CN106442770B - Refining metabolism small molecule marker relevant to idiopathic male infertility and its detection method and application - Google Patents
Refining metabolism small molecule marker relevant to idiopathic male infertility and its detection method and application Download PDFInfo
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- CN106442770B CN106442770B CN201610802618.9A CN201610802618A CN106442770B CN 106442770 B CN106442770 B CN 106442770B CN 201610802618 A CN201610802618 A CN 201610802618A CN 106442770 B CN106442770 B CN 106442770B
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Abstract
The invention belongs to analytical chemistry and clinical medicine domain, refining metabolism small molecule marker relevant to idiopathic male infertility and its detection method and application are disclosed.The marker is that refining is metabolized small molecule acetylcarnitine and/or tryptophan, 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 small to be related to refining metabolism relevant to idiopathic male infertility
Molecular marker and its detection method and application based on UPLC-Q exactive MS.
Background technique
It is counted according to WHO, there are about 15% couple at child-bearing age by the puzzlement of infertility in the whole world.China due to large population base,
Infertile patient numbers are in newly-married couple far more than million, because infertility caused by male factor is higher than 50%.Male sterility
In still there is about 40%~75% to can not find reason, referred to as idiopathic male infertility disease at present.Idiopathic male infertility causes pole
For serious Disease Spectrum.
In fact, idiopathic male infertility diagnosis is extremely difficult.The standard of WHO male sterility is that man and wife lives together 1 year after marriage
More than, contraceptives are not taken, and the infertile person in the wife's side is caused due to the bridegroom's or husband's side.However, the diagnostic criteria lived together 1 year, is indulged in significantly
The time that early treatment and intervention are carried out to male sterility is missed；Due to many men and wives of practical reasons do not ensure that it is stringent same
It occupies 1 year, so that last male sterility judgement becomes extremely difficult；And in order to exclude wife's side reason, the wife's side also needs to carry out
Detailed inspection brings heavy economy and medical burden.Traditional seminal parameters inspection only focuses on sperm quantity, movement
The conventional parameters such as degree, semen volume, pH and liquefying time cannot comprehensively react whole situations of sperm.Moreover, male sterility passes through
Often show as Sperm parameters Non Apparent Abnormality, but also traditional seminal parameters inspection can not efficient diagnosis male not
It educates.In addition to this because being influenced by factors such as abstinence times, Sperm parameters conventional analysis result is also embodied by larger
Fluctuation.Thus, clinical diagnosis generally requires to bring burden to doctor and patient with reference to multiple semen routine analysis.Cause
And clinically need the new diagnostic method for idiopathic male infertility.
Metabolism group (Metabolomics/Metabonomics) is grow up phase late 1990s one new
Set up schools section, it be by investigate biosystem be genetically changed it is stimulated or disturbance after, the variation of metabolite or its with
The variation of time, to study a science of biosystem.So-called metabolism group (Metabolome) is the downstream product of genome
It is also final product, is some participation organism metabolism, the small molecule for maintaining organism normal function and growth and development
The set of object is closed, mainly endogenous small molecule of the relative molecular mass less than 1000, these endogenous metabolism small molecules are related to
Glycometabolism, energetic supersession, lipid metaboli, amino acid metabolism, nucleic acid metabolism, coenzyme metabolism etc..
Organism under normal condition is a complete system, and the metabolin in biological fluid, cell and tissue is in
One stable equilibrium state.Pathological change will have occurred due to heredity or the day after tomorrow in body, this balance is just broken, generation
It thanks product and metabolic process and also produces corresponding variation.Metabolism small molecule is understood in lysis by metabonomic analysis
Variation, people can be helped to find related biomarker (biomarker), can be with the diagnosis of aided disease, can also be with
The metabolic pathway for helping people to be related to by small-molecule substance itself understands the pathogenesis of disease and provides spy for medicament research and development
Anisotropic target.In recent years, metabolism group achieves in the early diagnosis of disease many in the research of mankind's various diseases
The research achievement being of great significance, such as cardiovascular disease, diabetes and cancer, correlative theses are published in academic journal
On " Nature ", " Nature medicine ", " Journal of hepatology " and " Cancer research ", show
Metabolism small molecule huge potentiality and value in diagnosing human disease.The problem of basic reason of male sterility is sperm.
Refining as the liquid component of sperm can be well reflected sperm quality change, and have it is noninvasive, easy to collect is excellent
Point.However, also not using application of the metabonomic analysis refining metabolism small molecule in the diagnostic monitoring of idiopathic male infertility
It is paid close attention to accordingly.
The common technology of metabolism group research at present includes liquid chromatograph mass spectrography (LC-MS), chromatography of gases-mass spectrum connection
With (GC-MS) and nuclear magnetic resonance technique (NMR).Nuclear magnetic resonance technique feature is to component to be measured without destruction, Sample pretreatment letter
It is single, but sensitivity is lower；Gas chromatography-mass spectrography has preferable sensitivity and reproducibility, but generally to use derivatization
Method carries out pre-treatment to sample, so that experimental procedure becomes complicated.And LC-MS has sample process simple, high sensitivity is faced
The practical feature of bed.UPLC-Q exactive MS is the combination of high resolution mass spectrum of new generation Yu ultra high efficiency liquid phase, is had
Compared to the stronger sensitivity of traditional LC-MS, specificity and stability.So carrying out refining using UPLC-Q exactive MSS
It is metabolized the metabonomic analysis of small molecule, if stable special refining metabolism relevant to idiopathic male infertility morbidity can be found
Small molecule researches and develops the UPLC-Q exactive MS detection method of corresponding metabolism small molecule mark as biomarker,
It not only is in first place in the world in the field, can also create the economic benefit to attract people's attention, it is strong to China's male genetic is improved
Kang Shuiping also will be primary strong promotion.
Summary of the invention
The object of the present invention is to provide refinings relevant to idiopathic male infertility to be metabolized small molecule marker.
Another object of the present invention is to provide the detection method of above-mentioned refining metabolism small molecule marker.
A further object of the present invention is to provide the kit for detecting above-mentioned refining metabolism small molecule marker.
The purpose of the present invention is what is realized by following technical measures:
Refining relevant to idiopathic male infertility is metabolized small molecule marker, and refining is not metabolized small molecule second to the marker
Acylcarnitine and/or tryptophan.
The described refining metabolism small molecule marker is preparing answering in idiopathic male infertility diagnosis or monitoring reagent box
With.
A kind of kit diagnosed or monitor idiopathic male infertility, the kit contain detection refining metabolism small molecule second
The reagent of acylcarnitine and/or tryptophan.
The kit, the kit contain using UPLC-Q exactive MS method detection refining metabolism small molecule
The reagent of acetylcarnitine and/or tryptophan.
The kit, the kit contain following reagent:
Acetylcarnitine and/or tryptophan standards product；
Internal standard A: creatinine, valine, niacin, thymidine, glutaric acid, L- phenylpropylamine acid, N- acetaminophen, horse
The Isotopic Internal Standard (deuterium mark, aqueous solution) of one or more substances in 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: protein precipitation is used, and contains 100% methanol；
Reagent B: mobile phase is used, the water containing 0.1% formic acid；
Reagent C: mobile phase is used, the acetonitrile containing 0.1% formic acid；
Reagent D: redissolving and use, ultrapure water.
A method of refining relevant to idiopathic male infertility is metabolized small molecule marker as previously described for detection, should
Method uses UPLC-Q exactive MS method, detects the content that small molecule acetylcarnitine and/or tryptophan are metabolized in refining.
In above-mentioned detection method:
One, liquid-phase condition:
Liquid-phase chromatographic column is Hypersil GOLD C18 chromatographic column, and 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；(amount of mobile phase A and the amount of corresponding Mobile phase B totally 100% in each gradient, under
Together)
Input mode: 10 μ l of volume；
Two, Mass Spectrometry Conditions
It is analyzed using heating electrospray ionisation mode (HESI), positive ion mode spray voltage: 3.5kV；Anion
Mode spray voltage: 2.5kV；Capillary temperature under both of which: 250 DEG C, heter temperature: 425 DEG C, sheath gas air-flow: 50AU,
Assist gas air-flow: 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 refining sample 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 148 people of 148 people of idiopathic male infertility and normal healthy controls to clarify a diagnosis at random, totally 296 people.
A group: healthy control group (148 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 6-8 months；
4. without whole body major disease.
B group: idiopathic male infertility disease group (148 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 6-8 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 metabolism small molecule screening
And verifying
1. Sample pretreatment
1.1. 10 μ L refinings are taken, 10 μ L internal standard A are added, 10 μ L internal standard B are added, 10 μ L internal standard C are added, methanol (examination is added
Agent A) 40 μ L, vortex 30s, protein precipitation.
1.2. 4 DEG C of 16000g are centrifuged 15min in centrifuge, and supernatant is transferred to 1.5mL import EP pipe, and by supernatant
It is concentrated to dryness in centrifugal concentrating drying instrument at room temperature.
1.3. it is redissolved with 10 μ L ultrapure waters (reagent D), it is to be analyzed.
2. instrument detects
2.1. analysis instrument: UPLC Ultimate 3000system (Dionex) high performance liquid chromatograph；Q-
Exactive high-resolution mass spectrometer.
2.2. liquid-phase condition:
2.2.1. 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.
2.2.2 acetonitrile (examination of water (reagent B) and (B) of the mobile phase used for (A) containing 0.1% formic acid containing 0.1% formic acid
Agent C), flow velocity is 400 μ L/min.
2.2.3 instrument gradient are as follows: 0-3min 1%B, 3-10min 1% arrives 99%B, 10-13min 99%B, 13-
13.1min99% to 1%B, 13.1-17min 1%B.
2.2.4 input mode: 10 μ l of volume.
2.3. Mass Spectrometry Conditions
2.3.1 heating electrospray ionisation mode (HESI) is analyzed.
2.3.2 using heating electrospray ionisation mode (HESI), positive ion mode spray voltage: 3.5kV；Negative ion mode
Spray voltage: 2.5kV；Capillary temperature under both of which: 250 DEG C, heter temperature: 425 DEG C, sheath gas air-flow: 50AU, auxiliary
Gas air-flow: 13AU, blowback air air-flow: 0AU；Lens voltage: 60V.Using mode is swept entirely, scanning range: 70 arrive 1050m/z；Point
Resolution: 70000.
3. substance is qualitative
Metabolism small molecule is qualitative to compare Chromatographic information (retention time) and matter using with standard items acetylcarnitine and tryptophan
Spectrum information (accurate molecular weight), and compare the Chromatographic information of Isotopic Internal Standard standard items series in sample in real time with corrected retention time
Between.
4. data are analyzed:
Biomarker screening confirms key metabolites using using Multivariate Logistic Regression.
5. being metabolized the difference and diagnostic significance of small molecule in healthy control group, idiopathic male infertility group refining sample.
The corrected age, constitutional index, smoking and history of drinking history information, logistic regression find refining
The content increase of acetylcarnitine and tryptophan significantly reduces the generation of idiopathic male infertility.Using random crowd using above-mentioned
It is metabolized small molecule combinatorial and diagnoses idiopathic male infertility, sensitivity 93.33%, specificity 86.67%, below ROC curve
Product is 0.9822, 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, which is also prepared for, a kind of can diagnose or monitor idiopathic male infertility
Kit, the kit contains the examination for detecting relevant to idiopathic male infertility refining metabolism small molecule marker
Agent, comprising being stabilized and the standard items and assistant analysis of detectable acetylcarnitine and tryptophan in measurement subject's refining
Internal standard standard items series.Diagnostic kit can also include a set of refining metabolism small molecule extract and used in chromatograph reagent and
Equipment.
Beneficial effects of the present invention:
The present inventor compares in normal control and idiopathic male infertility refining by using UPLC-Q exactive MS
Metabolism small molecule, it was found that exist in refining can be used for assessing whether with idiopathic male infertility, with diagnostic value
Refining is metabolized the UPLC-Q exactive MS's of the combination of small molecule marker and refining metabolism small molecule marker detection
Using 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 refining metabolism small molecule is evaluated as idiopathic male infertility:
(1) refining metabolism small molecule is a kind of new biomarkers, is associated with disease outcome by force, not only stable, micro-
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) refining metabolism small molecule marker provided by the invention can be used as the diagnosis marker of idiopathic male infertility,
It, to provide foundation for the further testing in depth testing of clinician, can be quickly quasi- in early stage auxiliary diagnosis idiopathic male infertility
The morbid state and coincident with severity degree of condition, the control prece offer support for taking more personalized in time for really grasping patient, delay
With prevention progression of disease.
(3) present invention is verified using the refining sample of idiopathic male infertility and the random crowd of normal healthy controls, it was demonstrated that
Acetylcarnitine and tryptophan levels have higher sensitivity and specificity in diagnosis idiopathic male infertility in refining, can make
For marker use.
(4) present invention uses tight, multistage verifying and appraisement system, and initial stage screens a variety of refining generations by preliminary experiment
Thank to small molecule, carry out independent crowd's verifying using UPLC-Q exactive MS, ensure that the refining metabolism biological marker 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 refining acetylcarnitine and tryptophan significantly reduces the generation of idiopathic male infertility.aIt does not adjust
The single factor test Logistic regression result of Confounding Factor.bIt is polynary after adjusting age, constitutional index, smoking and history of drinking history
Logistic regression result.
Fig. 2 is metabolized small molecule detection level fluctuation (mean ± standard deviation).
Fig. 3 Qualify Phase, the Normal group and idiopathic male infertility made of refining acetylcarnitine content information
ROC curve between group.
Fig. 4 Qualify Phase, using the Normal group and idiopathic male infertility group of the production of refining tryptophane information
Between ROC curve.
Fig. 5 Qualify Phase, the Normal group made of refining acetylcarnitine and tryptophane information and idiopathic
ROC curve between male sterility group.
Specific embodiment
The present invention will be further explained by examples below.
Embodiment 1: research object selection and group basis
This part research object examines idiopathic male infertility case and health from the head of affiliated hospital of Nanjing Medical University
Fertility control.Research contents and informed consent form obtain the approval of Ethics Committee of 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 148 idiopathic males
Sterile case and 148 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 refining metabolism small molecule biomarker.Specific sample group standard
It is as follows:
First stage screening stage
It is included in 148 people of 148 people of idiopathic male infertility and normal healthy controls to clarify a diagnosis at random, totally 296 people.
A group: healthy control group (148 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 6-8 months；
4. without whole body major disease.
B group: idiopathic male infertility disease group (148 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 6-8 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
1.1. 10 μ L refinings are taken, 10 μ L internal standard A are added, 10 μ L internal standard B are added, 10 μ L internal standard C are added, methanol (examination is added
Agent A) 40 μ L, vortex 30s, protein precipitation.
1.2. 4 DEG C of 16000g are centrifuged 15min in centrifuge, and supernatant is transferred to 1.5mL import EP pipe, and by supernatant
It is concentrated to dryness in centrifugal concentrating drying instrument at room temperature.
1.3. it is redissolved with 10 μ L ultrapure waters (reagent D), it is to be analyzed.
2. instrument detects
2.1. analysis instrument: UPLC Ultimate 3000system (Dionex) high performance liquid chromatograph；Q-
Exactive high-resolution mass spectrometer.
2.2. liquid-phase condition:
2.2.1 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.
2.2.2 acetonitrile (examination of water (reagent B) and (B) of the mobile phase used for (A) containing 0.1% formic acid containing 0.1% formic acid
Agent C), flow velocity is 400 μ L/min.
2.2.3 instrument gradient are as follows: 0-3min 1%B, 3-10min 1% arrives 99%B, 10-13min 99%B, 13-
13.1min99% to 1%B, 13.1-17min 1%B.
2.2.4 input mode: 10 μ l of volume.
2.3. Mass Spectrometry Conditions
2.3.1 heating electrospray ionisation mode (HESI) is analyzed.
2.3.2 using heating electrospray ionisation mode (HESI), positive ion mode spray voltage: 3.5kV；Negative ion mode
Spray voltage: 2.5kV；Capillary temperature under both of which: 250 DEG C, heter temperature: 425 DEG C, sheath gas air-flow: 50AU, auxiliary
Gas air-flow: 13AU, blowback air air-flow: 0AU；Lens voltage: 60V.Using mode is swept entirely, scanning range: 70 arrive 1050m/z；Point
Resolution: 70000.
3. substance is qualitative
Metabolism small molecule is qualitative to compare Chromatographic information (retention time) and matter using with standard items acetylcarnitine and tryptophan
Spectrum information (accurate molecular weight), and compare the Chromatographic information of Isotopic Internal Standard standard items series in sample in real time with corrected retention time
Between.
4. data are analyzed:
Biomarker screening confirms key metabolites using using Multivariate Logistic Regression.
5. being metabolized the difference and diagnostic significance of small molecule in healthy control group, idiopathic male infertility group refining sample.
The corrected age, constitutional index, smoking and history of drinking history information, logistic regression find refining
The content increase of acetylcarnitine and tryptophan significantly reduces the generation (Fig. 1) of idiopathic male infertility.
The stability analysis of 3 refining of embodiment metabolism small molecule
(interval time is evaluated to the stability of refining acetylcarnitine and tryptophan levels using the method for embodiment 2
It is 2 weeks).The results show that acetylcarnitine and tryptophan measurement horizontal stable (Fig. 2) in refining, have as diagnosis/monitoring mark
The characteristic of object.
Embodiment 4 is metabolized diagnosis of the small molecule combinatorial 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 seminal plasma samples detection acetylcarnitines and tryptophan of 15 controls draw ROC curve with this and assess the sensitivity of diagnosis and special
Property, and then evaluation capacity of this 2 metabolism small molecule levels to idiopathic male infertility in assessment detection refining.
The sensitivity of acetylcarnitine is 93.33%, specificity 80.00%, and area is 0.8933 (Fig. 3) under ROC curve；
Tryptophan sensitivity is 80.00%, specificity 73.33%, and area is 0.8267 (Fig. 4) under ROC curve.
The sensitivity for combining acetylcarnitine and tryptophan is 93.33%, specificity 86.67%, and area is under ROC curve
0.9822 (Fig. 5).
So combining acetylcarnitine and tryptophan has the ability for preferably diagnosing idiopathic male infertility.
Production of the embodiment 5 for idiopathic male infertility refining metabolism small molecule detection and diagnosis kit
Determine in normal control and idiopathic male infertility refining 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 metabolism small molecule, as whether be idiopathic male infertility diagnosis index.It is preferred that will
The quantity of the correspondence refining metabolism small molecule filtered out is controlled at 2, this is the optimization made on the basis of preliminary experiment
It simplifies.It is metabolized small molecule using this 2 refinings, preferable sensitivity and specificity can be not only ensured, but also cost can be saved, mitigate
The burden of patient, moreover it is possible to reduce detection time, have the advantages that quick, accurate, economy, be convenient for clinical promotion and application, adopt certainly
With wherein 1 marker can also be with more preferable using 2 marker effects.This kit includes a collection of refining metabolism small molecule inspection
It surveys and uses reagent and consumptive material, wherein the standard items that are qualitative and quantitatively using acetylcarnitine and tryptophan of metabolism small molecule, auxiliary point
Analysis uses internal standard A: creatinine, valine, niacin, thymidine, glutaric acid, L- phenylpropylamine acid, N- acetaminophen, horse urine
The deuterium mark Isotopic Internal Standard of sour eight kinds of substances.Internal standard B: the deuterium mark Isotopic Internal Standard of pentadecanoic acid.Internal standard C: lignoceric acid
Deuterium mark Isotopic Internal Standard.It is other that there are also mating reverse chromatograms column (the Hypersil GOLD C18 chromatographies for being used for UPLC chromatographic isolation
Column, 100mm × 2.1mm, 1.9 μm of partial size), the reagent (100% methanol) for precipitating γ-seminoprotcin, the reagent for mobile phase
(water containing 0.1% formic acid and containing 0.1% formic acid acetonitrile), (100% is ultrapure for the reagent for extracting metabolism small molecule
Water).The value of this kit is only 10 μ l refinings of needs, can be detected the content of refining metabolism small molecule marker, then leads to
Content diagnosis idiopathic male infertility is crossed, and is easy to carry out dynamic monitoring and observation therapeutic effect.
Specific kit forms are as follows:
Acetylcarnitine standard items
Tryptophan standards product
Internal standard A (creatinine, valine, niacin, thymidine, glutaric acid, L- phenylpropylamine acid, N- acetaminophen, horse
The deuterium mark Isotopic Internal Standard aqueous solution of eight kinds of substances of uric acid)
Internal standard B (the deuterium mark Isotopic Internal Standard methanol solution of pentadecanoic acid)
Internal standard C (the deuterium mark Isotopic Internal Standard methanol solution 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 (100% methanol)
Reagent B (water containing 0.1% formic acid)
Reagent C (acetonitrile containing 0.1% formic acid)
Reagent D (100% ultrapure water).
Leading reference
Asiago,V.M.,L.Z.Alvarado,N.Shanaiah,G.A.Gowda,K.Owusu-Sarfo,
R.A.Ballas,and D.Raftery.2010.Early detection of recurrent breast cancer
using metabolite profiling.Cancer Res 70:8309-8318.
Brindle,J.T.,H.Antti,E.Holmes,G.Tranter,J.K.Nicholson,H.W.Bethell,
S.Clarke,P.M.Schofield,E.McKilligin,D.E.Mosedale,and D.J.Grainger.2002.Rapid
and noninvasive diagnosis of the presence and severity of coronary heart
disease using1H-NMR-based metabonomics.Nat Med 8:1439-1444.
Jodar,M.,A.Soler-Ventura,R.Oliva,Molecular Biology of Reproduction,
Development Research Group.2016.Semen proteomics and male infertility.J
Proteomics.[Epub ahead of print]
Dunn,W.B.,D.I.Broadhurst,H.J.Atherton,R.Goodacre,and
J.L.Griffin.2011.Systems level studies of mammalian metabolomes:the roles of
mass spectrometry and nuclear magnetic resonance spectroscopy.Chemical
Society reviews 40:387-426.
Glinski,M.,and W.Weckwerth.2006.The role of mass spectrometry in
plant systems biology.Mass spectrometry reviews 25:173-214.
Godin,J.P.,L.B.Fay,and G.Hopfgartner.2007.Liquid chromatography
combined with mass spectrometry for 13C isotopic analysis in life science
research.Mass spectrometry reviews 26:751-774.
Locasale,J.W.,A.R.Grassian,T.Melman,C.A.Lyssiotis,K.R.Mattaini,
A.J.Bass,G.Heffron,C.M.Metallo,T.Muranen,H.Sharfi,A.T.Sasaki,D.Anastasiou,
E.Mullarky,N.I.Vokes,M.Sasaki,R.Beroukhim,G.Stephanopoulos,A.H.Ligon,
M.Meyerson,A.L.Richardson,L.Chin,G.Wagner,J.M.Asara,J.S.Brugge,L.C.Cantley,
and M.G.Vander Heiden.2011.Phosphoglycerate dehydrogenase diverts glycolytic
flux and contributes to oncogenesis.Nat Genet 43:869-874.
Munger,J.,B.D.Bennett,A.Parikh,X.J.Feng,J.McArdle,H.A.Rabitz,T.Shenk,
and J.D.Rabinowitz.2008.Systems-level metabolic flux profiling identifies
fatty acid synthesis as a target for antiviral therapy.Nat Biotechnol 26:
1179-1186.
Nicholson,J.K.,J.Connelly,J.C.Lindon,and E.Holmes.2002.Metabonomics:a
platform for studying drug toxicity and gene function.Nat Rev Drug Discov 1:
153-161.
Soga,T.,M.Sugimoto,M.Honma,M.Mori,K.Igarashi,K.Kashikura,S.Ikeda,
A.Hirayama,T.Yamamoto,H.Yoshida,M.Otsuka,S.Tsuji,Y.Yatomi,T.Sakuragawa,
H.Watanabe,K.Nihei,T.Saito,S.Kawata,H.Suzuki,M.Tomita,and
M.Suematsu.2011.Serum metabolomics reveals gamma-glutamyl dipeptides as
biomarkers for discrimination among different forms of liver disease.Journal
of hepatology55:896-905.
Sreekumar,A.,L.M.Poisson,T.M.Rajendiran,A.P.Khan,Q.Cao,J.Yu,B.Laxman,
R.Mehra,R.J.Lonigro,Y.Li,M.K.Nyati,A.Ahsan,S.Kalyana-Sundaram,B.Han,X.Cao,
J.Byun,G.S.Omenn,D.Ghosh,S.Pennathur,D.C.Alexander,A.Berger,J.R.Shuster,
J.T.Wei,S.Varambally,C.Beecher,and A.M.Chinnaiyan.2009.Metabolomic profiles
delineate potential role for sarcosine in prostate cancer progression.Nature
457:910-914.
Suhre,K.,S.Y.Shin,A.K.Petersen,R.P.Mohney,D.Meredith,B.Wagele,
E.Altmaier,P.Deloukas,J.Erdmann,E.Grundberg,C.J.Hammond,M.H.de Angelis,
G.Kastenmuller,A.Kottgen,F.Kronenberg,M.Mangino,C.Meisinger,T.Meitinger,
H.W.Mewes,M.V.Milburn,C.Prehn,J.Raffler,J.S.Ried,W.Romisch-Margl,N.J.Samani,
K.S.Small,H.E.Wichmann,G.Zhai,T.Illig,T.D.Spector,J.Adamski,N.Soranzo,and
C.Gieger.2011.Human metabolic individuality in biomedical and pharmaceutical
research.Nature 477:54-60.
Wang,J.,P.Alexander,L.Wu,R.Hammer,O.Cleaver,and
S.L.McKnight.2009.Dependence of mouse embryonic stem cells on threonine
catabolism.Science325:435-439.
Wang,T.J.,M.G.Larson,R.S.Vasan,S.Cheng,E.P.Rhee,E.McCabe,G.D.Lewis,
C.S.Fox,P.F.Jacques,C.Fernandez,C.J.O'Donnell,S.A.Carr,V.K.Mootha,J.C.Florez,
A.Souza,O.Melander,C.B.Clish,and R.E.Gerszten.2011a.Metabolite profiles and
the risk of developing diabetes.Nat Med 17:448-453.
Wang,Z.,E.Klipfell,B.J.Bennett,R.Koeth,B.S.Levison,B.Dugar,
A.E.Feldstein,E.B.Britt,X.Fu,Y.M.Chung,Y.Wu,P.Schauer,J.D.Smith,H.Allayee,
W.H.Tang,J.A.DiDonato,A.J.Lusis,and S.L.Hazen.2011b.Gut flora metabolism of
phosphatidylcholine promotes cardiovascular disease.Nature 472:57-63.
Zhang,Y.,Y.Dai,J.Wen,W.Zhang,A.Grenz,H.Sun,L.Tao,G.Lu,D.C.Alexander,
M.V.Milburn,L.Carter-Dawson,D.E.Lewis,H.K.Eltzschig,R.E.Kellems,
M.R.Blackburn,H.S.Juneja,and Y.Xia.2011.Detrimental effects of adenosine
signaling in sickle cell disease.Nat Med 17:79-86.
Claims (1)
1. refining metabolism small molecule marker relevant to idiopathic male infertility is preparing idiopathic male infertility diagnosis or prison
Application in test agent box, the marker are that refining is metabolized small molecule acetylcarnitine and tryptophan.
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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 |
| CN105765383A (en) * | 2013-10-28 | 2016-07-13 | 唾液科技有限公司 | Salivary biomarker for cancer, method and device for assaying same, and method for determining salivary biomarker for cancer |
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| CN102449161A (en) * | 2009-03-31 | 2012-05-09 | 梅塔博隆公司 | Biomarkers related to insulin resistance and methods using the same |
| 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 |
| CN105765383A (en) * | 2013-10-28 | 2016-07-13 | 唾液科技有限公司 | Salivary biomarker for cancer, method and device for assaying same, and method for determining salivary biomarker for cancer |
| CN105784982A (en) * | 2016-04-19 | 2016-07-20 | 上海市计划生育科学研究所 | Application of seminal plasma unsaturated fatty acid metabolites |
Non-Patent Citations (1)
| Title |
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| Urinary phytoestrogen levels related to idiopathic male infertility in Chinese men;Yankai Xia 等;《Environment International》;20130629;第59卷;161-167 |
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