WO2019201216A1

WO2019201216A1 - Combination of dodecanoic acid and prostaglandin e2 as auxiliary diagnostic marker of macrosomia and application thereof

Info

Publication number: WO2019201216A1
Application number: PCT/CN2019/082754
Authority: WO
Inventors: 陈敏健; 赵子平; 韦筱淇; 曹易之; 庄婷钰; 黄艳倩; 戴礼瑞; 张逸; 王擎; 张鹏鹏; 张静; 裴雨洋; 胡艳辉; 孙蓉丽; 陆春城; 吴炜; 夏彦恺; 王心如
Original assignee: 南京医科大学
Priority date: 2018-04-16
Filing date: 2019-04-15
Publication date: 2019-10-24
Also published as: CN108872424A

Abstract

Disclosed is a combination of dodecanoic acid and prostaglandin E2 acting as an auxiliary diagnostic marker of macrosomia and an application thereof, relating to the fields of analytical chemistry and clinical medicine, wherein the marker is metabolic small molecular dodecanoic acid and prostaglandin E2 of serum during pregnancy. The marker adopts the UPLC-Q exactive MS method for detection, has better sensitivity and specificity, and is capable of being used for the early diagnosis or monitoring of macrosomia, thereby having a better promotional value in clinical applications.

Description

Combination of dodecanoic acid and prostaglandin E2 as a diagnostic marker for giant children and its application

Field of invention

The invention belongs to the field of analytical chemistry and clinical medicine, and relates to the combination of dodecanoic acid and prostaglandin E2 as a macroscopic auxiliary diagnostic marker and application thereof, and particularly based on UPLC-Q exactive MS detection of metabolic small molecule markers related to macrosogenesis. Combination of objects and their applications.

Background technique,

Macrosomia is a newborn that points to a weight of 4 kg or more within 1 hour of birth. The occurrence of huge children is harmful to both mother and baby. A huge child is one of the important factors in dystocia, and it also increases the risk of heart malformation. Now the study finds that the chances of a large child growing up with obesity are greater, and it will become a susceptible population of diabetes, hypertension and other diseases.

The early diagnosis of giant children is generally based on some of the mother's susceptible medical characteristics such as diabetes, obesity and expired pregnancy. However, for people who do not have the above characteristics, the early diagnosis of giant children needs to rely on B-ultrasound detection. However, the B-ultrasound detection time is longer, the inspection price is higher, and some pregnant women are more worried about the radiation of the B-ultrasound examination. All of these have made the diagnosis of the giant child difficult and brought a serious financial burden to the family, so it is urgent to find a new method of diagnosis.

Metabolomics/Metabonomics is a new discipline developed in the late 1990s by examining the changes in its metabolites or their changes over time after a genetic system has been genetically altered or stimulated or disturbed. To study a science of biological systems. The metabolome is the downstream product and final product of the genome. It is a collection of small molecular compounds involved in the metabolism of the organism and maintaining the normal function and growth of the organism. It is mainly an endogenous small molecule with a relative molecular mass of less than 1000. These endogenous metabolic small molecules involve sugar metabolism, energy metabolism, lipid metabolism, amino acid metabolism, nucleic acid metabolism, and coenzyme metabolism.

The organism in its normal state is a complete system in which the metabolites in biological fluids, cells and tissues are in a stable equilibrium. The pathological changes occurred in the body due to genetic or acquired causes, and this balance was broken, and metabolites and metabolic processes also changed accordingly. Metabolomics analysis of changes in metabolic small molecules in the disease process can help people find relevant biomarkers to aid in the diagnosis of diseases, and also help people understand the disease through the metabolic pathways involved in small molecules themselves. Pathogenesis and provide specific targets for drug development. In recent years, metabolomics has made many significant research results in the early diagnosis of diseases in human diseases, such as cardiovascular disease, diabetes and cancer. Related papers published in the academic journals Nature, Nature medicine, Journal of hepatology and Cancer research show the great potential and value of small metabolic molecules in the diagnosis of human diseases.

Current techniques for metabolomics research include liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR). The characteristics of NMR technology are that the components to be tested are not damaged, the sample preparation is simple, but the sensitivity is low; gas chromatography-mass spectrometry has good sensitivity and reproducibility, but the sample is pretreated by derivatization method. , making the experimental steps complicated. LC-MS has the characteristics of simple sample processing, high sensitivity and strong clinical applicability. UPLC-Q exactive MS is a combination of a new generation of high resolution mass spectrometry and ultra high performance liquid phase, which has greater sensitivity, specificity and stability than traditional LC-MS. Therefore, UPLC-Q exactive MSS is used for metabolomics analysis of small molecules of metabolism. If a stable small molecule of specific serum metabolism related to macrosomia is detected as a biomarker, UPLC- of the metabolic small molecule marker of the corresponding disease is developed. Q exactive MS detection method is not only an international leader in this field, but also can create eye-catching economic benefits. It will also be a powerful boost to improve the maternal and child health level in China.

Summary of the invention

The object of the present invention is to provide a relevant combination of serum metabolic small molecule markers for the diagnosis of a large number of children during the diagnosis.

Another object of the present invention is to provide a method for detecting the above small molecule markers based on the UPLC-Q exactive MS method.

Still another object of the present invention is to provide a kit for detecting and diagnosing chromatographic mass spectrometry for the above-described serum metabolic small molecule marker.

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

A small molecule marker of serum metabolism during pregnancy associated with a giant child, which is a serum metabolism of the small molecule dodecanoic acid and prostaglandin E2.

The use of the serum metabolic small molecule marker during pregnancy for the preparation of a large early diagnosis or monitoring reagent.

A kit for early diagnosis or monitoring of a large child, the kit comprising a reagent for detecting a small molecule marker of dopanoic acid and/or prostaglandin E2 during pregnancy.

The kit comprises a reagent for detecting a small molecule marker dodecanoic acid and/or prostaglandin E2 during pregnancy by UPLC-Q exactive MS method.

The kit comprising the following reagents:

Dodecanoic acid and/or prostaglandin E2 standards;

Carbon 13-labeled dodecanoic acid stable isotope internal standard;

Carbon-13-labeled prostaglandin E2 stable isotope internal standard.

The kit further comprising:

Hypersil GOLD C18 column;

Reagent A: used for precipitation of protein, containing 100% methanol;

Reagent B: water for mobile phase containing 0.1% formic acid;

Reagent C: acetonitrile containing 0.1% formic acid for mobile phase;

Reagent D: For reconstitution, ultrapure water.

A method for detecting a small-molecular marker of serum metabolism in pregnancy associated with a giant child, characterized in that the method uses UPLC-Q exactive MS method to detect a small molecule marker dodecanoic acid and/or prostaglandin E2 during pregnancy. content.

The method described, wherein:

First, the liquid phase conditions:

The liquid chromatography column is a Hypersil GOLD C18 column with a column temperature of 40 ° C;

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 μL/min;

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;

Injection method: volume 10μl;

Second, the mass spectrometry conditions

1. Heating electrospray ionization mode (HESI) for analysis;

2. Positive ion mode spray voltage: positive mode, injection voltage is 3.5kV, negative mode, injection voltage is 2.5kV; for two modes, capillary temperature is 300 ° C, heater temperature is 425 ° C, sheath gas flow is 50 AU, The auxiliary air flow is 13 AU, the backflush gas flow is 0 AU, the full scan analysis (70 to 1,050 m/z), and the resolution is set to 70,000.

The invention is described in detail as follows:

The inventors collected standard-compliant pregnant blood samples by standard operating procedure (SOP), systematically collected complete population basic information and clinical data, and analyzed by UPLC-Q exactive MS based metabolomics method.

Specifically, the experimental methods of research mainly include the following parts:

I. Research object selection and grouping basis

First stage screening stage

A total of 97 pregnant women were randomly included.

1. Age between 23 and 36 years old;

2. The gestational age is less than or equal to 41 weeks;

3. Contains huge cases of children (baby birth weight ≥ 4kg, 16 people).

Second stage verification stage

A total of 30 pregnant women were included in the case and control.

Group A: healthy control group (15 people, 2.5kg ≤ baby birth weight <4kg):

1. Age between 23 and 36 years old;

2. The gestational age is less than or equal to 41 weeks;

3. No major systemic diseases.

Group B: large group (15 people, baby birth weight ≥ 4kg):

1. Age between 23 and 36 years old;

2. The gestational age is less than or equal to 41 weeks;

3. No major systemic diseases.

Second, UPLC-Q exactive MS metabolomics analysis and screening and verification of metabolic small molecules for macrodiagnosis

Sample pretreatment

1.1. Fresh pregnant blood was centrifuged at 3000 rpm for 5 min in a centrifuge, and 100 μl of the supernatant was dispensed into a clean 1.5 ml EP tube.

1.2.50 μl of serum was added with 50 μl of water and 300 μl of methanol (Reagent A) to precipitate the protein.

1.3. Pipette the supernatant, dry with nitrogen and dry with vacuum.

1.4. Reconstitute the dried material (Reagent D).

2. Instrument detection

2.1. Analytical instrument: UPLC Ultimate 3000 system (Dionex) high performance liquid chromatography; Q-Exactive high resolution mass spectrometer.

2.2. Liquid phase conditions:

The liquid chromatography column was a Hypersil GOLD C18 column (100 mm x 2.1 mm, particle size 1.9 μm, Thermo Scientific, Germany) with a column temperature of 40 °C.

The mobile phases used were (A) water containing 0.1% formic acid (Reagent B) and (B) Acetonitrile containing 0.1% formic acid (Reagent C).

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 (B refers to mobile phase B, the amount of mobile phase A in each gradient is 100% of the corresponding amount of mobile phase B, the same below).

Injection method: volume 10μl;

2.3. Mass spectrometry conditions

Heating electrospray ionization (HESI) for analysis;

Positive ion mode spray voltage: positive mode, injection voltage is 3.5kV, negative mode, injection voltage is 2.5kV; for both modes, capillary temperature is 300°C, heater temperature is 425°C, sheath gas flow is 50AU, auxiliary gas The flow rate is 13 AU, the backflush gas flow is 0 AU, the full scan analysis (70 to 1,050 m/z), and the resolution is set to 70,000.

3. Material characterization

Metabolic small molecule qualitative alignment of chromatographic information (retention time) and mass spectrometry information (precise molecular weight, isotope distribution and MS/MS fragmentation information) with standard dodecanoic acid and prostaglandin E2, and real-time comparison of isotope internal standards in samples Chromatographic information (retention time) of the standard (carbon 13-labeled dodecanoic acid, carbon-13-labeled prostaglandin E2 stable isotope internal standard).

4. Data analysis:

Biomarker Screening Multiple linear regression was used to screen robust birth weight-related metabolite combinations, and multivariate logistic regression was used to identify key metabolite combinations.

5. Differences and diagnostic significance of small metabolic molecules in serum samples from healthy control group and giant child group.

After adjusting for BMI and gestational age at the end of pregnancy, multiple linear regression analysis showed that the combination of dodecanoic acid and prostaglandin E2 in pregnancy was positively correlated with the birth weight of the offspring. After adjusting for BMI and gestational age at the end of pregnancy, multivariate logistic regression analysis showed that the combination of serum dodecanoic acid and prostaglandin E2 increased significantly during the pregnancy. The independent population was applied to the diagnosis of giant infants with the combination of metabolic small molecules. The sensitivity was 80.00%, the specificity was 80.00%, and the area under the ROC curve was 0.8978, which has a high diagnostic value.

Third, the diagnostic kit preparation method

Based on the above series of experimental results, the inventors have also prepared a diagnostic kit that can be used for dynamic monitoring of giant children, which comprises determining the stable and detectable dodecanoic acid in the serum of a subject during pregnancy. And prostaglandin E2 stable isotope internal standard and standard for dodecanoic acid and prostaglandin E2. The diagnostic kit also includes a set of reagents and equipment for small molecule extraction and chromatographic separation of serum metabolism.

The beneficial effects of the invention:

The present inventors used UPLC-Q exactive MS to compare metabolic small molecules in the serum of normal controls and large mothers during pregnancy, and found that there are serum metabolic small molecule markers in the serum during pregnancy that can be used to evaluate whether the mother has a large child and has diagnostic value. The combination of the substance and the application of the UPLC-Q exactive MS for detecting the small molecule marker of serum metabolism has developed a huge diagnostic and monitoring kit for clinical application.

The superiority of the present invention for using serum metabolic small molecules during pregnancy as a marker for juvenile evaluation is as follows:

(1) Serum metabolism Small molecule is a novel biomarker, which is strongly associated with disease outcomes. It is not only stable, minimally invasive, easy to detect, but also quantitatively accurate, which will greatly improve the sensitivity and specificity of large-scale diagnosis. The successful development of small molecule biomarkers will open up a new situation for the prevention and treatment of adverse birth outcomes, and provide reference for the development of biomarkers for other diseases.

(2) The serum metabolic small molecule marker provided by the invention can be used as a diagnostic marker for giant children, and can detect a giant child in a small invasive manner at an early stage, thereby providing a basis for further deep examination by a clinician, and quickly and accurately grasping the disease of the patient. State and severity of illness, timely and more personalized prevention and treatment programs to support, delay and prevent disease progression.

(3) The present invention verifies the maternal serum samples of the giant and healthy control populations, and proves that the combination of the dodecanoic acid and prostaglandin E2 in the serum during pregnancy has higher sensitivity and specificity in detecting large children, and can be used as a marker. Use.

(4) The invention adopts a strict and multi-stage verification and evaluation system, and initially selects a plurality of serum metabolic small molecules through preliminary experiments, and uses UPLC-Q exactive MS to perform independent population verification, thereby ensuring the serum metabolic biomarkers and diagnostic methods. Reliability.

(5) UPLC-Q exactive MS technical sample processing is simple, instrumental analysis is fast and accurate, and has a high clinical diagnostic practical value.

DRAWINGS

Figure 1 shows the birth weight of the first phase population and the control.

The top and bottom of the box diagram represent the seventy-fifth and twenty-fifth percentiles, respectively. The upper to the lower of the box diagram represent the maximum to the minimum, (-) is the median, and (+) is the average.

Figure 2 shows the birth weight of the second-stage population and the control, as shown in Figure 1.

In the screening phase of Figure 3, after adjusting for BMI and gestational age at the end of pregnancy, multiple linear regression and multiple logistic regression analysis consistently found that the combination of serum dodecanoic acid and prostaglandin E2 during pregnancy significantly increased the risk of progeny being a large child.

Figure 4 Metabolic small molecule combination detection level volatility (mean ± standard deviation).

In the verification phase of Figure 5, the ROC curve between the normal control group and the giant group was prepared using information on the combination of serum metabolic small molecules during pregnancy.

detailed description

The invention is further illustrated by the following examples.

Example 1 Study object selection and grouping basis

The inventors collected blood samples of pregnant children and children of normal children who met the requirements from the Nanjing Maternal and Child Health Hospital affiliated to Nanjing Medical University (the difference in birth weight between giant and control) is shown in Figure 1, Figure 2). Weighing newborns after birth, if the body weight is equal to or greater than 4 kg within 1 hour after birth, it is diagnosed as "macro". The first stage randomly included 97 maternal samples that met the requirements, of which 15 were large children; the second stage included 30 maternal samples, 15 of which were compared with 15 large children, as a small molecule of blood metabolism during pregnancy. Screening test subjects for markers. The specific sample classification criteria are as follows:

First stage screening stage

A total of 97 pregnant women were randomly included.

4. Age between 23 and 36 years old;

5. The gestational age is less than or equal to 41 weeks;

6. Contains huge cases of children (baby birth weight ≥ 4kg, 16 people).

Second stage verification stage

A total of 30 pregnant women were included in the case and control.

Group A: healthy control group (15 people, 2.5kg ≤ baby birth weight <4kg):

4. Age between 23 and 36 years old;

5. The gestational age is less than or equal to 41 weeks;

6. No major systemic diseases.

Group B: large group (15 people, baby birth weight ≥ 4kg):

4. Age between 23 and 36 years old;

5. The gestational age is less than or equal to 41 weeks;

6. No major systemic diseases.

Example 2 UPLC-MS Metabolomics Mutant Biomarker Screening

Sample pretreatment

1.3. Pipette the supernatant, dry with nitrogen and dry with vacuum.

1.4. Reconstitute the dried material (Reagent D).

2. Instrument detection

2.2. Liquid phase conditions:

The instrument gradient was: 0-3 min 1% B, 3-10 min 1% to 99% B, 10-13 min 99% B, 13-13.1 min 99% to 1% B, 13.1-17 min 1% B.

Injection method: volume 10μl;

2.3. Mass spectrometry conditions

1. Heating electrospray ionization mode (HESI) for analysis;

3. Material characterization

4. Data analysis:

After adjusting for BMI and gestational age at the end of pregnancy, multiple linear regression analysis showed that the combination of serum dodecanoic acid and prostaglandin E2 during pregnancy was positively correlated with the birth weight of the offspring. After adjusting for BMI and gestational age at the end of pregnancy, multivariate logistic regression analysis found that an increase in the combination of serum dodecanoic acid and prostaglandin E2 during pregnancy significantly increased the risk of offspring being large (Figure 3).

Example 3 Stability Analysis of Serum Metabolism Small Molecules

The stability of the combined levels of serum dodecanoic acid and prostaglandin E2 during maternal pregnancy was evaluated by the method of Example 2 (interval of 2 weeks). The results showed that the combination of dodecanoic acid and prostaglandin E2 in serum was stable (Fig. 4) and possessed characteristics as a diagnostic/monitoring marker.

Example 4 Diagnosis of giant children by small molecule combination of metabolism

According to the UPLC-Q exactive MS metabolomics method described above, the present inventors measured the ROC curve and evaluated the sensitivity of the test by detecting dodecanoic acid and prostaglandin E2 in serum samples of 15 independent and 15 controls of the independent population. Specificity, and then to assess the detection of the level of these two small molecules in the serum for the auxiliary diagnosis of giant children.

Figure 5 shows that the sensitivity of the combination of dodecanoic acid and prostaglandin E2 is 80.00%, the specificity is 86.67%, and the area under the ROC curve is 0.8267, which has a high diagnostic value.

Therefore, the combination of dodecanoic acid and prostaglandin E2 has a better effect in assisting the diagnosis of giant children.

Example 5 Preparation of a Small Molecule Detection and Diagnostic Kit for Giant Serum Metabolism

First, the UPLC-Q exactive MS method was used to determine the high-abundance metabolic small molecules in the serum of normal and large children. Then, in the UPLC-Q exactive MS-based metabolomics technique, metabolic small molecules associated with giants are screened as indicators for detecting whether they are large children and the degree of diagnosis. Finally, the number of corresponding small serum metabolism molecules selected was controlled to two, which is the optimized reduction based on the pre-experiment. The kit includes a batch of reagents and consumables for detecting small molecules of serum metabolism during pregnancy, wherein the qualitative and quantitative metabolism of small molecules are based on the standard of dodecanoic acid and prostaglandin E2, and the auxiliary quantitative and auxiliary qualitative use of carbon 13 labeled twelve A stable isotope internal standard for alkanoic acid, carbon-13-labeled prostaglandin E2. Others are a reversed-phase column for HPLC separation (Hypersil GOLD C18 column, 100mm x 2.1mm, particle size 1.9μm), reagent for precipitating serum proteins (100% methanol), reagents for mobile phase (Water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid), a reagent for extracting small molecules (100% ultrapure water). The value of this kit is that only 100μl of pregnant serum is needed, and the content of serum metabolic small molecule markers can be detected. Then, the combined detection of large children can be used for early auxiliary diagnosis, and it is easy to dynamically monitor and observe the therapeutic effect.

The specific kits are composed as follows:

Dodecanoic acid standard

Prostaglandin E2 standard

Carbon 13-labeled dodecanoic acid stable isotope internal standard

Carbon-13-labeled prostaglandin E2 stable isotope internal standard

Column (Thermo 100mm × 2.1mm, particle size 1.9μm, Hypersil GOLD C18 column)

Reagent A (containing 100% methanol)

Reagent B (water containing 0.1% formic acid)

Reagent C (acetonitrile containing 0.1% formic acid)

Reagent D (100% ultrapure water).

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Claims

A small molecule marker of serum metabolism during pregnancy associated with a giant child, characterized in that the marker is a serum metabolizing small molecule dodecanoic acid and prostaglandin E2.
The use of the serum metabolic small molecule marker of pregnancy according to claim 1 for the preparation of a macroscopic early diagnosis or monitoring reagent.
A kit for early diagnosis or monitoring of a large child, characterized in that the kit contains a reagent for detecting a small molecule marker of dopanoic acid and/or prostaglandin E2 during pregnancy.
The kit according to claim 3, characterized in that the kit contains a reagent for detecting the serum metabolism small molecule marker dodecanoic acid and/or prostaglandin E2 by the UPLC-Q exactive MS method.
The kit according to claim 4, characterized in that the kit contains the following reagents:

Dodecanoic acid and/or prostaglandin E2 standards;

Carbon 13-labeled dodecanoic acid stable isotope internal standard;

Carbon-13-labeled prostaglandin E2 stable isotope internal standard.
The kit according to claim 5, characterized in that the kit further comprises:

Hypersil GOLD C18 column;

Reagent A: used for precipitation of protein, containing 100% methanol;

Reagent B: water for mobile phase containing 0.1% formic acid;

Reagent C: acetonitrile containing 0.1% formic acid for mobile phase;

Reagent D: For reconstitution, ultrapure water.
A method for detecting a large-scale serum metabolic small molecule marker associated with a giant child according to claim 1, characterized in that the method uses UPLC-Q exactive MS method to detect serum metabolic small molecule marker dodecanoic acid during pregnancy / or the content of prostaglandin E2.
The method of claim 7 wherein:

First, the liquid phase conditions:

The liquid chromatography column is a Hypersil GOLD C18 column with a column temperature of 40 ° C;

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 μL/min;

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;

Injection method: volume 10μl;

Second, the mass spectrometry conditions

1. Heating electrospray ionization mode (HESI) for analysis;

2. Positive ion mode spray voltage: positive mode, injection voltage is 3.5kV, negative mode, injection voltage is 2.5kV; for two modes, capillary temperature is 300 ° C, heater temperature is 425 ° C, sheath gas flow is 50 AU, The auxiliary air flow is 13 AU, the backflush gas flow is 0 AU, the full scan analysis (70 to 1,050 m/z), and the resolution is set to 70,000.