CN109187792B

CN109187792B - Tryptophanol and xanthosine in urine as diagnostic markers of idiopathic male infertility and application thereof

Info

Publication number: CN109187792B
Application number: CN201811131373.7A
Authority: CN
Inventors: 陈敏健; 丁兴旺; 管昱盛; 张怡云; 周昆; 徐菠; 庄婷钰; 张晓玲; 韩秀梅; 胡艳辉
Original assignee: Nanjing Medical University
Current assignee: Nanjing Medical University
Priority date: 2018-09-27
Filing date: 2018-09-27
Publication date: 2022-04-08
Anticipated expiration: 2038-09-27
Also published as: CN109187792A

Abstract

The invention belongs to the fields of analytical chemistry and clinical medicine, and discloses tryptophanol and xanthosine in urine as idiopathic male sterility diagnostic markers and application thereof. The marker is tryptophanol and xanthosine, has high sensitivity and specificity in diagnosing idiopathic male sterility, and can be used for preparing diagnostic or monitoring reagent for idiopathic male sterility.

Description

Tryptophanol and xanthosine in urine as diagnostic markers of idiopathic male infertility and application thereof

Technical Field

The invention belongs to the fields of analytical chemistry and clinical medicine, and relates to tryptophanol and xanthosine related to idiopathic male infertility in urine as biomarkers and a detection method and application thereof based on UPLC-Q active MS.

Background

At present, the number of people who are infertile and infertile in newly married couples in China is far more than one million. The current research considers that genetic factors such as the sequence change of AZF gene region of Y chromosome, the copy number change of DAZ gene, the sequence change of sex hormone receptor gene and the like, and environmental factors such as harmful factor exposure, nutrition, living habits and the like are all related to male sterility. It is noted that a significant portion of male infertility is manifested as unexplained etiology, i.e., idiopathic male infertility, which makes it extremely difficult to diagnose idiopathic male infertility and delays the timing of treatment. The WHO diagnosis standard of male infertility is that the couple lives together for more than 1 year after marriage, and no contraceptive measures are taken, so that the male causes the female to be infertile. However, for practical reasons many couples cannot guarantee strict co-existence for 1 year, so that the last judgment of male sterility becomes very difficult; the observation time of the same residence is as long as 1 year, so that the time for early treatment and intervention of male infertility is greatly delayed; in order to eliminate the reason for the female, the female also needs to perform detailed examination, which brings heavy economic and medical burden. The existing male sterility related examination depends on the conventional semen routine examination, and only focuses on the routine parameters of the number of sperms, the motility, the semen volume, the pH value, the liquefaction time and the like. The conventional analysis result of the conventional semen parameters shows larger fluctuation because of the influence of factors such as abstinence time and the like. Therefore, clinical diagnosis often requires reference to multiple semen routine analyses, which puts a burden on doctors and patients. More importantly, conventional semen parameter examination does not fully reflect the entire condition of the semen. Therefore, male infertility is often manifested as no obvious abnormality in conventional semen parameters, and the traditional semen parameter examination cannot effectively diagnose male infertility. Thus, there is a clinical need for new diagnostic methods for idiopathic male infertility.

Metabolomics (metabolomics) is another new important research field of post-genomics, transcriptomics and proteomics, which is the science of studying the changes of all metabolites produced by a biological system under external stimuli, and focuses on the changes of small molecule metabolites with molecular weight less than 1000 in the metabolic cycle, which reflect the changes of the body's response to disease states or environmental stimuli. Metabonomics can systematically review, analyze and explore small molecular biomarkers produced by cells, tissues and organs and changes thereof, so that the material basis of functional phenotype change can be more intuitively understood, the objects relate to metabolic networks of organisms such as lipid metabolism, sugar metabolism, amino acid metabolism, nucleic acid metabolism, coenzyme metabolism and the like, and the metabonomics are closer to phenotype omics than genomics, proteomics and transcriptomics. Metabonomics can greatly reduce the sampling trauma of patients because the analyzed samples can be urine non-invasive samples. Metabonomics shows extremely high application potential and value in the diagnosis of complex diseases, and has the characteristics of high sensitivity and stability. Metabonomics finds out specific biomarkers from the whole metabolic components of organisms by analyzing and researching the whole metabolic components of the organisms, and can diagnose clinical diseases timely, accurately, highly sensitively and specifically. At present, metabonomics is mainly applied to coronary heart disease, liver disease, diabetes, hypertension, obesity and tumor in the aspect of disease diagnosis. It is worth noting that urine is a biological sample which is easy to obtain clinically, and has the advantages of being noninvasive and large in size. Is particularly suitable for diagnosing and screening diseases. However, the application of the urine metabolism small molecules in the diagnosis and monitoring of idiopathic male infertility by using metabonomics analysis has not received corresponding attention.

UPLC-Q active MS is a combination of a new generation of high resolution mass spectrum and an ultra-high performance liquid phase, and has higher sensitivity, specificity and stability compared with the traditional LC-MS. Therefore, the UPLC-Q active MS is adopted to carry out metabonomic analysis of urine metabolism micromolecules, if stable specific urine metabolism micromolecules related to the onset of idiopathic male infertility can be found as biomarkers, and a UPLC-Q active MS detection method of corresponding metabolism micromolecule markers is developed, the UPLC-Q active MS detection method is in an international leading position in the field, can create remarkable economic benefits, and has wide significance for promoting the male reproductive health in China.

Disclosure of Invention

The invention aims to provide urine tryptophanol and xanthosine markers related to idiopathic male infertility.

The invention also aims to provide a detection method of the urine tryptophanol and xanthosine markers.

Still another object of the present invention is to provide a kit for detecting the above-mentioned urinary tryptophanol and xanthosine markers.

The aim of the invention is achieved by the following technical measures:

urine markers related to idiopathic male infertility are urine tryptophanol and xanthosine.

The urine tryptophanol and xanthosine markers are applied to the preparation of a diagnostic or monitoring kit for idiopathic male infertility.

A kit for diagnosing or monitoring idiopathic male infertility, the kit comprising reagents for detecting tryptophanol and xanthosine in urine.

The kit contains a reagent for detecting tryptophanol and xanthosine in urine by using a UPLC-Q active MS method.

The kit comprises the following reagents:

tryptophanol and xanthosine standards;

internal standard A: isotope internal standard (deuterium standard, water solution) of one or more substances of creatinine, valine, nicotinic acid, thymine, glutaric acid, L-phenylalanine, N-acetyl-p-aminophenol and hippuric acid;

internal standard B: isotopic internal standards of pentadecanoic acid (deuterium standard, methanol solution);

internal standard C: isotopic internal standard of tetracosanoic acid (deuterium standard, methanol solution).

Further, the kit further comprises:

hypersil GOLD C18 chromatography column;

reagent A: precipitating protein with 100% methanol;

and (3) reagent B: for the mobile phase, water containing 0.1% formic acid;

and (3) reagent C: the mobile phase is acetonitrile containing 0.1 percent of formic acid;

and (3) reagent D: for reconstitution, ultra pure water.

A method for detecting tryptophanol and xanthosine markers in urine related to idiopathic male infertility adopts UPLC-Q active MS method to detect tryptophanol and xanthosine content in urine.

The detection method comprises the following steps:

firstly, liquid phase conditions:

the liquid chromatographic column is Hypersil GOLD C18 chromatographic column, and the column temperature is 40 ℃;

the mobile phase A is water containing 0.1% formic acid, the mobile phase B is acetonitrile containing 0.1% formic acid, and the flow rate is 400 μ L/min;

the instrument gradient was: 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; (the amount of mobile phase A in each gradient is 100% of the amount of the corresponding mobile phase B, the same applies hereinafter)

And (3) sample introduction mode: volume 5. mu.l;

second, Mass Spectrometry Condition

Analysis was performed using a heating electrospray ionization (HESI) mode, positive ion mode spray voltage: 3.5 kV; negative ion mode spray voltage: 2.5 kV; capillary temperature in two modes: 250 ℃, heater temperature: 425 ℃, sheath gas flow: 50AU, auxiliary gas flow: 13AU, reverse air flow: 0 AU; lens voltage: 60V; adopting a full-scanning mode, wherein the scanning range is as follows: 70 to 1050 m/z; resolution ratio: 70000.

the invention is described in detail below:

the inventor collects urine samples meeting the standard by a Standard Operation Program (SOP), and the system collects complete basic information and clinical data of people and adopts a metabolic group method based on UPLC-Q active MS for analysis.

The experimental method of research mainly includes the following parts:

first, research object selection and grouping basis

First stage screening stage

Specifically diagnosed idiopathic male infertility 607 and healthy control 430 were randomized, for a total of 1037.

Group A: healthy control group (430 people):

1. age between 19 and 39 years;

2. a body mass index between 17 and 31;

3. a male with healthy reproductive capacity and having healthy offspring after 6-8 months;

4. there is no serious disease of the whole body.

Group B: idiopathic male infertility disease group (607 human):

1. age matched to control group;

2. the body mass index is matched with a control group;

3. a male with no success in attempting pregnancy for 12 months and a spouse without infertility disorder;

4. there is no clear cause of male infertility;

5. the smoking and drinking history is matched with the control group;

6. matching nationality with a control group;

7. there is no serious disease of the whole body.

Second phase verification phase

Specifically diagnosed idiopathic male infertility 15 and healthy controls 15 were included, for a total of 30.

Group A: healthy control group (15 persons):

1. age between 24 and 36 years;

2. a body mass index between 19 and 24;

4. there is no serious disease of the whole body.

Group B: idiopathic male infertility disease group (15 people):

1. age matched to control group;

2. the body mass index is matched with a control group;

4. there is no clear cause of male infertility;

5. the smoking and drinking history is matched with the control group;

6. matching nationality with a control group;

7. there is no serious disease of the whole body.

II, screening and verifying urine biomarkers tryptophanol and xanthosine for UPLC-Q active MS metabonomics analysis and idiopathic male sterility diagnosis

1. Sample pretreatment

1.1. Add 10. mu.L of internal standard A to 300. mu.L of urine, add 10. mu.L of internal standard B, add 10. mu.L of internal standard C, add 900. mu.L of methanol (reagent A), and vortex for 30 s.

1.2. The supernatant was transferred to a 1.5mL inlet EP tube after centrifugation at 16000g in a centrifuge at 4 ℃ for 15min and concentrated to dryness in a centrifugal concentration desiccator at room temperature.

1.3. Reconstituted with 5. mu.L of ultrapure water (reagent D) and ready for analysis.

2. Instrumental detection

2.1. An analytical instrument: UPLC Ultimate 3000system (dionex) hplc; q-active high resolution mass spectrometer.

2.2. Liquid phase conditions:

2.2.1. the liquid chromatography column was a Hypersil GOLD C18 column (100 mm. times.2.1 mm, particle size 1.9 μm, Thermo Scientific, Germany) at a column temperature of 40 ℃.

2.2.2 the mobile phases used were (A) water containing 0.1% formic acid (reagent B) and (B) acetonitrile containing 0.1% formic acid (reagent C) at a flow rate of 400. mu.L/min.

2.2.3 Instrument gradient: 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.

2.2.4 sample introduction mode: volume 5. mu.l.

2.3. Conditions of Mass Spectrometry

2.3.1 analysis by heated electrospray ionization (HESI).

2.3.2 using the heating electrospray ionization mode (HESI), positive ion mode spray voltage: 3.5 kV; negative ion mode spray voltage: 2.5 kV; capillary temperature in two modes: 250 ℃, heater temperature: 425 ℃, sheath gas flow: 50AU, auxiliary gas flow: 13AU, reverse air flow: 0 AU; lens voltage: 60V. Adopting a full-scanning mode, wherein the scanning range is as follows: 70 to 1050 m/z; resolution ratio: 70000.

3. characterization of the substance

The biomarker characterization adopts the comparison of chromatographic information (retention time) and mass spectrum information (accurate molecular weight) with the tryptophanol and xanthosine standard substances, and compares the chromatographic information of isotope internal standard substance series in a sample in real time to correct the retention time.

4. And (3) data analysis:

biomarker screening key metabolites were confirmed using multiple Logistic regression.

5. The difference and the diagnostic significance of tryptophanol and xanthosine in urine of a healthy control group and an idiopathic male sterility group.

After correcting the information of age, body mass index, smoking and drinking history, multivariate Logistic regression analysis finds that the contents of tryptophanol and xanthosine in the urine sample are closely related to idiopathic male infertility. The metabolic small molecule combination is applied to random population to diagnose idiopathic male infertility, the sensitivity is 93.33%, the specificity is 93.33%, the area under an ROC curve is 0.9822, and the method has high auxiliary diagnosis value.

Third, preparation method of diagnostic kit

Based on the above series of experimental results, the present inventors also prepared a kit capable of diagnosing or monitoring idiopathic male infertility, the kit comprising reagents for detecting urine biomarkers tryptophanol and xanthosine associated with idiopathic male infertility, comprising standards for measuring tryptophanol and xanthosine stably present and detectable in urine of a subject and a series of internal standards for aiding analysis. The diagnostic kit can also comprise a set of reagents and devices for urine tryptophanol and xanthosine extraction and chromatographic separation.

The invention has the beneficial effects that:

the invention discloses a UPLC-Q active MS, which is used for comparing metabolic small molecules in normal control and idiopathic male sterility urine, finds out the combination of urine tryptophanol and xanthosine which can be used for evaluating whether the urine suffers from idiopathic male sterility and has diagnostic value and the application of the UPLC-Q active MS for detecting the urine tryptophanol and the xanthosine, and develops the idiopathic male sterility diagnosis and monitoring kit which can be conveniently applied clinically.

The invention adopts urine metabolism micromolecules as markers for evaluating idiopathic male sterility, and has the advantages that:

(1) the urine metabolism micromolecules are novel biomarkers, are strongly associated with disease fates, are stable, minimally invasive and easy to detect, are accurate in quantification, and can greatly improve the sensitivity and specificity of idiopathic male infertility diagnosis.

(2) The urine metabolism micromolecule marker provided by the invention can be used as a diagnostic marker of idiopathic male infertility, and can assist in diagnosing the idiopathic male infertility at an early stage, thereby providing a basis for further deep inspection of a clinician, providing support for rapidly and accurately mastering the disease state and the disease severity of a patient, timely adopting a more personalized prevention and treatment scheme, and delaying and stopping disease progression.

(3) The urine sample of the idiopathic male infertility and the healthy control random population is adopted for verification, and the levels of tryptophanol and xanthosine in the urine are proved to have higher sensitivity and specificity in the diagnosis of the idiopathic male infertility and can be used as markers.

(4) The invention adopts a strict and multistage verification and evaluation system, screens a plurality of urine metabolism micromolecules through preliminary experiments at the initial stage, and uses UPLC-Q active MS to verify independent population, thereby ensuring the reliability of the urine metabolism biomarker and the diagnosis method.

(5) The UPLC-Q active MS technology has the advantages of simple sample processing, rapid and accurate instrument analysis and higher clinical diagnosis practical value.

Drawings

In the screening stage of fig. 1, information of corrected age, body mass index, smoking and drinking history and multivariate Logistic regression analysis find that urine tryptophanol and xanthosine are closely related to idiopathic male infertility.^aThe one-way Logistic regression results for confounding factors were not adjusted.^bAdjusting multiple Logistic regression results after age, body mass index, smoking and drinking history.

FIG. 2 Metabolic assay level volatility (mean. + -. standard deviation).

FIG. 3 shows ROC curve between normal control group and idiopathic male infertility group prepared by using urine tryptophanol content information in the verification stage.

FIG. 4 shows ROC curve between normal control group and idiopathic male infertility group prepared by urine xanthosine content information at verification stage.

FIG. 5 shows the ROC curve between the normal control group and the idiopathic male infertility group prepared by using the information of the contents of tryptophanol and xanthosine in urine at the verification stage.

Detailed Description

The invention is further illustrated by the following examples.

Example 1: subject selection and grouping basis

The study subjects were from the first-visit idiopathic male infertility case and healthy fertility control of the subsidiary hospital of the Nanjing medical university. The research content and the informed consent were approved by the ethical committee of the university of medical Nanjing, and were in compliance with the requirements of the relevant regulations. Cases and controls signed informed consent after understanding the content. All subjects performed a complete physical examination and completed a questionnaire that included personal basic data, lifestyle habits, occupational and environmental exposure, genetic risk factors, sexual and reproductive function, disease history, and physical activity. In the first stage, 607 cases of idiopathic male infertility and 430 healthy controls were included as required; 15 cases of idiopathic male infertility and 15 healthy controls meeting the requirements at the second stage were used as screening subjects of urine biomarkers of idiopathic male infertility. The specific sample classification criteria are as follows:

first stage screening stage

Group A: healthy control group (430 people):

1. age between 19 and 39 years;

2. a body mass index between 17 and 31;

4. there is no serious disease of the whole body.

Group B: idiopathic male infertility disease group (607 human):

1. age matched to control group;

2. the body mass index is matched with a control group;

4. there is no clear cause of male infertility;

5. the smoking and drinking history is matched with the control group;

6. matching nationality with a control group;

7. there is no serious disease of the whole body.

Second phase verification phase

Group A: healthy control group (15 persons):

1. age between 24 and 36 years;

2. a body mass index between 19 and 24;

4. there is no serious disease of the whole body.

Group B: idiopathic male infertility disease group (15 people):

1. age matched to control group;

2. the body mass index is matched with a control group;

4. there is no clear cause of male infertility;

5. the smoking and drinking history is matched with the control group;

6. matching nationality with a control group;

7. there is no serious disease of the whole body.

Example 2: UPLC-MS metabonomics idiopathic male sterility biomarker screening

1. Sample pretreatment

1.1. Add 10. mu.L of internal standard A to 300. mu.L of urine, add 10. mu.L of internal standard B, add 10. mu.L of internal standard C, add 40. mu.L of methanol (reagent A), and vortex for 30 s.

2. Instrumental detection

2.2. Liquid phase conditions:

2.2.1 the liquid chromatography column was a Hypersil GOLD C18 column (100 mm. times.2.1 mm, particle size 1.9 μm, Thermo Scientific, Germany) at a column temperature of 40 ℃.

2.2.4 sample introduction mode: volume 5. mu.l.

2.3. Conditions of Mass Spectrometry

2.3.1 analysis by heated electrospray ionization (HESI).

3. characterization of the substance

4. And (3) data analysis:

5. The differences of tryptophanol and xanthosine in urine samples of the healthy control group and the idiopathic male sterility group and the diagnostic significance.

After correcting the information of age, body mass index, smoking and drinking history, multivariate Logistic regression analysis finds that urine tryptophanol and xanthosine are closely related to idiopathic male infertility (figure 1).

Example 3 stability analysis of tryptophanol and xanthosine in urine

The stability of tryptophanol and xanthosine levels in urine was evaluated using the method of example 2 (interval of 2 weeks). The results show that the levels of tryptophanol and xanthosine measured in urine are stable (fig. 2), and the urine has the characteristics of being used as a diagnosis/monitoring marker.

Example 4 diagnosis of idiopathic male infertility by combination of tryptophanol and xanthosine in urine

According to the UPLC-Q active MS metabonomics method, the inventor detects tryptophan and xanthosine by urine samples of 15 random population cases and 15 control cases, draws an ROC curve and evaluates the sensitivity and specificity of diagnosis, and further evaluates the diagnostic ability of detecting the 2 substance levels in urine on idiopathic male infertility.

The sensitivity of tryptophanol was 80.00%, the specificity was 86.67%, and the area under the ROC curve was 0.9689 (FIG. 3); the sensitivity of xanthosine was 80.00%, the specificity was 80.00%, and the area under the ROC curve was 0.8978 (FIG. 4).

The sensitivity of the combination of tryptophanol and xanthosine was 93.33%, the specificity was 93.33%, and the area under the ROC curve was 0.9822 (FIG. 5).

Therefore, the combination of tryptanthrin and xanthosine has better capability of diagnosing idiopathic male infertility.

EXAMPLE 5 preparation of detection and diagnostic kit for tryptophanol and xanthosine in idiopathic male infertility urine

Firstly, determining metabolic small molecules with higher abundance in normal control and idiopathic male infertility urine by using a UPLC-Q active MS method. Then, biomarkers related to the idiopathic male sterility are screened by the UPLC-Q active MS-based metabonomics technology as a diagnostic index of whether the idiopathic male sterility is detected. The number of corresponding urine biomarkers screened is preferably controlled to 2, which is a reduction of the optimization made on a preliminary experiment basis. The 2 biomarkers tryptophanol and xanthosine in the urine are adopted, so that the sensitivity and specificity can be better guaranteed, the cost can be saved, the burden of a patient can be reduced, the detection time can be shortened, the method has the advantages of rapidness, accuracy and economy, the clinical popularization and use are convenient, 1 of the markers can be adopted, and the effect is better by adopting the 2 markers. The kit comprises a batch of reagent and consumable for detecting the tryptophanol and the xanthosine in urine, wherein the qualitative and quantitative detection of the biomarker adopts tryptophanol and xanthosine standard substances, and the auxiliary analysis adopts an internal standard A: the deuterium labeled isotope internal standard of eight substances of creatinine, valine, nicotinic acid, thymine, glutaric acid, L-phenylalanine, N-acetyl-p-aminophenol and hippuric acid. Internal standard B: deuterium-labelled isotopic internal standard of pentadecanoic acid. Internal standard C: deuterium-labelled isotopic internal standard of tetracosanoic acid. Other examples are a reversed phase chromatographic column (Hypersil GOLD C18 column, 100 mm. times.2.1 mm, particle size 1.9 μm), a reagent for precipitating urine protein (100% methanol), a reagent for mobile phase (0.1% formic acid in water and 0.1% formic acid in acetonitrile), and a reagent for extraction of tryptophanol and xanthosine (100% ultrapure water) for UPLC chromatographic separation. The value of the kit is that the contents of tryptophanol and xanthosine markers in urine can be detected only by 300 mul of urine, then idiopathic male infertility is diagnosed by the contents, and dynamic monitoring and observation of treatment effects are easy to carry out.

The specific kit comprises the following components:

tryptophanol standard

Standard xanthosine

Internal standard A (deuterium isotope internal standard water solution of eight substances of creatinine, valine, nicotinic acid, thymine, glutaric acid, L-phenylalanine, N-acetyl-p-aminophenol and hippuric acid)

Internal standard B (methanol solution with deuterium standard isotope of pentadecanoic acid)

Internal standard C (methanol solution with deuterium isotope internal standard of tetracosanoic acid)

Further, the method may further comprise:

chromatographic column (Thermo 100mm X2.1 mm, particle size 1.9 μm, 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).

Primary references

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 using ¹H-NMR-based metabonomics.Nat Med 8:1439-1444.

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.

Jessica,D.Tracey,G.,R.Mark,W.Christos,G.,S,and Matthew,B.,C.,2016.The use of metabolomics to monitor simultaneous changes in metabolic variables following supramaximal low volume high intensity exercise.Metabolomics 12:7.

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 ofphosphatidylcholine promotes cardiovascular disease.Nature 472:57-63.

Zeng J,Huang X,Zhou L,Tan Y,Hu C,Wang X,Niu J,Wang H,Lin X,Yin P.Metabolomics Identifies Biomarker Pattern for Early Diagnosis of Hepatocellular Carcinoma:from Diethylnitrosamine Treated Rats to Patients.2015.Sci Rep 5:16101.

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. Application of tryptophanol and xanthosine in preparing diagnostic or monitoring reagent for idiopathic male infertility is provided.