CN112730638A - Diabetes combined myocardial infarction metabolism marker, detection reagent and kit - Google Patents
Diabetes combined myocardial infarction metabolism marker, detection reagent and kit Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention discloses a metabolic marker for diabetic combined myocardial infarction, which comprises the following components: dihydroceramide 22:0, dihydroceramide 8:0, phosphorylated dihydroceramide 18:0, ceramide 18:1, and phosphorylated dihydroceramide 16: 0. The invention also provides a detection reagent and a kit. The invention can be used for diagnosing diabetes combined myocardial infarction, and improves the convenience and standardization degree of diagnosis.
Description
Technical Field
The invention relates to the technical field of biochemistry. More specifically, the invention relates to a marker, a detection reagent and a kit for diabetes complicated with myocardial infarction metabolism.
Background
Diabetes is a metabolic disease characterized by chronic hyperglycemia, with the pathogenesis of insulin resistance and beta cell dysfunction. Patients with diabetes are at 2-4 times higher risk of cardiovascular disease than non-diabetic patients, with myocardial infarction with diabetes mortality 2.3 times higher than non-diabetic patients. At present, cardiovascular complications are the leading cause of death and disability of diabetes, and the prevention and treatment of diabetes and myocardial infarction is an important measure for improving the life quality of diabetics and reducing the death rate of diabetes. Therefore, there is a need for a metabolic marker, a detection reagent and a kit for diabetes complicated with myocardial infarction.
Disclosure of Invention
An object of the present invention is to provide a metabolic marker, a detection reagent and a kit for diabetic myocardial infarction, which can be used for diagnosing diabetic myocardial infarction, and improve the convenience and standardization of diagnosis.
To achieve these objects and other advantages in accordance with the purpose of the invention, according to one aspect of the present invention, there is provided a diabetes-associated myocardial infarction metabolic marker, comprising:
dihydroceramide 22:0, dihydroceramide 8:0, phosphorylated dihydroceramide 18:0, ceramide 18:1, and phosphorylated dihydroceramide 16: 0.
According to another aspect of the invention, detection reagents are also provided, including reagents for detecting the diabetes combined myocardial infarction metabolic markers.
Further, the detection reagent comprises a standard curve stock solution and an internal standard stock solution which are suitable for the liquid chromatography-mass spectrometry technology.
Further, the standard curve stock solution of the detection reagent comprises a ceramide 16:0 solution, a ceramide 18:0 solution, a ceramide 20:0 solution, a ceramide 22:0 solution, a ceramide 24:1 solution and a ceramide 24:0 solution.
Further, the detection reagent and the internal standard stock solution comprise a ceramide 17:0 solution.
Further, the detection reagent also comprises a blank matrix.
Further, the detection reagent and the blank matrix are bovine serum albumin solution.
According to still another aspect of the present invention, there is also provided a kit characterized by comprising the detection reagent.
The invention at least comprises the following beneficial effects:
the metabolic marker exists in serum, and is convenient to analyze and measure. The detection reagent and the kit can be used for diagnosing diabetes combined myocardial infarction, improve the convenience and the standardization degree of the diagnosis of the diabetes combined myocardial infarction and provide reference for preventing and treating the diabetes combined myocardial infarction.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIGS. 1-5 are various sphingolipid metabolite calibration curves; FIG. 1 shows a standard curve (weight coefficients: 1/x, r) of ceramide 16:0(Cer 16:0)20.9942); FIG. 2 shows a standard curve of ceramide 18:0(Cer 18:0) (weight coefficients: 1/x, r)20.9940); FIG. 3 shows a standard curve of ceramide 20:0(Cer 20:0) (weight coefficients: 1/x, r)20.9955); FIG. 4 shows a ceramide 22:0(Cer 22:0) standard curve (weight coefficient: 1/x, r)20.9900); fig. 5 is a ceramide 24:0(Cer 24:0) standard curve (weight factor: 1/x, r2 ═ 0.9916);
FIG. 6 is a plot of sphingolipid metabolites with significant differences between diabetic vs diabetic infarcts (potential biomarkers in box line); a, quantitative sphingosine of a standard curve; b, other sphingolipids quantified according to the Cer 18:0 standard curve.
Detailed Description
The present invention is described in further detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Embodiments of the present application provide markers of diabetes complicated with myocardial infarction metabolism, including: dihydroceramide 22:0, dihydroceramide 8:0, phosphorylated dihydroceramide 18:0, ceramide 18:1, and phosphorylated dihydroceramide 16:0, i.e., dhCer 22:0, dhCer 8:0, dhCer 18:0:1P, Cer 18:1, dhCer 16:0: 1P. Also comprises one or more of ceramide 16:0, ceramide 14:0, hexosylceramide 16:0, phosphorylated ceramide 12:0 and hexosylceramide 24:0, namely Cer 16:0, Cer 14:0, HexCer 16:0, Cer 12:0:1P, HexCer 24: 0.
The embodiment of the application also provides a detection reagent, which comprises a reagent for detecting the diabetes combined myocardial infarction metabolic marker, and any reagent capable of detecting the metabolic marker.
In other embodiments, the standard curve stock solution and the internal standard stock solution suitable for the LC-MS technology, the standard curve stock solution and the internal standard stock solution capable of using the LC-MS technology for metabolic markers, and the LC-MS technology can be a high performance liquid chromatograph and a triple quadrupole mass spectrometer.
In other embodiments, the standard curve stock solution comprises a ceramide 16:0 solution, a ceramide 18:0 solution, a ceramide 20:0 solution, a ceramide 22:0 solution, a ceramide 24:1 solution, and a ceramide 24:0 solution. Cer 16:0, Cer 18:0, Cer 20:0, Cer 22:0, Cer 24:1 and Cer 24:0 are quantitatively analyzed by a standard curve method, and other sphingolipid metabolites are substituted into a Cer 18:0 standard curve for quantitative analysis.
In other embodiments, the internal standard stock comprises a ceramide 17:0 solution.
In other embodiments, a blank matrix is also included.
In other embodiments, the blank matrix is a bovine serum albumin solution.
Embodiments of the present application also provide kits comprising the detection reagents described in the above embodiments.
The following is a specific example:
1. purpose(s) to
Establishing a sphingomyelinomics analysis method based on a liquid chromatography-mass spectrometry technology, analyzing serum samples of patients with asthma and diabetes combined myocardial infarction, and searching for potential biomarkers related to diseases according to statistical analysis and metabonomics potential biomarker screening standards.
2. Sample preparation: the diabetes combined myocardial infarction related samples comprise: diabetes and diabetes combined myocardial infarction samples and the like. The number of samples in each group is shown in Table 1.
TABLE 1 sample grouping information
3. Laboratory instruments and materials
A high performance liquid chromatograph (Agilent 1290 RRLC system, Agilent Technologies, inc. santa Clara, CA) equipped with a high pressure pump, an autosampler, a degasser, a column oven; triple quadrupole mass spectrometer (Agilent 6490 Triple Quad mass spectrometer, Agilent Technologies, inc. santa Clara, CA) equipped with an electrospray ion source (ESI); masshunter 3.0 data processing system. ORTEX-2GENE vortex mixer, Scientific Industry; METLER TOLEDO AG135 model electronic balance, Mettler, Switzerland.
Sphingolipid standards (Cer 16_0, Cer 18_0, Cer 20_0, Cer 22_0, Cer 24_1, Cer 24_0, Cer 17_0), Avanti corporation, usa; deionized water, Hangzhou Waha group Co; methanol, mass spec grade, Fisher corporation; methyl tert-butyl ether, chromatographic grade, Fisher corporation; chloroform, chromatographic grade, Beijing Tong GuangZhou Fine chemical Co; ammonium formate, mass spec grade, Sigma company; formic acid, ms grade, Sigma company.
4. The experimental method comprises the following steps:
4.1 analytical methods
4.1.1 chromatographic conditions
A chromatographic column: peeke SPECTRA C8SR (150 mm. times.3.0 mm, 3 μm); mobile phase A: water (containing 0.1% formic acid, 1mmol/L ammonium formate), mobile phase B: methanol (containing 0.1% formic acid, 1mmol/L ammonium formate); flow rate: 0.5 mL/min; column temperature: 40 ℃; sample introduction amount: 10 μ L, gradient elution conditions: 80% -100% B for 0-10 min; 10-18min 100% B; pre-balancing: and 6 min.
4.1.2 Mass Spectrometry conditions
Detecting in positive ion mode of AJS ESI source: temperature of the drying gas: 280 ℃; flow rate of drying gas: 14L/min; the speed of the atomization gas flow: 30 psi; temperature of sheath gas: 250 ℃; flow rate of sheath gas: 11L/min; capillary voltage: 4000V; auxiliary nozzle voltage: 1500V.
54 sphingosine metabolites can be quantitatively analyzed by using a segmented Multiple Reaction Monitoring (MRM) mode, and retention time, quantitative ion pair and related mass spectrum parameters of each sphingosine metabolite are shown in a table 2.
TABLE 2 segmentation, retention time, quantitative ion pairs and associated Mass Spectrometry parameters for each sphingolipid metabolite
4.2 detection reagent
4.2.1 preparation of stock solutions
Each of Cer 16:0, Cer 18:0, Cer 20:0, Cer 22:0, Cer 24:1, and Cer 24:0 was precisely weighed at about 5mg, and the weighed substances were placed in a 5mL volumetric flask, and the volume was measured using chloroform: methanol (1:1, v/v) is dissolved and diluted to the scale, and standard yeast stock solutions with the concentration of 1mg/mL are respectively prepared.
4.2.2 preparation of stock solutions
Cer 17:0 about 5mg was precisely weighed, and placed in a 5mL volumetric flask, and the contents of chloroform: methanol (1:1, v/v) was dissolved and diluted to the mark to prepare an internal standard stock solution with a concentration of 1 mg/mL.
4.2.3 preparation of working solution with standard curve
Standard curve sample working solutions are prepared by using the stock solutions (1mg/mL) of the standard curve samples, and are diluted step by step through methanol solutions to finally prepare the standard curve working solutions (W1-W10, 100 times of the actual concentration in serum) with the concentrations of 20, 50, 100, 200, 500, 1000, 2000, 5000, 10000 and 20000 ng/mL.
4.2.4 preparation of internal standard working solution
10 μ g/mL internal standard working solution: accurately sucking 10 mu L of 1mg/mL stock solution, adding 990 mu L of methanol solution, and uniformly mixing to obtain 10 mu g/mL internal standard working solution. And (3) putting 500 mu L of internal standard working solution (10 mu g/mL) into a 5mL volumetric flask, diluting with a methanol solution, fixing the volume, and uniformly mixing to obtain 1000ng/mL of internal standard working solution (10 times of the concentration in serum).
4.3 preparation of blank matrix
Accurately weighing 100mg of bovine serum albumin in a 10mL volumetric flask, dissolving with normal saline, and fixing the volume to the scale to obtain the blank matrix.
4.4 matrix Standard Curve sample preparation
10 mu L of each concentration standard curve working solution W1-W10 is transferred by using a pipette gun and is respectively added into 990 mu L of blank matrix, and after uniform mixing, 1mL of matrix standard curve samples S1-S10(0.2, 0.5, 1, 2, 5, 10, 20, 50, 100 and 200ng/mL) are obtained, subpackaged and numbered, and stored at-80 ℃ for later use.
4.5 sample pretreatment
Precisely sucking 100 μ L human serum sample (or matrix standard curve sample), adding 10 μ L internal standard solution, and vortex mixing. Adding 1.5mL of methanol and 5mL of methyl tert-butyl ether, performing high-power vortex for 15min, adding 1.5mL of deionized water, performing phase separation, performing centrifugation at 4500rpm for 10min, absorbing the upper organic phase, placing in another glass tube, performing nitrogen blow-drying, and re-dissolving with 100 mu L of mobile phase B.
4.6 quality control sample preparation
And (3) absorbing part of serum, mixing the serum and the sample, and performing post-preparation analysis according to a sample pretreatment method, wherein the number of quality control samples is more than 5% of the total analysis samples, and calculating the peak area RSD value of each sphingolipid metabolite in the quality control samples to perform quality control.
4.7 data analysis
Quantitative analysis was performed on 54 sphingolipid metabolites using Agilent MassHunter quantitative analysis software, where Cer 16:0, Cer 18:0, Cer 20:0, Cer 22:0, Cer 24:1, Cer 24:0 were quantitatively analyzed by a standard curve method (weight coefficient: 1/x), and other sphingolipid metabolites were quantitatively analyzed by substituting into the Cer 18:0 standard curve. Comparison of significance differences between groups was performed using independent sample t-test in SPSS 19.0 statistical analysis software (P < 0.05), potential biomarker extraction was performed using SIMCA P14.0 metabolomics analysis software (VIP value greater than 1; Jack-knife value greater than 0; absolute value of Pcor in S-plot greater than 0.58).
5. The experimental results are as follows:
5.1 Standard Curve and Linear Range for Each sphingolipid metabolite
Taking a substrate standard curve sample of each sphingolipid metabolite series concentration, continuously sampling from low concentration to high concentration, taking the concentration (X) of a measured object in the substrate as a horizontal coordinate, taking the peak area ratio (Y) of the measured object and an internal standard substance as a vertical coordinate, and calculating a standard curve by using Mass Hunter workshop Software Quantitative Analysis (Version B.03.02) to obtain a substrate standard curve linear equation and a linear correlation coefficient value r2(see FIGS. 1-5). The results show that the ratio of the peak area to the internal standard peak area is good in linearity with concentration, and the correlation coefficient r is2Are all greater than 0.99.
5.2 quality control sample analysis
The number of human serum samples is 40, 6 needles of quality control samples (accounting for more than 5 percent of the total number of the samples) are analyzed in the experiment in total, the peak area RSD of each sphingolipid metabolite quantitatively analyzed in the quality control samples is calculated to be less than 30 percent and accords with the analysis requirement of metabonomics, and the peak area RSD value of each sphingolipid metabolite control sample is shown in table 3.
TABLE 3 Peak area RSD of each sphingolipid metabolite in quality control samples
Note: hex is hexosylceramide, P is phosphorylated ceramide, HexSph is hexosylsphingosine (hexosylsphingosine)
Respectively carrying out quantitative analysis on samples of each group such as a diabetes group, a diabetes myocardial infarction and the like, carrying out comparison among the groups, judging the difference among sphingolipid metabolite groups through independent sample t test, and searching potential biomarkers among the groups through analysis of SIMCA P software OPLS-DA. Sphingolipid metabolites with significant differences (p < 0.05) from t-test among groups are shown in Table 4, sphingolipid metabolites that meet the criteria for potential biomarker screening are shown in Table 5, and a histogram of sphingolipid content with significant differences among groups is shown in FIG. 6.
TABLE 4 sphingolipid metabolites with significant differences between groups of diabetic myocardial infarction-related human serum
TABLE 5 latent biomarkers between groups of diabetic myocardial infarction-related human serum
6. To summarize:
sphingomyelinomics research based on the liquid chromatography-mass spectrometry technology finds and obtains metabolic markers among diabetic vs diabetic myocardial infarction groups through the analysis of biomedicine and the screening of potential biomarkers. The research result shows that 10 ceramide metabolites have significant difference among groups (t test, p is less than 0.05), wherein 5 ceramide metabolites (dihydroceramide 22:0, dihydroceramide 8:0, phosphorylated dihydroceramide 18:0, ceramide 18:1 and phosphorylated dihydroceramide 16:0) meet the potential biomarker screening standard, the content of the 5 potential biomarkers in the diabetic myocardial infarction group is significantly increased, the diagnosis significance of the diabetic complication myocardial infarction is realized, and the diagnosis method can be used for diagnosing clinical diabetic complication CVD. Detection reagents are also provided based on metabolic markers.
The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. The application, modification and variation of the diabetes combined myocardial and myocardial infarction metabolic marker, the detection reagent and the kit of the invention are obvious to those skilled in the art.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention may be embodied with additional modifications as would be readily apparent to those skilled in the art, and the invention is thus not limited to the details given herein and to the illustrations shown and described without departing from the generic concept as defined by the claims and their equivalents.
Claims (8)
1. Diabetes complicated myocardial infarction metabolic marker, which is characterized by comprising:
dihydroceramide 22:0, dihydroceramide 8:0, phosphorylated dihydroceramide 18:0, ceramide 18:1, and phosphorylated dihydroceramide 16: 0.
2. A detection reagent comprising a reagent for detecting the metabolic marker of diabetes complicated with myocardial infarction according to claim 1.
3. The detection reagent of claim 2, comprising standard curve stock and internal standard stock suitable for use in a LC-MS technique.
4. The detection reagent of claim 3, wherein the standard curve stock solution comprises a ceramide 16:0 solution, a ceramide 18:0 solution, a ceramide 20:0 solution, a ceramide 22:0 solution, a ceramide 24:1 solution, and a ceramide 24:0 solution.
5. The detection reagent of claim 3, wherein the internal standard stock solution comprises a ceramide 17:0 solution.
6. The detection reagent of claim 3, further comprising a blank matrix.
7. The detection reagent of claim 6, wherein the blank matrix is a bovine serum albumin solution.
8. A kit comprising the detection reagent according to any one of claims 2 to 7.
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| CN113433254A (en) * | 2021-08-27 | 2021-09-24 | 宝枫生物科技（北京）有限公司 | Biomarker for diagnosing cerebral infarction of patient with leukoencephalopathy and application of biomarker |
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| CN116087485B (en) * | 2021-11-08 | 2023-10-20 | 中国医学科学院阜外医院 | Biomarkers for early prediction of cardiovascular disease (CVD) in diabetics and uses thereof |
| CN115356490A (en) * | 2022-08-17 | 2022-11-18 | 北京大学第三医院（北京大学第三临床医学院） | Biomarker for diagnosing type II diabetes, kit and application thereof |
| CN115356490B (en) * | 2022-08-17 | 2023-05-05 | 北京大学第三医院（北京大学第三临床医学院） | Biomarker for diagnosis of type II diabetes mellitus, kit and application thereof |
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