CN111830142A - Use of ceramides for the production of a kit for assessing the risk of an adverse event in a heart failure patient - Google Patents
Use of ceramides for the production of a kit for assessing the risk of an adverse event in a heart failure patient Download PDFInfo
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Abstract
The invention discloses application of a product for detecting ceramide in preparation of a kit; the kit is used for at least one of the following (a1) - (a 3): (a1) screening or aiding in the diagnosis of heart failure patients; (a2) assessing or aiding in assessing the risk of prognosis for a heart failure patient; (a3) assessing or aiding in assessing the risk of an adverse event occurring in a heart failure patient. The results of the study show that the ratio of plasma ceramide cer (d18:1/16:0)/cer (d18:1/24:0) is an important predictor of the occurrence of adverse events of heart failure in patients with end-stage heart failure, and is independent of the lipid markers currently used. This helps identify high risk patients that require more aggressive therapeutic intervention.
Description
Technical Field
The present invention relates to the use of ceramides for the preparation of a kit for assessing the risk of an adverse event in a heart failure patient.
Background
Heart failure, as the terminal stage of various cardiovascular diseases, is a progressive disease with a high incidence of disease and a high rate of hospitalization. At present, no good biomarker is available for predicting the poor prognosis of the heart failure, and energy metabolism change in the process of the heart failure occurrence and development is likely to indicate different stages of disease development, and under the conditions that more markers are still needed for auxiliary judgment on the prognosis of the heart failure prediction at present and the pathological mechanism is uncertain, no effective method is available for reducing the morbidity and mortality of the heart failure patients, and a new marker is urgently needed for assisting diagnosis and treatment and clinical management of the heart failure.
Disclosure of Invention
The invention aims to provide a marker for evaluating the prognosis risk of a heart failure patient.
The invention provides application of a product for detecting ceramide in preparation of a kit; the kit is used for at least one of the following (a1) - (a 3):
(a1) screening or aiding in the diagnosis of heart failure patients;
(a2) assessing or aiding in assessing the risk of prognosis for a heart failure patient;
(a3) assessing or aiding in assessing the risk of an adverse event occurring in a heart failure patient;
the invention also protects a kit comprising a product for detecting ceramide, and the kit is used for at least one of the following (a1) - (a 3):
(a1) screening or aiding in the diagnosis of heart failure patients;
(a2) assessing or aiding in assessing the risk of prognosis for a heart failure patient;
(a3) assessing or aiding in assessing the risk of an adverse event occurring in a heart failure patient;
the invention also protects the application of ceramide as a marker in screening or auxiliary diagnosis of heart failure patients.
The invention also protects the application of ceramide as a marker in the assessment or auxiliary assessment of the prognostic risk of heart failure patients.
The invention also protects the use of ceramides as markers for assessing or aiding in the assessment of the risk of an adverse event in a heart failure patient.
The ceramides described in the present invention include the following 7 classes: ceramides (d18:1/14:0), ceramides (d18:1/16:0), ceramides (d18:1/18:0), ceramides (d18:1/20:0), ceramides (d18:1/22:0), ceramides (d18:1/24:0) and ceramides (d18:1/24: 1).
Still further, the ceramides include the following 4 classes: ceramides (d18:1/16:0), ceramides (d18:1/18:0), ceramides (d18:1/20:0) and ceramides (d18:1/24: 1).
Or, the ceramide includes the following 4 classes: ceramides (d18:1/16:0) and ceramides (d18:1/24: 0).
The patients with heart failure in the invention particularly refer to patients with end-Stage heart failure (Stage C/D) classified according to the 2013 American Heart Association (AHA) guidelines for the management of acute and chronic heart failure.
The risk of adverse events occurring in a heart failure patient in the present invention refers to the risk of all-cause death and/or adverse events occurring in a heart transplant after discharge from a hospital in said heart failure patient.
The kit of the invention comprises a product for detecting the ceramide content in a biological sample of a healthy population or a heart failure patient.
The biological sample is a blood sample, a serum sample, a plasma sample or a blood lipid fraction or a blood lipoprotein fraction obtained therefrom;
the product for detecting the ceramide content in the biological sample of the healthy population or the heart failure patient comprises reagents (such as ceramide standard and the like) required for extracting the ceramide in the biological sample and determining by LC-MS/MS.
The kit is used for screening or assisting in diagnosing the patients with heart failure; the kit also comprises a carrier recorded with the following judgment standards: if the contents of ceramide (d18:1/16:0), ceramide (d18:1/18:0), ceramide (d18:1/20:0) and ceramide (d18:1/24:0) in the biological sample of the patient are all obviously higher than those of the healthy population, the patient is judged or suspected to be the patient with heart failure; and if the contents of the ceramide (d18:1/16:0), the ceramide (d18:1/18:0), the ceramide (d18:1/20:0) and the ceramide (d18:1/24:0) in the biological sample of the patient do not have significant difference with those of healthy people, judging that the patient is not or is suspected to be not the heart failure patient.
The content of ceramide (d18:1/16:0), ceramide (d18:1/18:0), ceramide (d18:1/20:0) and ceramide (d18:1/24:0) in the biological sample of the healthy population can be obtained by the skilled person by means of the prior art. The amounts of ceramide (d18:1/16:0), ceramide (d18:1/18:0), ceramide (d18:1/20:0) and ceramide (d18:1/24:0) in biological samples from healthy persons can be found in the following references: tarasov K, Ekroos K, Suoniemi M, et al molecular lipid identified by silicon Simvastatin and PCSK9Deficiency [ J ]. The Journal of clinical Endocrinology and Metabolim, 2013,99(1):45-52.)
The kit is used for evaluating or assisting in evaluating the prognosis risk of a heart failure patient, or evaluating or assisting in evaluating the risk of the heart failure patient of generating an adverse event; the kit also comprises a carrier recorded with the following judgment standards:
the measured concentration values of ceramide (d18:1/16:0) and ceramide (d18:1/24:0) were calculated according to the following formula (d18:1/16:0)/(d18:1/24:0), and the ratio was calculated, and then the cut-off value (approximately SE + SP-1) was obtained from the product under the ROC curve (data result was treated with SPSS 20.0); and if the cut-off value of the ratio is greater than or equal to 0.245, judging that the patient to be tested is a high-risk patient with all-cause death and heart transplantation.
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FIG. 1 is a comparison of the concentration of different types of ceramide in healthy individuals and in patients with heart failure.
FIG. 2 is a graph showing the comparison of the levels of various types of ceramide with the level of adverse events occurring in patients with end-stage heart failure.
Detailed Description
The present invention is described below with reference to specific embodiments, but the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The method for detecting ceramide in a biological sample (including a blood sample, a serum sample, a plasma sample or a blood lipid fraction or a blood lipoprotein fraction obtained therefrom) in the following examples was carried out as follows:
detection was performed on an ultrahighpressure liquid chromatography tandem mass spectrometry system Ultimate 3000 tandem TSQ Quantiva triple quadrupole mass spectrometer (Thermo Fisher Corp.). The mass and counts of the detected peaks obtained by mass spectrometry were converted into a list of corresponding classes of ceramide concentrations. A standard curve (calibration curve) was generated to determine the dynamic range of quantitation for each class of ceramide detected.
A strict threshold was applied to separate the background noise from the actual ceramide peak. Each sample was controlled and accepted only if the acceptance criteria were met. The masses and counts of the peaks detected are converted into a list of the names of the corresponding classes of ceramides, and the concentrations are obtained from the sample volumes, using a standard curve for quantification according to the ratio of the ceramides measured to the internal standard. The standard curve was prepared by mixing known amounts of internal reference (see table 1 below) with various types of ceramide standards (see table 2 below).
TABLE 1
| Species of internal reference | Goods number | Brand |
| C18 ceramide-d7(d18:1-d7/18:0) | 860676 | Avanti Polar Lipids |
| C24 ceramide-d 7(d18:1-d7/24:0) | 860678 | Avanti Polar Lipids |
| C24:1 ceramide-d 7(d18:1/24:1(15Z)) | 860679 | Avanti Polar Lipids |
TABLE 2
The sample extraction procedure was as follows:
1. preparation of standards
1) The primary mother liquor (5mM) was dissolved in chloroform/methanol 1:2(v/v) and separately in methanol solution to make 100uM stock solution, which was stored at-80 ℃ for two years or more.
2) Preparing a 10uM standard: the standard substance-ethanol mixed solution is prepared according to the volume ratio of 7:3 (one part of each of seven standard substances with 7 times of volume: 3 times of volume of ethanol solution), and the solution with the concentration of 10uM mixed standard substance can be obtained.
2. Preparing internal ginseng:
1) preparing a 20uM internal reference mixture: the internal reference and ethanol mixed solution is prepared according to the volume ratio of 3:2(3 times of volume of each of three internal references: 2 times of volume of ethanol solution), and the solution with the internal reference concentration of 20uM can be obtained.
3. Preparing a standard curve:
1) and (3) taking 40 mu L of ethanol solution containing 20uM of internal reference, placing the ethanol solution into a chromatographic flask, blowing nitrogen, and redissolving the ethanol solution into 800 mu L of methanol/acetonitrile mixed solution to obtain methanol/acetonitrile mixed solution containing 1uM of internal reference.
2) 80 μ L of 10uM standard mixture was placed in a chromatographic flask, purged with nitrogen, and redissolved in 100 μ L of 1uM internal reference in methanol/acetonitrile mixture to give 8uM mixed standard-S13 (highest concentration standard).
3) 14 chromatographic flasks were taken and labeled Blank, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, respectively. 50 μ L of a methanol/acetonitrile mixture containing 2uM of internal reference was added to each of the chromatography flasks of Blank, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, and S13.
4) Add 100. mu.L of 8uM mixed standard to the chromatographic flask of S13, and perform gradient dilution to the subsequent chromatographic flasks S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S13 (50. mu.L each) to obtain a standard curve. The concentrations of the corresponding standard curves in the S1-S13 chromatographic vials were, in order, 1.593nM, 3.906nM, 7.812nM, 15.625nM, 31.25nM, 62.5nM, 125nM, 250nM, 500nM, 1000nM, 2000nM, 4000nM, 8000 nM.
4. Sample treatment:
1) thawing the sample/quality control plasma; adding 20 μ L of plasma into a 12 × 75mm glass test tube, and adding 300 μ L of methanol containing 20 μ L of 2 μ M internal standard;
2) shaking for 2s, stopping shaking for 8s, centrifuging at 3000g and 4 deg.C for 15 min;
3) transferring the supernatant into a test tube with the diameter of 10 multiplied by 75mm, and blowing nitrogen; redissolving in 40 μ L methanol acetonitrile mixed solution (1:1, v/v), shaking for 2s, stopping shaking for 8s, shaking, transferring into a chromatographic bottle, and waiting for loading.
5. And (3) chromatographic column treatment:
when the chromatographic column is replaced, the ceramide detection is firstly washed by 50% B solution for 20 minutes, then by 100% B solution for 5 minutes, and finally by 80% B solution for 10 minutes.
Sample extracts (20. mu.L) were loaded onto a column attached to an ACQUITY UPLC BEH phenyl Column (3mm inner diameter X100 mm with 1.7 μm particles) at a column temperature of 40 ℃ Mobile phase A was 0.2% formic acid, 1uM aqueous ammonium formate solutionThe mobile phase B was 0.2% formic acid, 1uM ammonium formate in methanol, at a flow rate of 0.3mL/min, with a gradient as shown in Table 3. The ion source temperature was set at 300 ℃; the specific parameters are analyzed by using Xcaliibur 4.0 operating software, a standard curve is constructed by plotting the peak area ratio of the external standard/internal standard of the ceramide against the actual concentration, and the content of each corresponding type of ceramide in the sample is calculated according to the standard curve and the peak area ratio of the ceramide/internal standard in the sample. The linear range of ceramides is 0.95-8000nM, standard curve r2>0.995。
TABLE 3 mobile phase gradient setup
Example 1
After 568 days of the average follow-up visit of 255 patients with heart failure (HF patients), 45 patients died and 4 patients had heart transplants. The outcome events were all-cause death, heart transplantation. Higher levels of Cer (d18:1/16:0/d18:1/24:0) ratio in heart failure patients were significantly associated with all-cause mortality and with cardiac transplantation events, (log rank < 0.005).
As shown in fig. 1, the levels of different types of ceramides were compared between healthy persons (n-40) matched by age and sex and HF patients (n-40). Wherein, the Cer (d18:1/16:0), the Cer (d18:1/18:0), the Cer (d18:1/20:0) and the Cer (d18:1/24:1) have significant differences.
As shown in FIG. 2, it is a scatter diagram of the difference in ceramide levels between different types in patients with end-stage heart failure, wherein the group of adverse events in which Cer (d18:1/16:0) is higher (P < 0.005) and Cer (d18:1/24:0) is lower (P < 0.05) indicates that different types of ceramides act differently during the development of heart failure.
As shown in Table 1, after the mean 568 days of follow-up visit, 45 patients died, and 4 heart transplantation fate events were all-cause death and heart transplantation in 374 patients with heart failure.
TABLE 4 results of multiple COX regression analysis of specific ceramide ratios in patients with StageC/D Heart failure
In the study, the risk ratio of cer (D18:1/16:0)/cer (D18:1/24:0) after the ratio adjustment is 1.99 (95% CI, 1.01-0.047), namely, the risk of death and heart transplantation of the patients with Stage C/D heart failure is increased by 1.99 times for every increase of one unit.
The multiple Cox regression analysis in table 1, performed using the sps software v.22.0, performed a log10 transformation on the ceramide data for heart failure patients.
The above results demonstrate that: in end-stage heart failure patients, the ratio of plasma ceramide cer (d18:1/16:0)/cer (d18:1/24:0) is an important predictor of the occurrence of adverse events of heart failure, and is independent of the lipid markers currently used. This helps identify high risk patients that require more aggressive therapeutic intervention.
Claims (10)
1. The application of the product for detecting ceramide in the preparation of the kit; the kit is used for at least one of the following (a1) - (a 3):
(a1) screening or aiding in the diagnosis of heart failure patients;
(a2) assessing or aiding in assessing the risk of prognosis for a heart failure patient;
(a3) assessing or aiding in assessing the risk of an adverse event occurring in a heart failure patient.
2. A kit comprising a product for detecting ceramide, the use of which is at least one of the following (a1) - (a 3):
(a1) screening or aiding in the diagnosis of heart failure patients;
(a2) assessing or aiding in assessing the risk of prognosis for a heart failure patient;
(a3) assessing or aiding in assessing the risk of an adverse event occurring in a heart failure patient.
3. The use or kit according to claim 1 or 2, characterized in that: the ceramides include the following 7 classes: ceramides (d18:1/14:0), ceramides (d18:1/16:0), ceramides (d18:1/18:0), ceramides (d18:1/20:0), ceramides (d18:1/22:0), ceramides (d18:1/24:0) and ceramides (d18:1/24: 1).
4. The use or kit according to claim 3, characterized in that:
the ceramides include the following 4 classes: ceramides (d18:1/16:0), ceramides (d18:1/18:0), ceramides (d18:1/20:0), and ceramides (d18:1/24: 1).
Or, the ceramide includes the following class 2: ceramides (d18:1/16:0) and ceramides (d18:1/24: 0).
5. The use or kit according to any one of claims 1 to 4, wherein: the heart failure patients refer to end-stage heart failure patients divided according to the 2013 American Heart Association acute and chronic heart failure management guidelines.
6. The use or kit according to any one of claims 1 to 5, wherein: the risk of an adverse event occurring in a heart failure patient refers to the risk of all-cause death and/or the occurrence of an adverse event of a heart transplant after discharge from a hospital of the heart failure patient.
7. The use or kit according to any one of claims 1 to 6, wherein: the kit comprises a product for detecting the ceramide content in a biological sample of a healthy population or a heart failure patient;
the biological sample is a blood sample, a serum sample, a plasma sample or a blood lipid fraction or a blood lipoprotein fraction obtained therefrom;
the product for detecting the ceramide content in the biological sample of the healthy population or the heart failure patient comprises reagents required for extracting the ceramide in the biological sample and determining by LC-MS/MS.
8. The use or kit according to any one of claims 1 to 7, wherein: the kit is used for screening or assisting in diagnosing the patients with heart failure; the kit also comprises a carrier recorded with the following judgment standards: if the contents of ceramide (d18:1/16:0), ceramide (d18:1/18:0), ceramide (d18:1/20:0) and ceramide (d18:1/24:0) in the biological sample of the patient are all obviously higher than those of the healthy population, the patient is judged or suspected to be the patient with heart failure; and if the contents of the ceramide (d18:1/16:0), the ceramide (d18:1/18:0), the ceramide (d18:1/20:0) and the ceramide (d18:1/24:0) in the biological sample of the patient do not have significant difference with those of healthy people, judging that the patient is not or is suspected to be not the heart failure patient.
9. The use or kit according to any one of claims 1 to 7, wherein:
the kit is used for evaluating or assisting in evaluating the prognosis risk of a heart failure patient, or evaluating or assisting in evaluating the risk of the heart failure patient of generating an adverse event; the kit also comprises a carrier recorded with the following judgment standards:
the concentration values of ceramide (d18:1/16:0) and ceramide (d18:1/24:0) thus measured were calculated according to the following formula (d18:1/16:0)/(d18:1/24:0), and the cut-off value was obtained from the area under the ROC curve; and if the cut-off value of the ratio is greater than or equal to 0.245, judging that the patient to be tested is a high-risk patient with all-cause death and heart transplantation.
10. Use of ceramide as a marker for at least one of the following (b1) to (b 3):
b1) screening or auxiliary diagnosis of patients with heart failure.
b2) Use in assessing or aiding in assessing the risk of prognosis for a heart failure patient;
b3) use in assessing or aiding in assessing the risk of a heart failure patient for developing an adverse event.
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| CN112986454A (en) * | 2021-05-18 | 2021-06-18 | 天津云检医疗器械有限公司 | Serum marker of acute myocardial infarction, kit and application |
| CN113533754A (en) * | 2021-07-12 | 2021-10-22 | 北京市心肺血管疾病研究所 | Application of ceramide in preparation of kit for evaluating risk of adverse event of hypertensive patient |
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| CN114813986A (en) * | 2022-01-26 | 2022-07-29 | 北京市心肺血管疾病研究所 | Lipid metabolite group for predicting high-risk type dilated cardiomyopathy |
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| CN113782177A (en) * | 2020-12-02 | 2021-12-10 | 北京市心肺血管疾病研究所 | Data processing device and system for identifying and diagnosing plaque stability of coronary heart disease patient |
| CN112505228A (en) * | 2021-01-04 | 2021-03-16 | 深圳市绘云生物科技有限公司 | Metabolic marker combination for assessing cardiovascular disease risk of subject and application thereof |
| WO2022144028A1 (en) * | 2021-01-04 | 2022-07-07 | 深圳市绘云生物科技有限公司 | Metabolic marker combination for assessing risk of developing cardiovascular disease in subject, and application thereof |
| CN112505228B (en) * | 2021-01-04 | 2022-10-21 | 深圳市绘云生物科技有限公司 | Metabolic marker combination for assessing cardiovascular disease risk of subject and application thereof |
| CN112986454A (en) * | 2021-05-18 | 2021-06-18 | 天津云检医疗器械有限公司 | Serum marker of acute myocardial infarction, kit and application |
| CN113533754A (en) * | 2021-07-12 | 2021-10-22 | 北京市心肺血管疾病研究所 | Application of ceramide in preparation of kit for evaluating risk of adverse event of hypertensive patient |
| CN114137193A (en) * | 2021-11-22 | 2022-03-04 | 湖南豪思生物科技有限公司 | Kit for evaluating coronary artery disease and application thereof |
| CN114813986A (en) * | 2022-01-26 | 2022-07-29 | 北京市心肺血管疾病研究所 | Lipid metabolite group for predicting high-risk type dilated cardiomyopathy |
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