Application of metabolic marker in preparation of product for monitoring CsA renal toxicity and product

Abstract

The invention discloses application of a metabolic marker in preparation of a product for monitoring CsA renal toxicity and the product, and the metabolic marker can be used for monitoring CsA renal toxicity and is high in sensitivity, rapid, convenient and accurate and reliable in result. The metabolic marker is one or more of the following substances: phospholipid 18: 1 / 20: 0, phospholipid 20: 1 / 14: 1, lysophospholipid 18: 3, sphingolipid d18: 0 / 16: 1OH, sphingolipid d18: 0 / 24: 1OH and monoacylglycerol 20: 4 / 0: 0 / 0: 0.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine detection, and in particular to application of a metabolic marker in preparation of a product for monitoring CsA nephrotoxicity and a product capable of effectively reducing diagnostic model probability of the metabolic marker, which is used for assisting treatment of polycyclic cyclosporine A nephrotoxicity. BACKGROUND

[0002] As a potent calcineurin inhibitor, cyclosporine A (CsA) has made a revolutionary breakthrough in the field of organ transplantation since it was applied to the clinic in 1976. The drug significantly improves the short-term graft survival rate of solid organ transplantation such as kidney, liver and lung, and makes the 1-year survival rate of kidney transplantation jump from 50%-60% to about 90%, which is hailed as a "milestone in the history of organ transplantation". However, it is regrettable that the long-term survival rate of transplanted patients and organs has not been improved synchronously, and the 5-year survival rate of kidney transplantation still hovers around 60%. Existing studies have shown that 30%-50% of chronic transplanted kidney diseases are closely related to the nephrotoxicity of CsA, and this nephrotoxicity has become one of the main bottlenecks restricting the long-term efficacy of organ transplantation. At present, there is a lack of reliable indicators that can specifically diagnose CsA nephrotoxicity in the clinic, therefore, exploring early, sensitive and specific new biomarkers of CsA nephrotoxicity and constructing a CsA nephrotoxicity risk prediction model with high diagnostic efficiency have become key scientific problems to be solved in the current field of transplantation medicine.

[0003] Pharmacometabolomics realizes early prediction and dynamic monitoring of drug pharmacological action, efficacy or toxicity by systematically analyzing the dynamic changes of small molecule metabolites in biological fluids (such as blood, urine, sweat, etc.) before and after administration, and screening metabolites with significant differences. The technology has become an important tool for drug metabolism research and clinical safety evaluation. At present, the research on biomarkers of CsA nephrotoxicity has become a hot spot in this field, and with the advantages of high throughput and high sensitivity of pharmacometabolomics, it is expected to break through the limitations of traditional detection methods, efficiently identify specific biomarkers, and realize early warning of CsA nephrotoxicity, thereby providing a scientific basis for clinical precise medication and individualized treatment. SUMMARY

[0004] In order to overcome the defects of the prior art, the technical problem to be solved by the present application is to provide application of a metabolic marker in preparation of a product for monitoring CsA nephrotoxicity, which can be used for monitoring CsA nephrotoxicity and has high sensitivity, is fast and convenient, and the result is accurate and reliable.

[0005] The technical scheme of the present application is: the application of the metabolic marker in the preparation of a product for monitoring CsA nephrotoxicity, wherein the metabolic marker is one or several of the following substances: phospholipid 18:1 / 20:0, phospholipid 20:1 / 14:1, lysophospholipid 18:3, sphingolipid d18:0 / 16:1OH, sphingolipid d18:0 / 24:1OH, and monacylglycerol 20:4 / 0:0 / 0:0.

[0006] The metabolic marker of the present application can be used for monitoring CsA nephrotoxicity, has high sensitivity, is fast and convenient, and has accurate and reliable results, and can provide a basis for clinical decision-making, and at the same time, can provide a certain basis for subsequent basic research and clinical research, and has great application and research value.

[0007] The present application also provides a product for monitoring CsA nephrotoxicity, which is a compound product for reducing / increasing the level of the metabolic marker in plasma, and the product is food, a probiotic preparation, or a pharmaceutical preparation. BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 A basic flowchart of the CSA nephrotoxicity metabolomics research of the present application is shown.

[0009] Figure 2 A detection result of a sample high-resolution mass spectrometry in Example 1 of the present application is shown. Among them, Figure 2 A is a positive ion full scan total ion stream obtained by high-resolution mass spectrometry of a patient plasma sample; Figure 2 B is a negative ion full scan total ion stream obtained by high-resolution mass spectrometry of a patient plasma sample.

[0010] Figure 3 A multivariate statistical model diagram of a plasma sample in Example 1 of the present application is shown. Among them, Figure 3 A is a principal component analysis (PCA) model score diagram of plasma, Figure 3 B is an orthogonal partial least squares discriminant analysis (OPLS-DA) model score diagram of plasma. The results show that the normal renal function group and the CsA nephrotoxicity group can be well distinguished.

[0011] Figure 4 A column chart of the renal toxicity differential metabolites found from a patient plasma in Example 1 of the present application is shown. The results show that the renal toxicity markers have significant differences between the normal renal function group and the CsA nephrotoxicity group.

[0012] Figure 5The receiver operating characteristic (ROC) curve and area under the curve (AUC) value of the metabolic markers are shown in Example 2 of the present application. DETAILED DESCRIPTION

[0013] Figure 1 The basic flow chart of the CSA nephrotoxicity metabolomics study of the present application is shown.

[0014] The metabolic markers are one or more of the following: phospholipid 18:1 / 20:0, phospholipid 20:1 / 14:1, lysophospholipid 18:3, sphingolipid d18:0 / 16:1OH, sphingolipid d18:0 / 24:1OH, monacylglycerol 20:4 / 0:0 / 0:0.

[0015] The metabolic markers of the present application can be used to monitor CsA nephrotoxicity, with high sensitivity, fast and convenient, accurate and reliable results, which can provide a basis for clinical decision-making, and at the same time provide a certain basis for follow-up basic research and clinical research, with great application and research value.

[0016] Preferably, the biological sample used is plasma, serum, whole blood, whole blood dry blood spot, dry plasma spot, or urine.

[0017] Preferably, the level of metabolic markers in the biological sample is detected by one or more of the following methods: chromatography, spectroscopy, mass spectrometry, chemical analysis, immunization.

[0018] Preferably, the chromatography includes high performance liquid chromatography, thin layer chromatography, gas chromatography; the spectroscopy includes nuclear magnetic resonance spectroscopy, refractive index spectroscopy, ultraviolet spectroscopy, near infrared spectroscopy; the chemical analysis includes electrochemical analysis, radiochemical analysis.

[0019] Preferably, in the chromatography, the mobile phase: A mobile phase is acetonitrile-water (60:40, v / v) containing 10 mmol / L ammonium formate, and the D mobile phase is acetonitrile-isopropanol (10:90, v / v) containing 10 mmol / L ammonium formate; the gradient elution program for sample determination is: 0-11.0 min, 30% D-100% D; 11.0-13.0 min, 100% D; 13.0-13.2 min, 100% D-30% D; 13.2-18 min, 30% D. The flow rate is 0.25 mL / min; the chromatographic column is ACQUITY CSH C18 (1.7 μm, 2.1 x 100 mm), the column temperature is 45℃, and the injection volume is 5 μL.

[0020] Preferably, the mass spectrometry is high-resolution mass spectrometry, and the data is collected in ESI positive and negative ion Full scan-ddMS2 mode after gradient elution by a chromatographic column.

[0021] Preferably, in the mass spectrometry, the spray voltage is 3000V, the evaporation temperature is 400℃, the capillary temperature is 350℃, the S-lens RF is 50, the resolution of the primary full scan is 70000, the scan range is 114-1700m / z, the resolution of the secondary data-dependent scan is 17500, the AGC target is 1e 5 , the Maximun TT is 50ms, the positive ion mode NCE is 25, 30, and the negative ion mode NCE is 20, 24, 28.

[0022] Preferably, the pretreatment method of the biological sample before detection is as follows: 200μL of plasma is taken, 600μL of chloroform / methanol (volume ratio of 3:1) is added, 12000rpm centrifugation is performed at 4℃ for 5 minutes, 200μL of the upper solution is taken and concentrated by centrifugation, 100μL of isopropyl alcohol / acetonitrile (volume ratio of 1:1) is added to the dried sample, and the mixture is shaken and mixed for 40s; 12000rpm centrifugation is performed for 5 minutes, 80μL of the supernatant is taken and placed in a sample bottle, and then the sample is detected and analyzed by an LC-MS / MS system.

[0023] Also provided is a product prepared from the metabolic markers of the application for monitoring CsA nephrotoxicity, wherein the product is a compound preparation for reducing / increasing the level of the metabolic markers in plasma, and the product is food, probiotic preparation or pharmaceutical preparation.

[0024] Preferably, the metabolic markers phospholipid 18:1 / 20:0, phospholipid 20:1 / 14:1, lysophospholipid 18:3, sphingolipid d18:0 / 16:1OH, sphingolipid d18:0 / 24:1OH and monacylglycerol 20:4 / 0:0 / 0:0 in the plasma before and after the candidate food, probiotic preparation or pharmaceutical preparation intervention are detected, and whether the phospholipid 18:1 / 20:0, lysophospholipid 18:3, sphingolipid d18:0 / 16:1OH, sphingolipid d18:0 / 24:1OH, monacylglycerol 20:4 / 0:0 / 0:0 are increased and whether the phospholipid 20:1 / 14:1 is decreased are used as the standard for screening.

[0025] The embodiments of the application are described in detail below.

[0026] Example 1

[0027] Screening of metabolic markers in plasma of CsA nephrotoxicity patients and normal renal function group

[0028] (I) Case screening and sample collection:

[0029] 107 plasma samples from HC group and 62 plasma samples from CsA nephrotoxicity group were collected after CsA administration. A inclusion criteria: ① patients with allogeneic kidney transplantation, inflammatory myopathy, aplastic anemia; ② age 18-70 years old; ③ diagnosis of nephrotoxicity: meet the following diagnosis of acute and chronic kidney injury, and recover or alleviate after reducing, removing or replacing CsA administration. ④ diagnosis of acute kidney injury: serum creatinine increased by ≥0.3mg / dL(≥26.5μmol / L) within 48 hours, or serum creatinine increased to 1.5 times or more of the baseline value, and such increase was known or suspected to have occurred within the previous 7 days, or urine volume <0.5mL / (kg·h) for 6 hours. ⑤ diagnosis of chronic kidney injury: kidney injury or kidney function decline lasted at least 3 months. eGFR <60mL / (min·1.73m2), albuminuria >30mg / g; abnormal urinary sediment microscopy: abnormal urinary sediment such as red blood cell or white blood cell cast; imaging abnormalities: kidney injury can cause imaging abnormalities, such as polycystic kidney, hydronephrosis and small volume kidney with strong echo. B exclusion criteria: ① patients treated with CRRT due to kidney injury or failure; ② pregnant women, cancer patients or patients with a history of malignant tumor. All participants were from China-Japan Friendship Hospital. The age and gender of HC were matched with CsA nephrotoxicity patients to exclude metabolic differences caused by gender and age. Blood sampling time was in the morning on an empty stomach. The blood plasma samples of the subjects were collected and stored in a-80℃ refrigerator for lipidomic analysis.

[0030] (II) Main reagents:

[0031] LC / MS grade acetonitrile and methanol were purchased from Merck company, formic acid was purchased from CNW company. Other reagents were commercially available analytical pure. Deionized water was prepared by Milli-Q ultrapure water system of Millipore company.

[0032] (III) High-resolution mass spectrometry screening of plasma differential metabolites:

[0033] 3.1 Sample preparation:

[0034] Sample pretreatment: 200μL of plasma (normal kidney function group and CsA nephrotoxicity group) was added with 600μL of chloroform / methanol(3:1). Centrifuge at 12000rpm for 5min at 4℃, take 200μL of upper solution, and centrifuge to concentrate. Add 100μL of isopropyl alcohol / acetonitrile(1:1) to the dried sample, shake and mix for 40s. Centrifuge at 12000rpm for 5min, take 80μL of supernatant, and place it in a sample bottle for LC-MS / MS system detection and analysis

[0035] 3.2 Chromatography / mass spectrometry conditions:

[0036] QE-Orbitrap. The mobile phase A was acetonitrile-water (60:40, v / v) containing 10 mmol / L ammonium formate, and the mobile phase D was acetonitrile-isopropanol (10:90, v / v) containing 10 mmol / L ammonium formate. The gradient elution program for sample determination was as follows: 0-11.0 min, 30% D-100% D; 11.0-13.0 min, 100% D; 13.0-13.2 min, 100% D-30% D; 13.2-18 min, 30% D. The flow rate was 0.25 mL / min. The column was ACQUITY CSH C18 (1.7 μm, 2.1 x 100 mm), and the column temperature was 45°C. The injection volume was 5 μL. The data were collected in positive and negative ion modes under the conditions of an electrospray ion source (ESI) with a spray voltage of 3000 V, an evaporation temperature of 400°C, a capillary temperature of 350°C, and an S-lens RF of 50. The resolution of the full scan was 70000, and the scan range was 114-1700 m / z. The resolution of the data-dependent scan was 17500, the AGC target was 1e5, the maximum TT was 50 ms, the NCE in the positive ion mode was 25, 30, and the NCE in the negative ion mode was 20, 24, and 28.

[0037] The results of high-resolution mass spectrometry detection are shown in Figure 2

[0038] (Four) Data processing and statistical analysis:

[0039] The identification of endogenous metabolites was performed by obtaining the accurate mass number of each endogenous metabolite to the fifth decimal place using high-resolution mass spectrometry mzCloud, and identifying each endogenous metabolite by its molecular formula. Then, the raw format original data derived from Q Exactive LC-MS / MS were read using Lipid Search software, and the parameters such as retention time and mass-to-charge ratio were screened. Then, the peaks were aligned according to the retention time deviation of 0.2 min and the mass deviation of 5 ppm in different samples to make the identification more accurate. According to the parent ion and multi-stage mass spectrometry data in the sample, the structure of the lipid molecules and the adduction mode of the positive and negative ions were identified, and the quantitative results were normalized. The lipid data results were qualitatively and quantitatively analyzed. Subsequently, the MetaboAnalyst5.0 website was used to analyze the metabolite matrix, and the PCA and OPLS-DA model graphs were drawn to find the metabolic pattern differences and obvious classification trends between the normal renal function group and the CsA nephrotoxicity patients. Finally, the differential metabolic markers were selected according to the standards of VIP>1, P value<0.05, and FC≥1.2 or FC≤0.8.

[0040] (Five) Result analysis:

[0041] ​The total ion chromatogram of the metabolome of human plasma is shown in Figure 1. Figure 2 PCA and OPLS-DA were calculated by MetaboAnalyst 5.0 website. Figure 3 A) The results show the differences between the plasma of the normal group and the plasma of the CsA nephrotoxicity group. OPLS-DA( Figure 3 B) The results show that the plasma metabolites of the normal group and the CsA nephrotoxicity group can be completely separated. Endogenous substances with VIP>1, P value<0.05 and FC≥1.2 or FC≤0.8 were selected as the 6 main differential metabolites of the plasma for further analysis( Figure 4 ). Table 1 shows the change rate of the differential metabolites in human plasma. Compared with the normal group, the proportion of 5 endogenous substances in the CsA nephrotoxicity group is mainly increased, and the proportion of 1 endogenous substance is decreased, which further indicates that the metabolism and secretion of plasma lipids are affected after the occurrence of CsA nephrotoxicity, and the content of plasma lipids is obviously changed.

[0042] Table 1 Change rate of differential metabolites in human plasma obtained by OPLS-DA model

[0043] No Metabolite VIP P value Peak area ratio (%) Trend of change 1 Phosphatide 18:1 / 20:0 2.10 3.04E-23 161.28％ Up-regulated 2 Phosphatide 20:1 / 14:1 1.57 4.55E-13 78.36％ Down-regulated 3 Lysophosphatide 18:3 1.81 3.23E-09 124.77％ Up-regulated 4 Sphingolipid d18:0 / 16:1 OH 2.31 3.41E-30 181.61％ Up-regulated 5 Sphingolipid d18:0 / 24:1 OH 1.43 2.72E-11 122.05％ Up-regulated 6 Monoacylglycerol 20:4 / 0:0 / 0:0 1.73 1.00E-22 126.40％ Up-regulated

[0044] After consulting published literature, 6 plasma metabolite markers, phospholipid 18:1 / 20:0, phospholipid 20:1 / 14:1, lysophospholipid 18:3, sphingolipid d18:0 / 16:1OH, sphingolipid d18:0 / 24:1OH, and monacylglycerol 20:4 / 0:0 / 0:0, were found for the first time in the early diagnosis of CsA nephrotoxicity, which has great significance for the diagnosis and treatment of CsA nephrotoxicity.

[0045] Example Two

[0046] Diagnostic efficiency analysis of CsA nephrotoxicity markers

[0047] (I) Data statistics

[0048] For the patients with normal renal function and CsA nephrotoxicity, further based on the Receiver operation characteristic (ROC) curve, it was determined whether the six common metabolite markers, phospholipid 18:1 / 20:0, phospholipid 20:1 / 14:1, lysophospholipid 18:3, sphingolipid d18:0 / 16:1OH, sphingolipid d18:0 / 24:1OH, and monacylglycerol 20:4 / 0:0 / 0:0, could be used as early diagnostic biomarkers of CsA nephrotoxicity. The SPSS software was used to set the group as a state variable and the target plasma metabolite determination intensity as a test variable. After the ROC curve was calculated, the SPSS generated an output report containing the ROC curve graph and the Area under curve (AUC) value.

[0049] (B) Result analysis

[0050] The ROC curve analysis results of the patients with normal renal function and CsA nephrotoxicity showed that the AUC values of the six metabolites were all greater than 0.7, as shown in Table 2. Figure 5 Further, Table 2 shows that the Youden indices of the six metabolites were between 0.5 and 1, indicating that they were good in sample classification of the patients with CsA nephrotoxicity and the patients with normal renal function, and could effectively distinguish the patients with CsA nephrotoxicity from the patients with normal renal function.

[0051] Table 2 ROC parameters of the differential metabolites in human plasma

[0052]

[0053] The above is only the preferred embodiment of the present application, and does not limit the present application in any form. Any simple modification, equivalent change and modification made according to the technical essence of the present application to the above embodiment are still within the protection scope of the technical solution of the present application.

Claims

1. The application of metabolic markers in the preparation of products for monitoring CsA nephrotoxicity, characterized in that: The metabolic markers are one or more of the following substances: phospholipids 18:1 / 20:0, phospholipids 20:1 / 14:1, lysophospholipids 18:3, sphingolipids d18:0 / 16:1OH, sphingolipids d18:0 / 24:1OH, and monoacylglycerols 20:4 / 0:0 / 0:

0.

2. The application of the metabolic biomarker according to claim 1 in the preparation of products for monitoring CsA nephrotoxicity, characterized in that: The biological samples used are plasma, serum, whole blood, dried whole blood spots, dried plasma spots, or urine.

3. The application of the metabolic biomarker according to claim 2 in the preparation of products for monitoring CsA nephrotoxicity, characterized in that: The biological sample was tested for the levels of metabolic markers using one or more of the following methods: chromatography, spectroscopy, mass spectrometry, chemical analysis, and immunoassay.

4. The application of the metabolic biomarker according to claim 3 in the preparation of products for monitoring CsA nephrotoxicity, characterized in that: The chromatographic methods include high performance liquid chromatography, thin-layer chromatography, and gas chromatography; the spectroscopic methods include nuclear magnetic resonance spectroscopy, refractive index spectroscopy, ultraviolet spectroscopy, and near-infrared spectroscopy; and the chemical analysis methods include electrochemical analysis and radiochemical analysis.

5. The application of the metabolic biomarker according to claim 4 in the preparation of products for monitoring CsA nephrotoxicity, characterized in that: The mobile phases used in the chromatographic method are as follows: Mobile phase A is acetonitrile-water containing 10 mmol / L ammonium formate, with a volume ratio of 60:40; Mobile phase D is acetonitrile-isopropanol containing 10 mmol / L ammonium formate, with a volume ratio of 10:

90. The gradient elution program for sample determination is as follows: 0–11.0 min, 30%D–100%D; 11.0–13.0 min, 100%D; 13.0–13.2 min, 100%D–30%D; 13.2–18 min, 30%D; Flow rate: 0.25 mL / min; Column: ACQUITY CSH C18, 1.7 μm, 2.1 × 100 mm, column temperature: 45 °C, injection volume: 5 μL.

6. The application of the metabolic biomarker according to claim 4 in the preparation of products for monitoring CsA nephrotoxicity, characterized in that: The mass spectrometry method described is high-resolution mass spectrometry, which first uses a chromatographic column for gradient elution, and then acquires data in the ESI positive and negative ion Full scan-ddMS2 mode using an electrospray ionization source.

7. The application of the metabolic biomarker according to claim 6 in the preparation of products for monitoring CsA nephrotoxicity, characterized in that: In the mass spectrometry method described, the spray voltage is 3000V; the evaporation temperature is 400℃; the capillary temperature is 350℃; the S-lens RF is 50; the resolution of the first-stage full scan is 70000, and the scan range is 114-1700 m / z; the resolution of the second-stage data-dependent scan is: 17500, AGC target: 1e 5 Maximun TT: 50ms, positive ion mode NCE: 25, 30; negative ion mode NCE: 20, 24, 28.

8. The application of the metabolic biomarker according to claim 4 in the preparation of products for monitoring CsA nephrotoxicity, characterized in that: The pretreatment method for the biological samples before detection is as follows: [The text abruptly ends here, likely due to an incomplete sentence or a formatting error.] 200 μL of plasma was mixed with 600 μL of chloroform / methanol (volume ratio 3:1); the solution was added at 12000 rpm. Centrifuge at 4℃ for 5 minutes, take 200 μL of the supernatant and concentrate by centrifugation; add 100 μL of isopropanol / acetonitrile (volume ratio 1:1) to the dried sample, shake to mix for 40 s; centrifuge at 12000 rpm for 5 minutes, take 80 μL of the supernatant, place it in a sample vial, and analyze it using an LC-MS / MS system.

9. A product for preparing a metabolic marker in the monitoring of CsA nephrotoxicity, wherein the metabolic marker is the metabolic marker according to any one of claims 1-8, characterized in that: The product is a compound product that lowers / raises the levels of the metabolic markers in plasma, and the product is a food, probiotic preparation, or pharmaceutical preparation.

10. The article of the metabolic marker according to claim 9 in the preparation of products for monitoring CsA nephrotoxicity, characterized in that: The study detected metabolic markers in plasma before and after intervention with candidate foods, probiotic preparations, or drug preparations, including phospholipids 18:1 / 20:0, phospholipids 20:1 / 14:1, lysophospholipids 18:3, sphingolipids d18:0 / 16:1OH, sphingolipids d18:0 / 24:1OH, and monoacylglycerols 20:4 / 0:0 / 0:

0. The screening criteria were based on whether phospholipids 18:1 / 20:0, lysophospholipids 18:3, sphingolipids d18:0 / 16:1OH, sphingolipids d18:0 / 24:1OH, and monoacylglycerols 20:4 / 0:0 / 0:0 increased, and whether phospholipids 20:1 / 14:1 decreased.

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