CN112305123B - Application of small molecular substance in atherosclerotic cerebral infarction - Google Patents
Application of small molecular substance in atherosclerotic cerebral infarction Download PDFInfo
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- CN112305123B CN112305123B CN202011195866.4A CN202011195866A CN112305123B CN 112305123 B CN112305123 B CN 112305123B CN 202011195866 A CN202011195866 A CN 202011195866A CN 112305123 B CN112305123 B CN 112305123B
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Abstract
The invention relates to application of a small molecular substance in atherosclerotic cerebral infarction, in particular to application of a small molecular substance PE (P-18:0/18:2(9Z,12Z)) in atherosclerotic cerebral infarction.
Description
Technical Field
The invention belongs to the field of biomedicine, and relates to application of a small molecular substance in atherosclerotic cerebral infarction.
Background
The stroke is a disease with high morbidity, high disability rate, high mortality and high recurrence rate which seriously harms human health, and the stroke and heart disease and tumors always occupy the first 3 sites of death cause in China for nearly 30 years, and the ischemic stroke occupies 50-70 percent of the stroke, wherein the atherosclerotic thrombotic cerebral infarction is the most main type, 60-80 percent of the ischemic stroke is related to Atherosclerosis (AS), and the research on the pathogenesis of the atherosclerosis and the atherosclerotic cerebral infarction becomes the focus of research.
Metabonomics, an emerging subject and technology in the "post-genomics" era, is one of the most active fields of life science research in the world today. According to different research objects and purposes, metabonomics can be divided into the following research levels: (1) a metabolite target assay based on the quantification of a compound of interest, which is an assay for a specific component or components; (2) metabolic profiling based on simultaneous analysis of multiple metabolites in one or several metabolic pathways; (3) metabolite fingerprint analysis based on discrimination-free analysis; (4) qualitative and quantitative metabolomic analysis based on all metabolic components in a specific biological sample under defined conditions. Since the concept of metabonomics is proposed, the method has attracted great interest of scientists in various countries, and is widely applied to important fields of clinical medicine, pharmaceutical research, nutriology, food safety, environmental science, toxicology, plant microbiology and the like. The research on the correlation between the atherosclerotic cerebral infarction and the metabolite has important significance for revealing the pathogenesis of the atherosclerotic cerebral infarction and realizing early diagnosis and treatment of diseases.
Disclosure of Invention
In order to make up the defects of the prior art, the invention aims to provide a small molecular substance related to atherosclerotic cerebral infarction, and whether a patient has atherosclerotic cerebral infarction can be judged by detecting the small molecular substance, so that a new means is provided for early diagnosis of the atherosclerotic cerebral infarction.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides application of a reagent for detecting a small molecular substance in a sample in preparing a product for evaluating atherosclerotic cerebral infarction, wherein the small molecular substance is PE (P-18:0/18:2(9Z, 12Z)).
Further, the product comprises reagents for detecting the marker by chromatography, spectroscopy, mass spectrometry, chemical analysis.
Further, the product also includes reagents for processing the sample.
Further, the sample is selected from blood, plasma or serum.
Further, a decrease in the level of PE (P-18:0/18:2(9Z,12Z)) in the sample from the subject is indicative of the subject having, or being at risk of having, an atherosclerotic cerebral infarction.
Further, the subject sample is selected from blood, plasma or serum.
The invention provides a product for evaluating atherosclerotic cerebral infarction, which comprises a reagent for detecting a small-molecule substance PE (P-18:0/18:2(9Z,12Z)) in a sample.
Further, the product comprises a kit and a chip.
Further, the product indicates whether the subject has or is at risk of having an atherosclerotic cerebral infarction by detecting the level of a small molecule substance in a sample from the subject and comparing the level to a reference metabolite profile.
The invention provides the use of a small molecule substance in the manufacture of a product for identifying and assessing the effect of a medicament and/or surgical and/or physical therapy on anti-atherosclerotic cerebral infarction.
The invention has the advantages and beneficial effects that:
the invention discovers the micromolecular substance related to the atherosclerotic cerebral infarction for the first time, and can judge whether a subject has the atherosclerotic cerebral infarction and the risk of having the atherosclerotic cerebral infarction by detecting the level of the micromolecular substance so as to realize the diagnosis of the early stage of the atherosclerotic cerebral infarction, thereby carrying out intervention treatment at the early stage of the cerebral infarction and improving the life quality of patients.
Drawings
FIG. 1 is a statistical analysis diagram of OPLS-DA, wherein A is a statistical analysis diagram of reverse chromatographic positive ions; b is a reverse chromatogram anion statistical analysis chart; c is a hydrophilic chromatographic positive ion statistical analysis chart.
FIG. 2 is a graph of the levels of PE (P-18:0/18:2(9Z,12Z)) in different groups.
FIG. 3 is a graph of the diagnostic performance of PE (P-18:0/18:2(9Z,12Z)) as the test variable.
Detailed Description
The term "subject" is intended to include an animal capable of suffering or suffering from an atherosclerotic cerebral infarction. Examples of subjects include mammals, e.g., humans, non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the subject is a human, e.g., a human suffering from an atherosclerotic cerebral infarction, a human at risk of an atherosclerotic cerebral infarction, or a human potentially capable of suffering from an atherosclerotic cerebral infarction.
By "level" of a small molecule substance is meant the absolute or relative amount or concentration of said small molecule substance in a sample.
The term "sample" or "biological sample" means a biological material isolated from a subject. The biological sample may contain any biological material suitable for detecting a desired biomarker, and may comprise cellular material and/or non-cellular material from a subject. The sample may be isolated from any suitable biological fluid, such as, for example, blood, plasma, serum, urine, or cerebrospinal fluid (CSF). As a preferred embodiment, the sample is selected from blood, plasma, serum.
The small molecular substance of the atherosclerotic cerebral infarction is discovered by using a metabonomic spectrum analysis technology. In general, the metabolic profile of biological samples from human subjects diagnosed with atherosclerotic cerebral infarction and from one or more other groups of human subjects (e.g., control subjects with atherosclerosis) is determined. The metabolic profile of a biological sample from a subject having an atherosclerotic cerebral infarction is compared to the metabolic profile of a biological sample from one or more other groups of subjects. Molecules that are differentially present in the metabolic profile of a sample from a subject having an atherosclerotic cerebral infarction, including molecules that are differentially present at a statistically significant level, as compared to another group (e.g., a control subject having atherosclerosis) are identified as biomarkers to identify that group.
The small molecule species can be differentially present at any level, but is typically present at a level that is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or more; or generally at a level that is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% (i.e., absent). The biomarkers are preferably present differentially at levels of statistical significance (i.e. p-value less than 0.05 and/or q-value less than 0.10, as determined using the Welch's T-Test or the Wilcoxon's rank-sum Test).
In the present invention, a reference metabolite profile or reference level means a level of the small molecule substance that is indicative of a particular disease state, phenotype, or susceptibility to developing a particular disease state or phenotype. A "positive" reference level of a small molecule substance is intended to mean a level indicative of a particular disease state or phenotype. A "negative" reference level of a small molecule substance is intended to indicate a level without a particular disease state or phenotype. For example, an "atherosclerotic cerebral infarction positive reference level" of a small molecule substance means a level of a small molecule substance indicative of a positive diagnosis of an atherosclerotic cerebral infarction of the subject, and an "atherosclerotic cerebral infarction negative reference level" of a small molecule substance means a level of a small molecule substance indicative of a negative diagnosis of an atherosclerotic cerebral infarction of the subject. A "reference level" of a small molecule substance can be an absolute or relative amount or concentration of the small molecule substance, the presence or absence of the small molecule substance, a range of amounts or concentrations of the small molecule substance, a minimum and/or maximum amount or concentration of the small molecule substance, an average amount or concentration of the small molecule substance, and/or a median amount or concentration of the small molecule substance; also, in addition, a "reference level" of a combination of small molecule species may also be a ratio of absolute or relative amounts or concentrations of two or more small molecule species relative to each other. Appropriate positive and negative reference levels of a phenotypic small molecule substance for a particular disease state may be determined by measuring the level of the desired small molecule substance in one or more appropriate subjects, and such reference levels may be tailored for a particular population of subjects (e.g., the reference levels may be age-matched or gender-matched such that a comparison may be made between the level of the small molecule substance in a sample from a subject of a certain age or gender and the reference levels for a particular disease state, phenotype, or lack thereof in a group of ages or sexes). Such reference levels may also be tailored to the particular technique used to measure the level of small molecule species in the biological sample (e.g., LC-MS, GC-MS, etc.), where the level of small molecule species may vary based on the particular technique used.
In the present invention, "metabolite" or "small molecule substance" means organic molecules and inorganic molecules present in cells. The term does not include large macromolecules such as large proteins (e.g., proteins having a molecular weight of more than 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), large nucleic acids (e.g., nucleic acids having a molecular weight of more than 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), or large polysaccharides (e.g., polysaccharides having a molecular weight of more than 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000). Small molecule substances of cells are generally found free in solution in the cytoplasm or other organelles, such as mitochondria, where they form a pool of intermediates that can be further metabolized or used to produce macromolecules known as macromolecules. The term "small molecule substance" includes signal transduction molecules and intermediates in chemical reactions that convert energy derived from food into useful forms. Examples of small molecule substances include sugars, fatty acids, amino acids, nucleotides, intermediates formed during cellular processes, and other small molecule substances present within cells.
By "metabolic profile" or "small molecule profile" is meant the complete or partial listing of small molecules within a targeted cell, tissue, organ, organism, or portion thereof (e.g., cellular compartment). The list may include the amount and/or type of small molecules present. The "small molecule profile" can be determined using a single technique or a plurality of different techniques.
Identifying small molecule substances that are directed against atherosclerotic cerebral infarction allows for the diagnosis (or aids in the diagnosis) of disease in subjects exhibiting one or more symptoms consistent with the presence of an atherosclerotic cerebral infarction, and includes both a preliminary diagnosis in subjects not previously identified as having an atherosclerotic cerebral infarction and a diagnosis of recurrence of disease in subjects previously treated for an atherosclerotic cerebral infarction. A method of diagnosing (or aiding in diagnosing) whether a subject has an atherosclerotic cerebral infarction comprising:
analyzing a biological sample from a subject to determine the level of atherosclerotic cerebral infarcted small molecule species in the sample; and
comparing the level of the small molecule substance in the sample to an atherosclerotic cerebral infarction positive and/or an atherosclerotic cerebral infarction negative reference level of the small molecule substance to diagnose (or aid in diagnosing) whether the subject has an atherosclerotic cerebral infarction.
In a specific embodiment of the invention, the small molecule substance used comprises PE (P-18:0/18:2(9Z, 12Z)). When such a method is used to aid in the diagnosis of atherosclerotic cerebral infarction, the results of the method may be used in conjunction with other methods (or results thereof) that may be used to clinically determine whether a subject has an atherosclerotic cerebral infarction. The invention discovers the metabolic marker PE (P-18:0/18:2(9Z,12Z)) related to atherosclerotic cerebral infarction for the first time through extensive and intensive research. PE (P-18:0/18:2(9Z,12Z)) is significantly reduced in atherosclerotic cerebral infarcted patients compared to atherosclerotic patients.
In the present invention, any suitable method may be used to analyze a biological sample to determine the level of the small molecule substance in the sample. Suitable methods include chromatography (e.g., HPLC, gas chromatography, liquid chromatography), mass spectrometry (e.g., MS-MS), enzyme-linked immunosorbent assay (ELISA), antibody ligation, other immunochemical techniques, and combinations thereof. Further, the level of the one or more small molecule substances may be measured indirectly, for example by using an assay that measures the level of a compound (or compounds) that correlates with the level of the small molecule substance desired to be measured.
The Mass Spectrometry (MS) analysis mainly comprises liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), and the liquid chromatography is divided into ultra-high performance liquid chromatography (UPLC) and High Performance Liquid Chromatography (HPLC). Alternatively, the chromatogram can be optionally used in conjunction with time-of-flight (TOF), electrostatic Orbitrap (Orbitrap), ion trap time-of-flight (IT-TOF), quadrupole-time-of-flight (Q-TOF), and other mass spectra; for targeted metabonomics analysis, chromatography can be connected with mass spectra such as triple quadrupole (QQQ) or quadrupole ion Trap (Q-Trap) in series, and a multi-reaction monitor (MRM) is used for detection.
After determining the level of the small molecule substance in the sample, comparing the level to an atherosclerotic cerebral infarction positive and/or an atherosclerotic cerebral infarction negative reference level to diagnose or help diagnose whether the subject has an atherosclerotic cerebral infarction. Matching the level of the small molecule substance in the sample to an atherosclerotic cerebral infarction positive reference level (e.g., a level that is the same as, substantially the same as, above and/or below a minimum and/or maximum of, and/or within a range of the reference level) is indicative of a diagnosis that the subject has an atherosclerotic cerebral infarction. A match of the level of the small molecule substance in the sample to an atherosclerotic cerebral infarction negative reference level (e.g., a level that is the same as, substantially the same as, above, and/or below a minimum and/or maximum of, and/or within a range of a reference level) indicates that the subject is not diagnosed with an atherosclerotic cerebral infarction. Furthermore, the level of a small molecule substance that is differentially present (particularly at a statistically significant level) in the sample compared to an atherosclerotic cerebral infarction negative reference level is indicative of a diagnosis of an atherosclerotic cerebral infarction in the subject. The level of small molecule species differentially present (particularly at a statistically significant level) in the sample compared to an atherosclerotic cerebral infarction positive reference level is indicative of the subject not being diagnosed with an atherosclerotic cerebral infarction.
The level of the small molecule substance can be compared to an atherosclerotic cerebral infarction positive and/or an atherosclerotic cerebral infarction negative reference level using a variety of techniques including a simple comparison (e.g., a manual comparison) of the level of the small molecule substance in the biological sample to an atherosclerotic cerebral infarction positive and/or an atherosclerotic cerebral infarction negative reference level. The level of the small molecule substance in the biological sample may also be compared to an atherosclerotic cerebral infarction positive and/or an atherosclerotic cerebral infarction negative reference level using one or more statistical analyses (e.g., T-test, welch's T-test, Wilcoxon rank-sum test, Random Forest (Random Forest), T-score, Z-score) or using mathematical models (e.g., algorithms, statistical models, mixed effect models).
The invention provides a product for diagnosing atherosclerotic cerebral infarction, which comprises a reagent for detecting the level of a metabolite PE (P-18:0/18:2(9Z,12Z)) in a sample. The product may be in any form including, but not limited to, a kit, a chip.
As an alternative embodiment, the components of the kit may be packaged in one or more containers, such as one or more vials. In addition to the metabolite standards, the kit preferably further comprises a preservative or buffer for storage. In addition, the kit may contain instructions for use.
As an alternative embodiment, the chip has a reagent capable of detecting and/or quantifying one or more metabolites immobilized at predetermined locations on the substrate. As an illustrative example, the chip may be provided with reagents immobilized at discrete predetermined locations for detecting and quantifying the amount or concentration of PE (P-18:0/18:2(9Z,12Z)) in the sample; as described above, an increased level of this metabolite was found in a sample of a subject suffering from an atherosclerotic cerebral infarction. The chip may be configured such that a detectable output (e.g. a colour change) is provided only when the amount or concentration of the metabolite exceeds a threshold value selected or differentiated between the concentration of the metabolite indicative of an atherosclerotic subject and the amount or concentration of the metabolite indicative of a suffering from or susceptible to an atherosclerotic cerebral infarction. Thus, the presence of a detectable output (such as a color change) immediately indicates that the sample contains a significantly increased level of the metabolite, indicating that the subject is suffering from or susceptible to atherosclerotic cerebral infarction.
The most common global measure to judge the diagnostic efficacy of a small molecule substance or product is the area under the ROC curve (AUC). Conventionally, this area is always ≧ 0.5 (if not, the decision rule can be reversed to do so). The range of values was between 1.0 (test values that perfectly separated the two groups) and 0.5 (no significant distribution difference between the test values of the two groups). The area depends not only on a particular part of the line graph, such as the point closest to the diagonal or the sensitivity at 90% specificity, but also on the entire line graph. This is a quantitative, descriptive representation of how the ROC plot is close to perfect (area 1.0).
Overall assay sensitivity will depend on the specificity required to carry out the methods disclosed herein. In certain preferred settings, a specificity of 75% may be sufficient, and statistical methods and resulting algorithms may be based on this specificity requirement. In a preferred embodiment, the method for assessing an individual having an atherosclerotic cerebral infarction is 80%, 85%, or still preferably 90% or 95% based on specificity.
The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental methods in the examples, in which specific conditions are not specified, are generally carried out under conventional conditions.
Example screening and potency determination of metabolites associated with atherosclerotic cerebral infarction
1. Sample collection
Blood samples were collected from 21 patients with atherosclerotic cerebral infarction and 21 patients with atherosclerosis.
Atherosclerotic cerebral infarction group inclusion criteria:
1) the subject has signed an informed consent
2) Meets the acute cerebral infarction diagnosis standard of Chinese acute ischemic stroke diagnosis and treatment guidelines (2014 edition).
3) The etiological classification is atherosclerosis cerebral infarction.
4) Age 18-65 years old.
5)BMI 18.5-23.9kg/m2。
6) Blood routine: red blood cell count, MCHC, hemoglobin, white blood cell count, lymphocyte count, neutrophil count, monocyte count are in the normal range.
7) TG, TC, HDL-C, LDL-C, blood glucose, and glycated hemoglobin were in the normal range.
Exclusion criteria:
1) the combination of other diseases: nervous system diseases (past cerebral infarction, cerebral hemorrhage, multiple sclerosis, etc.); various chronic digestive system diseases, acute digestive system diseases within 3 months; circulatory disorders (coronary heart disease, heart failure, atrial fibrillation); respiratory diseases (chronic obstructive pulmonary disease, chronic bronchitis, asthma); metabolic diseases (obesity, hyperlipidemia, diabetes, metabolic syndrome, osteoporosis); urinary system diseases (chronic kidney disease, renal failure, kidney stones); hematological disorders (anemia); others (gout, depression, psychiatric disorders, chronic fatigue syndrome, fibromyalgia, food allergies, tumors).
2) The history of blood transfusion, operation and trauma of digestive system diseases.
3) Patients with abnormal electrocardiograms.
4) The following drugs were taken within 3 months: antibiotics, laxatives, clonazepam, sex hormone drugs, oral contraceptives, mesalamine, TNF-alpha inhibitors, immunosuppressants, antidepressants, PPIs, rupatadine, opioids, calcium agents, vitamin D, metformin, folic acid, beta-sympathetic inhalants, traditional Chinese medicines.
5) The probiotic preparation is administered within 3 months.
6) Antiplatelet and statins are applied before the disease.
7) Patients undergoing intravenous thrombolysis and endovascular intervention.
8) Pregnant or lactating women.
9) During this study, the patient had enrolled or planned to enroll in another clinical drug or device/interventional study.
The atherosclerotic group inclusion criteria were:
1) the subject has signed an informed consent.
2) Cervical vascular ultrasound and/or cervical vascular imaging is manifested as intracranial and extracranial vascular atherosclerosis.
3) Age 18-65 years old.
4)BMI 18.5-23.9kg/m2。
5) Blood routine: red blood cell count, MCHC, hemoglobin, white blood cell count, lymphocyte count, neutrophil count, monocyte count are in the normal range.
6) TG, TC, HDL-C, LDL-C, blood glucose, and glycated hemoglobin were in the normal range.
Exclusion criteria:
1) there are other diseases: nervous system diseases (cerebral infarction, cerebral hemorrhage, multiple sclerosis, etc.); various chronic digestive system diseases, acute digestive system diseases within 3 months; circulatory disorders (coronary heart disease, heart failure, atrial fibrillation); respiratory diseases (chronic obstructive pulmonary disease, chronic bronchitis, asthma); metabolic diseases (obesity, hyperlipidemia, diabetes, metabolic syndrome, osteoporosis); urinary system diseases (chronic kidney disease, renal failure, kidney stones); hematological disorders (anemia); others (gout, depression, psychiatric disorders, chronic fatigue syndrome, fibromyalgia, food allergies, tumors).
2) The history of blood transfusion, operation and trauma of digestive system diseases.
3) The electrocardiogram is abnormal.
4) The following drugs were taken within 3 months: antibiotics, laxatives, clonazepam, sex hormones, oral contraceptives, mesalamine, TNF-alpha inhibitors, immunosuppressants, antidepressants, PPIs, rupatadine, opioids, calcium agents, vitamin D, metformin, folic acid, beta-sympathetic inhalants, traditional Chinese medicines, antiplatelet drugs, and statins.
5) The probiotic preparation is administered within 3 months.
6) Pregnant or lactating women.
7) During this study, the subject has enrolled or is scheduled to enroll in another clinical drug or device/interventional study.
2. Non-targeted metabolomics detection
2.1 serum sample preparation
2.1.1 reverse phase chromatography method for processing serum samples
1) The plasma/serum samples were thawed on ice at 4 ℃ for 30-60 min.
2) Mu.l serum was taken to a labeled 1.5ml centrifuge tube and 300. mu.l methanol and 1ml methyl tert-butyl ether were added.
3) The protein was precipitated by shaking thoroughly for 15 s. Centrifuging at 12000rpm and 4 deg.C for 10min, collecting upper layer solution 100 μ l, placing in 200 μ l liner tube, and testing.
2.1.2 hydrophilic chromatography serum sample treatment method:
1) the plasma/serum samples were thawed on ice at 4 ℃ for 30-60 min.
2) Mu.l serum was taken to a labeled 1.5ml centrifuge tube and 150. mu.l acetonitrile was added.
3) The protein was precipitated by shaking thoroughly for 15 s. Centrifuging at 12000rpm and 4 deg.C for 10min, collecting upper layer solution 100 μ l, placing in 200 μ l liner tube, and testing.
2.2 chromatographic conditions
Chromatographic separation serum samples were analyzed by reverse phase chromatography and hydrophilic chromatography using U3000 flash liquid chromatography from Thermo Scientific.
2.2.1 reverse phase chromatographic separation conditions
Chromatography column waters UPLC HSS T3(1.8 μm 2.1mm 100 mm);
mobile phases a (acetonitrile/water 4:6, 0.1% formic acid, 10mM ammonium acetate) and B (acetonitrile/isopropanol 9:1, 0.1% formic acid, 10mM ammonium acetate);
elution procedure: see table 1;
flow rate: 0.3 ml/min;
the sample injection amount is 1.0 mu L;
column temperature: at 50 ℃.
TABLE 1C 18 reverse phase chromatography determination of elution procedure
2.2.1 conditions for hydrophilic chromatographic separation
Chromatography column waters UPLC BEH Amide (1.7 μm 2.1mm 100 mm);
mobile phases a (acetonitrile, 0.1% formic acid, 10mM ammonium acetate) and B (water, 0.1% formic acid, 10mM ammonium acetate);
elution procedure: see table 2;
flow rate: 0.3 ml/min;
sample introduction amount: 1.0 μ L;
column temperature: at 40 ℃.
TABLE 2 HILIC determination of polar Small molecule elution procedure
2.3 Mass Spectrometry conditions
Mass spectrometry uses a quadrupole rod orbited ion trap mass spectrometer equipped with a thermoelectric spray ion source. The voltages of the positive and negative ion sources were 3.7kV and 3.5kV, respectively. The capillary heating temperature was 320 ℃. The warp air pressure was 30psi and the assist air pressure was 10 psi. The evaporation temperature was 300 ℃ with volume heating. The tilted gas and the auxiliary gas are both nitrogen. The collision gas is nitrogen and the pressure is 1.5 mTorr. The first-order full scan parameters are: resolution 70000, automatic gain control target of 1 × 106Maximum isolation time 50ms, mass to charge ratio scan range 50-1500. The liquid system is controlled by Xcaliibur 2.2SP1.48 software, and both data acquisition and targeted metabolite quantitative processing are operated by the software.
3. Targeted metabonomic detection
3.1 serum sample processing method
1) Plasma samples were thawed by standing at 4 ℃ for 30 min.
2) A50. mu.l plasma sample was taken into a 1.5ml centrifuge tube, 150. mu.l methanol (containing indoleacetic acid-D2500 ppb, indolepropionic acid-D250 ppb) was added, and vortexed for 30 min.
3) Centrifuging at 12000rpm for 5min, collecting supernatant 100 μ l, placing in 200 μ l liner tube, and testing.
3.2 chromatographic conditions
The chromatographic separation adopts a Waters ACQUITY UPLC I-CLASS ultrahigh pressure liquid chromatographic system, and the chromatographic separation conditions are as follows:
chromatography column Waters UPLC BEH C8(1.7 μm 2.1mm 100 mm);
mobile phase A (water, 0.5Mm NH)4F) And B (methanol);
elution gradient: see table 3;
flow rate: 0.3 ml/min;
sample introduction amount: 1.0 μ L;
column temperature: at 45 ℃.
TABLE 3 elution procedure
3.3 Mass Spectrometry conditions
The mass spectrometer is a Waters XEVO TQ-XS type tandem quadrupole mass spectrometer. The voltage of the positive ion source is 3kv, and the voltage of the taper hole is 20V. The desolvation temperature is 550 ℃, and the source temperature is 150 ℃. The desolventizing air flow rate is 1000L/Hr, and the taper hole air flow rate is 7L/h.
3.4 Targeted Metabolic group data treatment
The peak area calculation of the targeted metabolome data adopts masslynx quantitative software, and the retention time allows the error to be 15 s. And the concentration calculation adopts a single-point isotope internal standard method to obtain a quantitative result.
4. Data processing
4.1 data quality control
To evaluate the stability and reproducibility of the system during sample collection, quality control samples were used. The quality control sample is obtained by transferring all samples into a fixed volume and uniformly mixing. The pretreatment method of the finger-controlled sample is the same as that of other samples. To obtain a reliable and reproducible metabolite, three factors need to be considered: 1) retention time, 2) signal strength, 3) mass accuracy. In the experiment, 5 blank sample balance chromatographic columns are adopted firstly, and then 3 quality control sample balance chromatographic columns are adopted. Then every 6-8 samples insert 1 quality control sample for monitoring the whole liquid quality system stability and repeatability. And simultaneously calculating the coefficient of variation value of the metabolic features extracted from the quality control samples, and deleting the metabolic features of which the coefficient of variation exceeds 15%.
4.2 PCA analysis
All collected data, no matter what separation mode or positive and negative ion mode, are processed by Progenetics QI software, and the steps include importing original data, aligning peaks, extracting peaks, normalizing, and finally forming a table of retention time, mass-to-charge ratio and peak intensity. The time for extracting peaks by the reversed phase chromatography and the hydrophilic chromatography is 1 to 16 and 1 to 12min in sequence. Various additive ions such as hydrogen and sodium are deconvoluted into each ion signature. Metabolite identification primary molecular weight matching was performed using the human metabolome database and the lipid database.
4.3OPLS-DA analysis
In order to obtain metabolite information which shows significant difference between the atherosclerotic cerebral infarction group (BL) and the atherosclerotic group (AS), statistical analysis was further performed on the two groups of samples by using a supervised multidimensional statistical method, namely partial least squares discriminant analysis (OPLS-DA).
Differentially expressed metabolites were searched for using the VIP (variable immunity in the project) value (threshold >1) of the OPLS-DA model in combination with the p-value of t-test (p < 0.05). The qualitative method of differential metabolites was: search the online database (HMDB) (compare mass to charge ratio m/z of mass spectra or exact molecular mass, error limit 0.01 Da).
4.4ROC analysis
According to the levels of the metabolites, a receiver operating characteristic curve (ROC) is drawn, two accurate confidence spaces are calculated, and the diagnostic efficacy of the differential metabolites is analyzed.
5. Results
The quality control result shows that the quality control samples are relatively gathered together, the system has good repeatability, and the acquired data can be further researched.
The results of the reverse chromatography positive ion, the reverse chromatography negative ion, and the hydrophilic chromatography positive ion are shown in table 4 and fig. 1, respectively.
TABLE 4 OPLS-DA analytical model parameters
Bioinformatic analysis results showed that the level of PE (P-18:0/18:2(9Z,12Z)) was significantly reduced in the atherosclerotic cerebral infarcted group compared to the atherosclerotic group (fig. 2).
The content of PE (P-18:0/18:2(9Z,12Z)) is used as a detection variable to judge the diagnosis efficiency, and the result shows that the area under the curve is 0.873, the cutoff value is 4110417.834, the sensitivity is 0.762, the specificity is 0.905 (figure 3), and the method has higher sensitivity, specificity and accuracy.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
Claims (7)
1. Use of a reagent for the detection of a small molecule substance in a sample selected from the group consisting of plasma and serum, in the manufacture of a product for the assessment of atherosclerotic cerebral infarction, wherein the small molecule substance is PE (P-18:0/18:2(9Z, 12Z)).
2. Use according to claim 1, wherein the product comprises reagents for the detection of markers by chromatography, spectroscopy, mass spectrometry, chemical analysis.
3. Use according to claim 1 or 2, wherein the product further comprises reagents for processing the sample.
4. The use according to claim 1, wherein a decrease in the level of PE (P-18:0/18:2(9Z,12Z)) in the sample from the subject is indicative of the subject having or being at risk of having an atherosclerotic cerebral infarction.
5. Use according to claim 1 or 2, wherein the product comprises a kit, a chip.
6. The use of claim 5, wherein the product is used to indicate whether a subject has, or is at risk for, an atherosclerotic cerebral infarction by detecting the level of a small molecule substance in a sample from the subject and comparing the level to a reference metabolite profile.
7. Use of a small molecule substance in a sample selected from plasma or serum for the manufacture of a product for identifying and assessing the effect of a pharmaceutical agent and/or a surgical and/or physical treatment on anti-atherosclerotic cerebral infarction, wherein the small molecule substance is PE (P-18:0/18:2(9Z, 12Z)).
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