking history
First Logistic regression result.
The horizontal fluctuation of Fig. 2 estrogen metabolism analyte detection (mean ± standard deviation).
Fig. 3 Qualify Phase, Normal group and idiopathic male using the production of urine methoxyestradiol content information
ROC curve between sterile group.
Fig. 4 Qualify Phase, the Normal group and idiopathic male infertility made of urine androstenedione content information
ROC curve between group.
Fig. 5 Qualify Phase, the Normal group made of urine methoxyestradiol and androstenedione content information and
ROC curve between idiopathic male infertility group.
Specific embodiment
The present invention will be further explained by examples below.
The selection of 1 research object of embodiment and group basis
This part research object examines idiopathic male infertility case and health from the head of affiliated hospital, Nanjing Medical University
Fertility control.Research contents and informed consent form obtain the approval of Ethics Committee, Nanjing Medical University, meet relevant laws and regulations
Requirement.Case and to impinge upon understand content after endorsed informed consent form.All research objects have carried out complete physique
It checks, and completing a includes personal basic data, living habit, occupation and environmental exposure, genetic risk factors, sexual function
With reproductive function, history of disease and the questionnaire of physical exertion.First stage incorporates satisfactory 607 idiopathic males
Sterile case and 430 normal healthy controls；The satisfactory 15 idiopathic male infertility cases of second stage and 15 health are right
According to the screening experiment object as idiopathic male infertility urine metabolism small molecule biomarker.Specific sample group standard
It is as follows:
First stage screening stage
It is included in 430 people of 607 people of idiopathic male infertility and normal healthy controls to clarify a diagnosis at random, totally 1037 people.
A group: healthy control group (430 people):
1. the age is between 19 to 39 years old；
2. constitutional index is between 17 to 31；
3. the male of fecundity health, and have healthy offspring after 5-8 months；
4. without whole body major disease.
B group: idiopathic male infertility disease group (607 people):
1. the age matches with control group；
2. constitutional index is matched with control group
3. pregnancy is attempted not succeed within 12 months, and spouse does not have the male of infertile disease；.
4. without the clear male sterility cause of disease；
5. Smoking And Drinking history is matched with control group；
6. nationality matches with control group；
7. without whole body major disease.
Second stage Qualify Phase
It is included in 15 people of 15 people of idiopathic male infertility and normal healthy controls to clarify a diagnosis, totally 30 people.
A group: healthy control group (15 people):
1. the age is between 24 to 36 years old；
2. constitutional index is between 19 to 24；
3. the male of fecundity health, and have healthy offspring after 5-8 months；
4. without whole body major disease.
B group: idiopathic male infertility disease group (15 people):
1. the age matches with control group；
2. constitutional index is matched with control group；
3. pregnancy is attempted not succeed within 12 months, and spouse does not have the male of infertile disease；
4. without the clear male sterility cause of disease；
5. Smoking And Drinking history is matched with control group；
6. nationality matches with control group；
7. without whole body major disease.
The screening of embodiment 2:UPLC-MS metabolism group idiopathic male infertility biomarker
1. Sample pretreatment
1. taking 300 μ L urines, 10 μ L internal standard A being added, 10 μ L internal standard B are added, 10 μ L internal standard C are added, 900 μ L of methanol is added
(reagent A), vortex 30s.
2. 4 DEG C of 16000g are centrifuged 15min in centrifuge, supernatant is transferred to 1.5mL import EP pipe, and supernatant is existed
It is concentrated to dryness in centrifugal concentrating drying instrument under room temperature.
3. it is redissolved with 10 μ L ultrapure waters (reagent D), it is to be analyzed.
2. instrument detects
1. analysis instrument: UPLC Ultimate 3000system (Dionex) high performance liquid chromatograph；Q-Exactive
High-resolution mass spectrometer.
2. liquid-phase condition:
2.1. liquid-phase chromatographic column be Hypersil GOLD C18 chromatographic column (100mm × 2.1mm, 1.9 μm of partial size, Thermo
Scientific, Germany), column temperature is 40 DEG C.
Water (reagent B) and (B) of 2.2 mobile phases used for (A) containing 0.1% formic acid contain the acetonitrile (reagent of 0.1% formic acid
C), flow velocity is 400 μ L/min.
2.3 instrument gradients are as follows: instrument gradient are as follows: 0-3min 1%B, 3-10min 1% arrives 99%B, 10-13min 99%
B, 13-13.1min 99% arrives 1%B, 13.1-17min 1%B.
2.4 input modes: 5 μ l of volume.
3. Mass Spectrometry Conditions
3.1 heating electrospray ionisation mode (HESI) are analyzed.
3.2 using heating electrospray ionisation mode (HESI), positive ion mode spray voltage: 3.5kV；Negative ion mode spray
Mist voltage: 2.5kV；Capillary temperature under both of which: 250 DEG C, heter temperature: 425 DEG C, sheath gas air-flow: 50AU assists gas
Air-flow: 13AU, blowback air air-flow: 0AU；Lens voltage: 60V.Using mode is swept entirely, scanning range: 70 arrive 1050m/z；It differentiates
Rate: 70000.
3. substance is qualitative
Estrogen metabolism object is qualitative to compare Chromatographic information (when reservation using with standard items methoxyestradiol and androstenedione
Between) and Information in Mass Spectra (accurate molecular weight), and compare the Chromatographic information of Isotopic Internal Standard standard items series in sample in real time with school
Positive retention time.
4. data are analyzed:
Biomarker screening confirms key metabolites using Multivariate Logistic Regression.
5. the difference and diagnostic significance of estrogen metabolism object in healthy control group, idiopathic male infertility group urine specimen.
The corrected age, constitutional index, smoking and history of drinking history information, logistic regression find urine
The content increase of methoxyestradiol and androstenedione significantly improves the generation (Fig. 1) of idiopathic male infertility.
The stability analysis of 3 urine estrogen metabolism object of embodiment
The stability of urine methoxyestradiol and Androstenedione levels is evaluated using the method for embodiment 2 (
It is 2 weeks every the time).The results show that methoxyestradiol and androstenedione measurement horizontal stable (Fig. 2) in urine, have conduct
Diagnosis/monitoring marker characteristic.
4 estrogen metabolism object of embodiment combines the diagnosis to idiopathic male infertility
According to above-mentioned UPLC-Q exactive MS metabolism group method, the present inventor by random 15 case of crowd and
The urine sample detection methoxyestradiol and androstenedione of 15 controls, draw ROC curve with this and assess the sensitivity of diagnosis
And specificity, and then evaluation capacity of this 2 estrogen metabolism object levels to idiopathic male infertility in assessment detection urine.
The sensitivity of methoxyestradiol is 86.67%, specificity 80.00%, and area is 0.9022 under ROC curve
(Fig. 3)；
Androstenedione sensitivity is 86.67%, specificity 86.67%, and area is 0.93333 (Fig. 4) under ROC curve；
The sensitivity for combining methoxyestradiol and androstenedione is 93.33%, specificity 93.33%, under ROC curve
Area is 0.9867 (Fig. 5).
So combining methoxyestradiol and androstenedione has the ability for preferably diagnosing idiopathic male infertility.
Embodiment 5 is used for the production of idiopathic male infertility urine estrogen metabolism analyte detection and diagnostic kit
Determine in normal control and idiopathic male infertility urine have by the method for UPLC-Q exactive MS first
There is the metabolism small molecule compared with high abundance.Then, it is wherein being sieved by the metabonomic technology based on UPLC-Q exactive MS
Select relevant to idiopathic male infertility estrogen metabolism object, as whether be idiopathic male infertility diagnosis index.Finally
The quantity of the correspondence urine estrogen metabolism object filtered out is controlled at 2, this be made on the basis of preliminary experiment it is optimal
That changes simplifies.Using this 2 estrogen metabolism objects, it can not only ensure preferable sensitivity and specificity, but also cost can be saved, subtract
The burden of light patient, moreover it is possible to reduce detection time, have the advantages that quick, accurate, economy, be convenient for clinical promotion and application, certainly
Use wherein that 1 marker can also be with more preferable using 2 marker effects.This kit includes a collection of urine estrogen metabolism
Analyte detection reagent and consumptive material wherein estrogen metabolism object qualitative and quantitatively use the mark of methoxyestradiol and androstenedione
Quasi- product, assistant analysis use internal standard A: creatinine, valine, niacin, thymidine, glutaric acid, L- phenylpropylamine acid, N- acetyl pair
The deuterium mark Isotopic Internal Standard of eight kinds of amino phenols, hippuric acid substances.Internal standard B: the deuterium mark Isotopic Internal Standard of pentadecanoic acid.Internal standard C: two
The deuterium mark Isotopic Internal Standard of tetradecanoic acid.It is other that there are also the mating reverse chromatograms column (Hypersil for being used for UPLC chromatographic isolation
GOLD C18 chromatographic column, 100mm × 2.1mm, 1.9 μm of partial size), (water containing 0.1% formic acid and contain for the reagent of mobile phase
The acetonitrile of 0.1% formic acid), for extracting the reagent (100% ultrapure water) of estrogen metabolism object.The value of this kit is
300 μ l urines are only needed, can be detected the content of urine estrogen metabolism object marker, then idiopathic male is diagnosed by content
Infertility, and it is easy to carry out dynamic monitoring and observation therapeutic effect.
Specific kit forms are as follows:
Methoxyestradiol standard items
Androstenedione standard items
Internal standard A: creatinine, valine, niacin, thymidine, glutaric acid, L- phenylpropylamine acid, N- acetaminophen, horse
The deuterium mark Isotopic Internal Standard of eight kinds of substances of uric acid
Internal standard B: the deuterium mark Isotopic Internal Standard of pentadecanoic acid
Internal standard C: the deuterium mark Isotopic Internal Standard of lignoceric acid
Further, can also contain:
Chromatographic column (Thermo 100mm × 2.1mm, 1.9 μm of partial size, Hypersil GOLD C18 chromatographic column)
Reagent A (contains 100% methanol)
Reagent B (water containing 0.1% formic acid)
Reagent C (acetonitrile containing 0.1% formic acid)
Reagent D (100% ultrapure water).
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Claims (1)
1. urine estrogen metabolism object marker is preparing the application in idiopathic male infertility diagnosis or monitoring reagent box, described
Urine estrogen metabolism object marker be urine estrogen metabolism object methoxyestradiol and androstenedione.
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| CN103630678A (en) * | 2013-03-29 | 2014-03-12 | 中国科学院城市环境研究所 | Biological marker of male infertility and application thereof |
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| CN103630678A (en) * | 2013-03-29 | 2014-03-12 | 中国科学院城市环境研究所 | Biological marker of male infertility and application thereof |
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