CN106290653A - The urine fatty acid metabolism thing mark relevant to idiopathic male infertility and detection method thereof and application - Google Patents
The urine fatty acid metabolism thing mark relevant to idiopathic male infertility and detection method thereof and application Download PDFInfo
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Abstract
The invention belongs to analytical chemistry and clinical medicine domain, disclose the urine fatty acid metabolism thing mark relevant to idiopathic male infertility and detection method thereof and application.This mark be this mark be urine fatty acid metabolism thing tetracosandioic acid and/or hexadecandioic acid (hexadecane diacid), use the detection of UPLC Q exactive MS method, this mark can be used for auxiliary diagnosis and the monitoring of idiopathic male infertility, has higher sensitivity and specificity, has clinical generalization value.
Description
Invention field
The invention belongs to analytical chemistry and clinical medicine domain, relate to the urine fatty acid relevant to idiopathic male infertility
Metabolite markers and detection method based on UPLC-Q exactive MS thereof and application.
Background technology
The whole world there are about the couple at child-bearing age of 15% and suffers sterile puzzlement.China is due to large population base, in newly-married couple
The patient numbers of sterility and infertility exceeds well over million, sterile is higher than 50% because of what male factor caused.Male sterility still has at present
About 40%～75% can not find reason, referred to as idiopathic male infertility disease.Idiopathic male infertility causes the most serious disease
Sick burden.
But, idiopathic male infertility diagnosis is extremely difficult.The standard of WHO male sterility be man and wife live together after marriage 1 year with
On, do not take contraceptives, owing to bridegroom's or husband's side reason causes the infertile person in the wife's side.But, the diagnostic criteria lived together 1 year, delays significantly
Male sterility carried out the time of early treatment and intervention；Due to practical reasons, a lot of man and wife does not ensures that strict living together
1 year so that last male sterility judges to become extremely difficult；And in order to get rid of wife's side reason, the wife's side also needs to carry out in detail
Thin inspection, brings heavy economy and medical burden.Traditional seminal parameters inspection only focus on sperm quantity, degrees of motion,
The conventional parameters such as semen volume, pH and liquefying time, it is impossible to react whole situations of seminal fluid all sidedly.Therefore, male sterility is frequent
Show as Sperm parameters Non Apparent Abnormality, also make traditional seminal parameters inspection can not efficient diagnosis male sterility.
In addition because being affected by factors such as abstinence time, Sperm parameters conventional analysis result is also embodied by bigger ripple
Dynamic property.Thus, clinical diagnosis generally requires with reference to repeatedly semen routine analysis, brings burden to doctor and patient.Thus, face
The new diagnostic method for idiopathic male infertility is needed badly on bed.
Metabolism group (Metabolomics/Metabonomics) is that grown up phase late 1990s is new
Set up schools section, and it is by investigating living things system after hereditary change or irriate or disturbance, the change of its metabolite or its with
The change of time, carrys out a science of postgraduate's objects system.So-called metabolism group (Metabolome) is the downstream product of genome
Also it is end product, is that some participate in organism metabolism, maintain organism normal function and the little molecularization of growth promoter
The set of compound, the mainly relative molecular mass little molecule of endogenous less than 1000, the little molecule of these endogenous metabolisms relates to
Carbohydrate metabolism, energy metabolism, lipid metabolism, amino acid metabolism, nucleic acid metabolism, coenzyme metabolism etc..
Organism under normal condition is a complete system, and the metabolite in biological fluid, cell and tissue is in
One stable poised state.Body there occurs pathological change due to heredity or the reason day after tomorrow, and this balance is just broken, generation
Thank to product and metabolic process also creates corresponding change.Little molecule metabolism is understood in lysis by metabonomic analysis
Change, people can be helped to find relevant biomarker (biomarker), can be with the diagnosis of aided disease, it is also possible to
People are helped to understand the pathogenesis of disease by the metabolic pathway that small-molecule substance itself relates to and provide spy for medicament research and development
The target of the opposite sex.In recent years, metabolism group achieves many in the research of mankind's various diseases in the early diagnosis of disease
The achievement in research being significant, such as cardiovascular disease, diabetes and cancer, correlative theses is published in academic journal
On " Nature ", " Nature medicine ", " Journal of hepatology " and " Cancer research ", represent
Potentiality that metabolism little molecule is huge in diagnosing human disease and value.It should be noted that urine is the most easily to obtain
The biological specimen obtained, has noinvasive, bulky advantage.It is particularly suitable for diagnosis and the examination of disease.But, use metabolism group
Analyze the application in the diagnostic monitoring of idiopathic male infertility of the urine metabolism little molecule to be paid close attention to the most accordingly.
Metabolism group research common technology includes liquid chromatograph mass spectrography (LC-MS), chromatography of gases-mass spectrum connection at present
With (GC-MS) and nuclear magnetic resonance technique (NMR).Nuclear magnetic resonance technique feature is without destroying to component to be measured, Sample pretreatment letter
Single, but sensitivity is relatively low；Gas chromatography-mass spectrography has preferable sensitivity and a repeatability, but derivatization to be used
Method carries out pre-treatment to sample so that experimental procedure becomes complicated.And LC-MS has sample process simply, highly sensitive, face
The feature that bed is practical.UPLC-Q exactive MS is a new generation's high resolution mass spectrum and the combination of ultra high efficiency liquid phase, has
Compare sensitivity, specificity and stability higher for traditional LC-MS.So using UPLC-Q exactive MS to carry out urine
Metabonomic analysis, if can find, the stable special urine metabolism little molecule relevant to idiopathic male infertility morbidity is as life
Thing mark, and research and develop the UPLC-Q exactive MS detection method of the little molecular marker of corresponding metabolism, not only in this field
It is in first place in the world, also can create the economic benefit attracted people's attention, to improving China's male reproductive health level also will be
The most strong promotion.
Summary of the invention
It is an object of the invention to provide the urine fatty acid metabolism thing mark relevant to idiopathic male infertility.
Another object of the present invention is to provide the detection method of above-mentioned urine fatty acid metabolism thing mark.
A further object of the present invention is to provide the test kit detecting above-mentioned urine fatty acid metabolism thing mark.
It is an object of the invention to be realized by following technical measures:
The urine fatty acid metabolism thing mark relevant to idiopathic male infertility, this mark is urine fatty acid metabolism
Thing tetracosandioic acid and/or hexadecandioic acid (hexadecane diacid).
Described urine fatty acid metabolism thing mark is in preparing idiopathic male infertility diagnosis or monitoring reagent box
Application.
A kind of diagnosis or the test kit of monitoring idiopathic male infertility, this test kit contains detection urine fatty acid metabolism thing
Tetracosandioic acid and/or the reagent of hexadecandioic acid (hexadecane diacid).
Described test kit, this test kit contains employing UPLC-Q exactive MS method detection urine fatty acid metabolism
Thing tetracosandioic acid and/or the reagent of hexadecandioic acid (hexadecane diacid).
Described test kit, this test kit contains following reagent:
Tetracosandioic acid standard substance
Hexadecandioic acid (hexadecane diacid) standard substance
Internal standard A: the acid of creatinine, valine, nicotinic acid, thymus pyrimidine, 1,3-propanedicarboxylic acid, L-phenylpropylamine, N-acetaminophen, horse
The Isotopic Internal Standard (deuterium mark, aqueous solution) of one or more materials 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, this test kit possibly together with:
Hypersil GOLD C18 chromatographic column；
Reagent A: containing 100% methanol (protein precipitation is used)；
Reagent B: the water (flowing is used mutually) containing the formic acid of 0.1%；
Reagent C: the acetonitrile (flowing is used mutually) containing the formic acid of 0.1%；
Reagent D: 100% ultra-pure water (redissolves and uses).
A kind of method detecting the above-mentioned urine fatty acid metabolism thing mark relevant to idiopathic male infertility, the party
Method uses UPLC-Q exactive MS method, detection urine fatty acid metabolism thing tetracosandioic acid and/or hexadecandioic acid (hexadecane diacid)
Content.
In the 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 is: 0-3min 1%B, 3-10min 1% to 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%, lower with)
Input mode: volume 5 μ l；
Two, Mass Spectrometry Conditions
Heating electron spray ionisation mode (HESI) is used to be analyzed, positive ion mode spray voltage: 3.5kV；Anion
Pattern spray voltage: 2.5kV；Capillary temperature under both of which: 250 DEG C, heter temperature: 425 DEG C, sheath entraining air stream: 50AU,
Auxiliary entraining air stream: 13AU, blowback air air-flow: 0AU；Lens voltage: 60V；Use and entirely sweep pattern, sweep limits: 70 arrive 1050m/
z；Resolution: 70000.
The present invention is described in detail as follows:
The present inventor gathers standard compliant urine specimen with S.O.P. (SOP), the crowd that systematic collection is complete
Back ground Information and clinical data, and have employed metabolism group method based on UPLC-Q exactive MS and be analyzed.
The experimental technique studied specifically mainly includes following components:
One, object of study selects and packet foundation
First stage screening stage
Include idiopathic male infertility 607 people and normal healthy controls 430 people clarified a diagnosis, totally 1037 people at random in.
A group: normal healthy controls group (430 people):
1. the age is between 19 to 39 years old；
2. constitutional index is between 17 to 31；
3. the male that reproductive performance is healthy, and had healthy offspring after 5-8 month；
4. without whole body major disease.
B group: idiopathic male infertility disease group (607 people):
1. the age mates with matched group；
2. constitutional index mates with matched group
3. attempt not success in conceived 12 months, and spouse does not has the male of infertile disease；.
4. without the clear and definite male sterility cause of disease；
5. Smoking And Drinking history is mated with matched group；
6. nationality mates with matched group；
7. without whole body major disease.
Second stage Qualify Phase
Include idiopathic male infertility 15 people and normal healthy controls 15 people clarified a diagnosis, totally 30 people in.
A group: normal healthy controls group (15 people):
1. the age is between 24 to 36 years old；
2. constitutional index is between 19 to 24；
3. the male that reproductive performance is healthy, and had healthy offspring after 5-8 month；
4. without whole body major disease.
B group: idiopathic male infertility disease group (15 people):
1. the age mates with matched group；
2. constitutional index mates with matched group；
3. attempt not success in conceived 12 months, and spouse does not has the male of infertile disease；
4. without the clear and definite male sterility cause of disease；
5. Smoking And Drinking history is mated with matched group；
6. nationality mates with matched group；
7. without whole body major disease.
Two, UPLC-Q exactive MS metabonomic analysis and idiopathic male infertility diagnosis fatty acid metabolism thing sieve
Choosing and checking
1. Sample pretreatment
1. take 300 μ L urines, add 10 μ L internal standards A, add 10 μ L internal standards B, add 10 μ L internal standards C, add methanol 900 μ L
(reagent A), vortex 30s.
2. in centrifuge, 4 DEG C of 16000g are centrifuged 15min, supernatant is transferred to 1.5mL import EP pipe, and is existed by supernatant
It is concentrated to dryness in centrifugal concentrating drying instrument under room temperature condition.
3. redissolve with 10 μ L ultra-pure water (reagent D), to be analyzed.
2. instrument detection
1. analytical tool: UPLC Ultimate 3000system (Dionex) high performance liquid chromatograph；Q-Exactive
High-resolution mass spectrometer.
2. liquid-phase condition:
2.1. liquid-phase chromatographic column is Hypersil GOLD C18 chromatographic column (100mm × 2.1mm, particle diameter 1.9 μm, Thermo
Scientific, Germany), column temperature is 40 DEG C.
2.2 flowings used are (A) water (reagent B) containing 0.1% formic acid and (B) acetonitrile (reagent containing 0.1% formic acid mutually
C), flow velocity is 400 μ L/min.
2.3 instrument gradients are: instrument gradient is: 0-3min 1%B, 3-10min 1% to 99%B, 10-13min 99%
B, 13-13.1min 99% to 1%B, 13.1-17min 1%B.
2.4 input modes: volume 5 μ l.
3. Mass Spectrometry Conditions
3.1 heating electron spray ionisation mode (HESI) are analyzed.
3.2 use heating electron spray ionisation mode (HESI), positive ion mode spray voltage: 3.5kV；Negative ion mode sprays
Mist voltage: 2.5kV；Capillary temperature under both of which: 250 DEG C, heter temperature: 425 DEG C, sheath entraining air stream: 50AU, assist gas
Air-flow: 13AU, blowback air air-flow: 0AU；Lens voltage: 60V.Use and entirely sweep pattern, sweep limits: 70 arrive 1050m/z；Differentiate
Rate: 70000.
3. material is qualitative
The qualitative employing of fatty acid metabolism thing and standard substance tetracosandioic acid and hexadecandioic acid (hexadecane diacid) comparison Chromatographic information are (during reservation
Between) and Information in Mass Spectra (accurate molecular weight), and in real time in comparison sample the Chromatographic information of Isotopic Internal Standard standard substance series with school
Positive retention time.
4. data analysis:
Biomarker screening uses Multivariate Logistic Regression to confirm key metabolites.
5. the difference of fatty acid metabolism thing and diagnostic significance in normal healthy controls group, idiopathic male infertility group urine specimen.
The information of corrected age, constitutional index, smoking and history of drinking history, logistic regression finds urine
The content increase of tetracosandioic acid and hexadecandioic acid (hexadecane diacid) significantly improves the generation of idiopathic male infertility.Random crowd is used to answer
With above-mentioned fatty acid metabolism thing combined diagnosis idiopathic male infertility, sensitivity is 93.33%, and specificity is 93.33%, ROC
Area under curve is 0.9822, has higher additive diagnostic value.
Three, diagnostic reagent box preparation method
According to above-mentioned series of experiments result, the present inventor is also prepared for one and can be used for idiopathic male infertility and dynamically supervise
The diagnostic kit surveyed, described diagnostic kit comprises stable existence and detectable tetracosandioic acid in mensuration experimenter's urine
Standard substance and the internal standard standard substance series of assistant analysis with hexadecandioic acid (hexadecane diacid).Diagnostic kit can also include a set of urine
Fatty acid metabolism thing extracts 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
The little molecule of metabolism, it was found that in urine exist can be used for assessing whether suffer from idiopathic male infertility, have auxiliary diagnosis valency
The urine fatty acid metabolism thing mark combination of value, and the UPLC-Q exactive of this fatty acid metabolism thing marker detection
The application of MS, develops and can be easy to the idiopathic male infertility diagnosis of clinical practice, monitoring reagent box.
The present invention uses the little molecule of urine metabolism to be advantageous in that as the mark of idiopathic male infertility evaluation:
(1) the little molecule of urine metabolism is a kind of new biomarkers, and it associates with disease outcome by force, not only stable, nothing
Wound, it is prone to detection, and quantitatively accurate, will be greatly improved the Sensitivity and Specificity of idiopathic male infertility diagnosis, such little point
Preventing and treating for idiopathic male infertility is started brand-new situation by the successful exploitation of sub-biomarker, for other diseases biological marker
The development of thing is offered reference.
(2) the little molecular marker of urine metabolism that the present invention provides can be used as the diagnosis marker of idiopathic male infertility,
Can in Check-up crowd the disease condition of clear and definite idiopathic male infertility, thus for the further testing in depth testing of clinician provide depend on
According to, for quick and precisely grasping morbid state and the coincident with severity degree of condition of patient, taking the control prece of more personalized to carry in time
For supporting, delay and stop progression of disease.
(3) present invention uses the urine specimen of idiopathic male infertility and the random crowd of normal healthy controls to verify, it was demonstrated that
In urine, tetracosandioic acid and hexadecandioic acid (hexadecane diacid) level have higher sensitivity and special in diagnosis idiopathic male infertility
Degree, can use as mark.
(4) present invention uses checking tight, multistage and appraisement system, and the initial stage screens multiple urine generation by preliminary experiment
Thanking to little molecule, application UPLC-Q exactive MS carries out independent crowd's checking, it is ensured that this urine metabolism biomarker and
The reliability of diagnostic method.
(5) UPLC-Q exactive MS technology sample process is simple, and Instrumental Analysis is accurate rapidly, has higher clinic
Diagnosis practical value.
Accompanying drawing explanation
Fig. 1 screening stage, the information of corrected age, constitutional index, smoking and history of drinking history, Multivariate Logistic Regression
Analyze the generation finding that the content increase of urine tetracosandioic acid and hexadecandioic acid (hexadecane diacid) significantly improves idiopathic male infertility.a
Do not adjust the single factor test Logistic regression result of Confounding Factor.bAfter adjusting age, constitutional index, smoking and history of drinking history many
Unit's Logistic regression result.
The horizontal undulatory property of Fig. 2 fatty acid metabolism analyte detection (mean ± standard deviation).
Fig. 3 Qualify Phase, the Normal group of employing urine tetracosandioic acid content information making and idiopathic male are not
Educate the ROC curve between group.
Fig. 4 Qualify Phase, the Normal group of employing urine hexadecandioic acid (hexadecane diacid) content information making and idiopathic male are not
Educate the ROC curve between group.
Fig. 5 Qualify Phase, use Normal group that urine tetracosandioic acid and hexadecandioic acid (hexadecane diacid) content information makes and
ROC curve between idiopathic male infertility group.
Detailed description of the invention
The invention will be further elaborated by the following examples.
Embodiment 1 object of study selects and packet foundation
This part object of study examines idiopathic male infertility case and health from the head of Affiliated Hospital of Nanjing Medical University
Fertility comparison.Research contents and Informed Consent Form all obtain the approval of Ethics Committee of Nanjing Medical University, meet relevant laws and regulations
Requirement.Case and to impinge upon understanding content after endorsed Informed Consent Form.All object of study have all carried out complete physique
Check, and complete portion and include individual's basic data, living habit, occupation and environmental exposure, genetic risk factors, sexual function
Questionnaire with reproductive function, history of disease and physical exertion.First stage incorporates satisfactory 607 example idiopathic male
Sterile case and 430 example normal healthy controls；The satisfactory 15 example idiopathic male infertility cases of second stage and 15 example health are right
According to the screening experiment object as the little Molecular biomarkers of idiopathic male infertility urine metabolism.Concrete sample group standard
As follows:
First stage screening stage
Include idiopathic male infertility 607 people and normal healthy controls 430 people clarified a diagnosis, totally 1037 people at random in.
A group: normal healthy controls group (430 people):
1. the age is between 19 to 39 years old；
2. constitutional index is between 17 to 31；
3. the male that reproductive performance is healthy, and had healthy offspring after 5-8 month；
4. without whole body major disease.
B group: idiopathic male infertility disease group (607 people):
1. the age mates with matched group；
2. constitutional index mates with matched group
3. attempt not success in conceived 12 months, and spouse does not has the male of infertile disease；.
4. without the clear and definite male sterility cause of disease；
5. Smoking And Drinking history is mated with matched group；
6. nationality mates with matched group；
7. without whole body major disease.
Second stage Qualify Phase
Include idiopathic male infertility 15 people and normal healthy controls 15 people clarified a diagnosis, totally 30 people in.
A group: normal healthy controls group (15 people):
1. the age is between 24 to 36 years old；
2. constitutional index is between 19 to 24；
3. the male that reproductive performance is healthy, and had healthy offspring after 5-8 month；
4. without whole body major disease.
B group: idiopathic male infertility disease group (15 people):
1. the age mates with matched group；
2. constitutional index mates with matched group；
3. attempt not success in conceived 12 months, and spouse does not has the male of infertile disease；
4. without the clear and definite male sterility cause of disease；
5. Smoking And Drinking history is mated with matched group；
6. nationality mates with matched group；
7. without whole body major disease.
Embodiment 2UPLC-MS metabolism group idiopathic male infertility biomarker screens
1. Sample pretreatment
1. take 300 μ L urines, add 10 μ L internal standards A, add 10 μ L internal standards B, add 10 μ L internal standards C, add methanol 900 μ L
(reagent A), vortex 30s.
2. in centrifuge, 4 DEG C of 16000g are centrifuged 15min, supernatant is transferred to 1.5mL import EP pipe, and is existed by supernatant
It is concentrated to dryness in centrifugal concentrating drying instrument under room temperature condition.
3. redissolve with 10 μ L ultra-pure water (reagent D), to be analyzed.
2. instrument detection
1. analytical tool: UPLC Ultimate 3000system (Dionex) high performance liquid chromatograph；Q-Exactive
High-resolution mass spectrometer.
2. liquid-phase condition:
2.1. liquid-phase chromatographic column is Hypersil GOLD C18 chromatographic column (100mm × 2.1mm, particle diameter 1.9 μm, Thermo
Scientific, Germany), column temperature is 40 DEG C.
2.2 flowings used are (A) water (reagent B) containing 0.1% formic acid and (B) acetonitrile (reagent containing 0.1% formic acid mutually
C), flow velocity is 400 μ L/min.
2.3 instrument gradients are: instrument gradient is: 0-3min 1%B, 3-10min 1% to 99%B, 10-13min 99%
B, 13-13.1min 99% to 1%B, 13.1-17min 1%B.
2.4 input modes: volume 5 μ l.
3. Mass Spectrometry Conditions
3.1 heating electron spray ionisation mode (HESI) are analyzed.
3.2 use heating electron spray ionisation mode (HESI), positive ion mode spray voltage: 3.5kV；Negative ion mode sprays
Mist voltage: 2.5kV；Capillary temperature under both of which: 250 DEG C, heter temperature: 425 DEG C, sheath entraining air stream: 50AU, assist gas
Air-flow: 13AU, blowback air air-flow: 0AU；Lens voltage: 60V.Use and entirely sweep pattern, sweep limits: 70 arrive 1050m/z；Differentiate
Rate: 70000.
3. material is qualitative
The qualitative employing of fatty acid metabolism thing and standard substance tetracosandioic acid and hexadecandioic acid (hexadecane diacid) comparison Chromatographic information are (during reservation
Between) and Information in Mass Spectra (accurate molecular weight), and in real time in comparison sample the Chromatographic information of Isotopic Internal Standard standard substance series with school
Positive retention time.
4. data analysis:
Biomarker screening uses Multivariate Logistic Regression to confirm key metabolites.
5. the difference of fatty acid metabolism thing and diagnostic significance in normal healthy controls group, idiopathic male infertility group urine specimen.
The information of corrected age, constitutional index, smoking and history of drinking history, logistic regression finds urine
The content increase of tetracosandioic acid and hexadecandioic acid (hexadecane diacid) significantly improves the generation (Fig. 1) of idiopathic male infertility.
The stability analysis of embodiment 3 urine fatty acid metabolism thing
Use embodiment 2 method the stability of urine tetracosandioic acid and hexadecandioic acid (hexadecane diacid) level is evaluated (
Interval is 2 weeks).Result shows, in urine, tetracosandioic acid and hexadecandioic acid (hexadecane diacid) measure horizontal stable (Fig. 2), possess conduct
The characteristic of diagnosis/monitoring mark.
The embodiment 4 fatty acid metabolism thing combination diagnosis to idiopathic male infertility
According to above-mentioned UPLC-Q exactive MS metabolism group method, the present inventor by random crowd 15 case and
The urine sample detection tetracosandioic acid of 15 comparisons and hexadecandioic acid (hexadecane diacid), draw ROC curve with this and assess the susceptiveness of diagnosis
And this 2 fatty acid metabolism thing levels evaluation capacity to idiopathic male infertility in specificity, and then assessment detection urine.
The sensitivity of tetracosandioic acid is 80.00%, and specificity is 80.00%, and under ROC curve, area is 0.8489 (figure
3)；
Hexadecandioic acid (hexadecane diacid) sensitivity is 80.00%, and specificity is 80.00%, and under ROC curve, area is 0.9289 (Fig. 4)；
The sensitivity of combination tetracosandioic acid and hexadecandioic acid (hexadecane diacid) is 93.33%, and specificity is 93.33%, under ROC curve
Area is 0.9822 (Fig. 5).
So combination tetracosandioic acid and hexadecandioic acid (hexadecane diacid) have the ability preferably diagnosing idiopathic male infertility.
Embodiment 5 is for idiopathic male infertility urine fatty acid metabolism analyte detection and the making of diagnostic kit
The method first passing through UPLC-Q exactive MS determines in normal control and idiopathic male infertility urine have
There is the little molecule of more abundant metabolism.Then, sieved by metabonomic technology based on UPLC-Q exactive MS wherein
Select the fatty acid metabolism thing relevant to idiopathic male infertility, as the diagnosis index being whether idiopathic male infertility.Finally
Controlling at 2 by the quantity of the corresponding urine fatty acid metabolism thing filtered out, this is the optimum made on the basis of preliminary experiment
That changes simplifies.Use this 2 fatty acid metabolism things, both can ensure preferable sensitivity and specificity, again can be cost-effective, subtract
The burden of light patient, moreover it is possible to reduce the detection time, there is quick, accurate, economic advantage, it is simple to clinical expansion uses, certainly
Use wherein 1 mark can also, use 2 mark effects more preferable.This test kit includes a collection of urine fatty acid metabolism
Analyte detection reagent and consumptive material, the wherein qualitative and quantitative employing tetracosandioic acid of fatty acid metabolism thing and the mark of hexadecandioic acid (hexadecane diacid)
Quasi-product, assistant analysis uses internal standard A: creatinine, valine, nicotinic acid, thymus pyrimidine, 1,3-propanedicarboxylic acid, L-phenylpropylamine are sour, N-acetyl pair
Amino phenols, the deuterium mark Isotopic Internal Standard of eight kinds of materials of hippuric acid.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.Other also has the supporting reverse chromatograms post (Hypersil for UPLC chromatographic isolation
GOLD C18 chromatographic column, 100mm × 2.1mm, particle diameter 1.9 μm), for the reagent (water of the formic acid containing 0.1% and containing of the phase that flows
The acetonitrile of the formic acid of 0.1%), for extracting the reagent (100% ultra-pure water) of fatty acid metabolism thing.The value of this test kit is
Have only to 300 μ l urines, the content of urine fatty acid metabolism thing mark can be detected, then diagnose idiopathic male by content
Sterile, and be prone to carry out dynamic monitoring and observe therapeutic effect.
Concrete test kit composition is as follows:
Tetracosandioic acid standard substance
Hexadecandioic acid (hexadecane diacid) standard substance
Internal standard A: the acid of creatinine, valine, nicotinic acid, thymus pyrimidine, 1,3-propanedicarboxylic acid, L-phenylpropylamine, N-acetaminophen, horse
The deuterium mark Isotopic Internal Standard of eight kinds of materials 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, it is also possible to contain:
Chromatographic column (Thermo 100mm × 2.1mm, particle diameter 1.9 μm, Hypersil GOLD C18 chromatographic column)
Reagent A (containing 100% methanol)
Reagent B (water of the formic acid containing 0.1%)
Reagent C (acetonitrile of the formic acid containing 0.1%)
Reagent D (100% ultra-pure water).
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Claims (8)
1. the urine fatty acid metabolism thing mark relevant to idiopathic male infertility, it is characterised in that this mark is urine fat
Fat acid metabolic thing tetracosandioic acid and/or hexadecandioic acid (hexadecane diacid).
2. the urine fatty acid metabolism thing mark described in claim 1 is preparing idiopathic male infertility diagnosis or monitoring reagent
Application in box.
3. a diagnosis or the test kit of monitoring idiopathic male infertility, it is characterised in that this test kit contains detection urine fat
Acid metabolic thing tetracosandioic acid and/or the reagent of hexadecandioic acid (hexadecane diacid).
Test kit the most according to claim 3, it is characterised in that this test kit contains employing UPLC-Q exactive MS
Method detection urine fatty acid metabolism thing tetracosandioic acid and/or the reagent of hexadecandioic acid (hexadecane diacid).
Test kit the most according to claim 4, it is characterised in that this test kit contains following reagent:
Tetracosandioic acid standard substance；
Hexadecandioic acid (hexadecane diacid) standard substance；
Internal standard A: the acid of creatinine, valine, nicotinic acid, thymus pyrimidine, 1,3-propanedicarboxylic acid, L-phenylpropylamine, N-acetaminophen, hippuric acid
In the Isotopic Internal Standard of one or more materials；
Internal standard B: the Isotopic Internal Standard of pentadecanoic acid；
Internal standard C: the Isotopic Internal Standard of lignoceric acid.
Test kit the most according to claim 5, it is characterised in that this test kit possibly together with:
Hypersil GOLD C18 chromatographic column；
Reagent A: containing 100% methanol；
Reagent B: containing the water of the formic acid of 0.1%；
Reagent C: containing the acetonitrile of the formic acid of 0.1%；
Reagent D: 100% ultra-pure water.
7. one kind is detected the urine fatty acid metabolism thing mark the most relevant to idiopathic male infertility
Method, it is characterised in that the method uses UPLC-Q exactive MS method, detects the urine fatty acid metabolism thing tetradecane two
Acid and/or the content of hexadecandioic acid (hexadecane diacid).
Method the most according to claim 7, it is characterised in that in the 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 is: 0-3min 1%B, 3-10min 1% to 99%B, 10-13min 99%B, 13-13.1min 99%
To 1%B, 13.1-17min 1%B；
Input mode: volume 5 μ l；
Two, Mass Spectrometry Conditions
Heating electron spray ionisation mode (HESI) is used to be analyzed, 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 entraining air stream: 50AU, auxiliary
Entraining air stream: 13AU, blowback air air-flow: 0AU；Lens voltage: 60V；Use and entirely sweep pattern, sweep limits: 70 arrive 1050m/z；Point
Resolution: 70000.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108872404A (en) * | 2017-05-09 | 2018-11-23 | 中国科学院上海生命科学研究院 | The application of overlength chain saturated fatty acid or its detection reagent in metabolic syndrome risk profile |
| CN108982721A (en) * | 2018-10-18 | 2018-12-11 | 云南中烟工业有限责任公司 | A kind of method that ultra high efficiency hydrophilic chromatographic quickly measures creatinine and uric acid in smoker urine |
| CN109187792A (en) * | 2018-09-27 | 2019-01-11 | 南京医科大学 | Tryptosol and xanthosine are as idiopathic male infertility diagnosis marker and its application in urine |
| CN109187793A (en) * | 2018-09-27 | 2019-01-11 | 南京医科大学 | 1- palmityl-SN- glycerine-phosphocholine and dodecanedioic acid are idiopathic male infertility diagnosis marker and its application |
| CN114414694A (en) * | 2022-01-21 | 2022-04-29 | 苏州南医大创新中心 | Molecular marker related to azoospermia and detection method and application thereof |
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| CN108872404A (en) * | 2017-05-09 | 2018-11-23 | 中国科学院上海生命科学研究院 | The application of overlength chain saturated fatty acid or its detection reagent in metabolic syndrome risk profile |
| CN109187792A (en) * | 2018-09-27 | 2019-01-11 | 南京医科大学 | Tryptosol and xanthosine are as idiopathic male infertility diagnosis marker and its application in urine |
| CN109187793A (en) * | 2018-09-27 | 2019-01-11 | 南京医科大学 | 1- palmityl-SN- glycerine-phosphocholine and dodecanedioic acid are idiopathic male infertility diagnosis marker and its application |
| CN109187792B (en) * | 2018-09-27 | 2022-04-08 | 南京医科大学 | Tryptophanol and xanthosine in urine as diagnostic markers of idiopathic male infertility and application thereof |
| CN108982721A (en) * | 2018-10-18 | 2018-12-11 | 云南中烟工业有限责任公司 | A kind of method that ultra high efficiency hydrophilic chromatographic quickly measures creatinine and uric acid in smoker urine |
| CN114414694A (en) * | 2022-01-21 | 2022-04-29 | 苏州南医大创新中心 | Molecular marker related to azoospermia and detection method and application thereof |
| CN114414694B (en) * | 2022-01-21 | 2023-04-28 | 苏州南医大创新中心 | Molecular marker related to azoospermia, and detection method and application thereof |
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