Application of metabolic marker in preparation of product for monitoring PB neurotoxicity and product

Abstract

The invention discloses application of a metabolic marker in preparation of a product for monitoring PB neurotoxicity and the product, which can be used for early diagnosis of PB neurotoxicity and have the advantages of high sensitivity, rapidness, convenience and accurate and reliable result. The metabolic marker comprises LysoPE 20: 4 / 0: 0, creatine, SM d18: 1 / 24: 0, SM d18: 0 / 24: 1, LysoPC 18: 0, palmitoyl sphingomyelin, phosphocholine, PC 14: 0 / 22: 1, creatinine, PC 18: 3 / 16: 0, SM d18: 1 / 16: 0, sphingosine, choline, LysoPC 18: 1, L-carnitine, palmitic acid, PC 18: 0 / 20: 4, 3-hydroxy-2-octyl glutaric acid, L-acetyl carnitine, LysoPC 20: 4, LysoPC 16: 0, and PC 20: 1 / 14: 1.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical testing, and in particular to an application of a metabolic marker in the preparation of a product for monitoring PB neurotoxicity, and a product capable of effectively reducing the probability of a metabolic marker diagnostic model, which is used for auxiliary treatment of polymyxin B neurotoxicity. Background Art

[0002] Polymyxin B (PB) entered clinical use in the 1950s for the treatment of Gram-negative bacterial infections. Due to the frequent occurrence of nephrotoxicity and neurotoxicity, the clinical application of PB gradually decreased in the 1970s. In recent years, the incidence of multidrug-resistant Gram-negative bacteria (MDR-GNB) infections has been on the rise, and PB has become the first-line drug for the treatment of MDR-GNB infections.

[0003] Clinically, the total incidence of PB neurotoxicity is about 14-28%, mainly manifested as confusion, drowsiness, dizziness, facial flushing, peripheral paresthesia, and even respiratory failure. For critically ill patients in a coma or deep sedation, the PB neurotoxicity effect is more hidden and difficult to be identified early. If intervention measures are not taken in time, it will seriously threaten the patient's life. Therefore, it is particularly important and urgent to find new markers of early, sensitive, specific, and non-invasive PB neurotoxicity and strengthen the monitoring of patients during PB medication.

[0004] Drug metabolomics analyzes the changes in small molecule metabolites (specifically compounds with a molecular weight of less than 1000 Da) in biological fluids (including key samples such as blood, urine, and sweat) before and after drug administration, and locks in metabolites with significant differences. It not only builds a solid bridge for early prediction and dynamic monitoring of pharmacological effects, but also becomes an indispensable tool in the field of drug metabolism, efficacy evaluation, and toxicity research. Relying on drug metabolomics technology, it is expected to efficiently and accurately discover PB neurotoxicity markers, achieve early diagnosis of adverse drug reactions, and provide more accurate and safe guidance for clinical drug use. Summary of the invention

[0005] In order to overcome the defects of the prior art, the technical problem to be solved by the present invention is to provide an application of a metabolic marker in the preparation of a product for monitoring PB neurotoxicity, which can be used for the early diagnosis of PB neurotoxicity with high sensitivity, rapidity and convenience, and accurate and reliable results.

[0006] The technical scheme of the present invention is: the use of such metabolic markers in the preparation of a product for monitoring PB neurotoxicity, wherein the metabolic markers include LysoPE 20:4 / 0:0, creatine, SM d18:1 / 24:0, SM d18:0 / 24:1, LysoPC 18:0, palmitoyl sphingomyelin, phosphorylcholine, PC 14:0 / 22:1, creatinine, PC 18:3 / 16:0, SM d18:1 / 16:0, sphingosine, choline, LysoPC 18:1, L-carnitine, palmitic acid, PC 18:0 / 20:4, 3-hydroxy-2-octylglutaric acid, L-acetylcarnitine, LysoPC 20:4, LysoPC 16:0, and PC 20:1 / 14:1.

[0007] The metabolic markers of the present invention can be effectively used for the early diagnosis of PB neurotoxicity, with high sensitivity, rapidity and convenience, accurate and reliable results, and can provide a basis for clinical decision-making. At the same time, it can provide a certain foundation for subsequent basic research and clinical research, and has great application and research value.

[0008] Also provided is a preparation of a metabolic marker in the preparation of a product for monitoring PB neurotoxicity, wherein the preparation is a composite preparation that reduces the probability of a metabolic marker diagnostic model in plasma, and the product is a food, a probiotic preparation or a pharmaceutical preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Shown is the basic flow chart of the PB neurotoxicity metabolomics study of the present invention.

[0010] Figure 2 Shown is the detection result of high-resolution mass spectrometry of the sample in Example 1 of the present invention.

[0011] in, Figure 2 A is the total ion current of negative ion full scan obtained by high-resolution mass spectrometry of rat plasma samples; Figure 2 B is the total ion current of positive ion full scan obtained by high-resolution mass spectrometry of rat plasma samples; Figure 2 C is the total ion current of the full scan of negative ions obtained by high-resolution mass spectrometry of rat brain samples; Figure 2 D is the total ion current of the positive ion full scan obtained by high-resolution mass spectrometry of rat brain samples.

[0012] Figure 3 Shown is a multivariate statistical model diagram of rat plasma and brain samples in Example 1 of the present invention.

[0013] in, Figure 3 A is the score diagram of the plasma principal component analysis (PCA) model. Figure 3B is a three-dimensional stereogram of the plasma partial least squares discriminant analysis (PLS-DA) model score. The results showed that healthy control (HC) and PB neurotoxic rats could be well distinguished; Figure 3 C is the brain PCA model score graph, Figure 3 D is a three-dimensional stereogram of the brain PLS-DA model score. The results showed that HC (healthy control) and PB neurotoxic rats could be well distinguished.

[0014] Figure 4 Shown is an intersection diagram of differential neurotoxic metabolites found in rat plasma and rat brain in Example 1 of the present invention.

[0015] Figure 5 Shown is the receiver operating characteristic curve (ROC) analysis result of the common metabolite markers in Example 2 of the present invention, including the ROC curve graph and the area under the curve (AUC) value.

[0016] Figure 6 It shows the ROC curve diagram and AUC analysis results of the shared metabolic marker fitting diagnostic model obtained by Logistic regression analysis in Example 3 of the present invention. DETAILED DESCRIPTION

[0017] Figure 1 Shown is the basic flow chart of the PB neurotoxicity metabolomics study of the present invention.

[0018] The application of this metabolic marker in the preparation of a product for monitoring PB neurotoxicity, the metabolic marker comprising LysoPE 20:4 / 0:0, creatine, SM d18:1 / 24:0, SM d18:0 / 24:1, LysoPC 18:0, palmitoyl sphingomyelin, phosphorylcholine, PC 14:0 / 22:1, creatinine, PC 18:3 / 16:0, SM d18:1 / 16:0, sphingosine, choline, LysoPC 18:1, L-carnitine, palmitic acid, PC 18:0 / 20:4, 3-hydroxy-2-octylglutaric acid, L-acetylcarnitine, LysoPC 20:4, LysoPC 16:0, PC 20:1 / 14:1.

[0019] The metabolic markers of the present invention can be effectively used for the early diagnosis of PB neurotoxicity, with high sensitivity, rapidity and convenience, accurate and reliable results, and can provide a basis for clinical decision-making. At the same time, it can provide a certain foundation for subsequent basic research and clinical research, and has great application and research value.

[0020] Preferably, the analysis method of the metabolic markers obtains a fitted regression curve through Logistic regression analysis:

[0021] Y=Logit(p)=20.15a-14.21b+76.60c-51.09d-0.99e-4.34

[0022] Where Y is the diagnosis probability, Logit(p) is the logistic regression function, and ae are LysoPE

[0023] 20:4 / 0:0, creatine, SM d18:1 / 24:0, SM d18:0 / 24:1, LysoPC 18:0.

[0024] Preferably, the biological sample is plasma, and the level of metabolite markers in the biological sample is detected by one or more of the following methods: chromatography, spectroscopy, mass spectrometry, chemical analysis, and immunology.

[0025] Preferably, the chromatography includes high performance liquid chromatography, thin layer chromatography, and gas chromatography; the spectroscopy includes nuclear magnetic resonance spectroscopy, refractive index spectroscopy, ultraviolet spectroscopy, and near infrared spectroscopy; and the chemical analysis includes electrochemical analysis and radiochemical analysis.

[0026] Preferably, the mobile phase in the chromatography method is: mobile phase A is an aqueous solution containing 0.1% formic acid and 2.5 mmol / L ammonium formate, and mobile phase D is acetonitrile; the gradient elution program for sample determination is: 0-1.0 min, 95% A; 1.0-5.0 min, 95%-40% A; 5.0-8.0 min, 40%-0% A; 8.0-11.0 min, 0% A; 11.0-14.0 min, 0%-40% A; 14.0-15.0 min, 40%-95% A; 15.0-18.0 min, 95% A, analysis time is 0 to 18 min, 5 μL is injected each time, the flow rate is 0.25 mL / min, the chromatographic column is: ACQUITY BEH C18 1.7 μm, 2.1×50 mm, and the chromatographic column temperature is 30°C.

[0027] Preferably, the mass spectrometry is a high-resolution mass spectrometry, which first uses a chromatographic column for gradient elution, and then collects data in an electrospray ion source ESI positive and negative ion Full scan-ddMS2 mode.

[0028] Preferably, in the mass spectrometry, spray voltage: 3000 V; evaporation temperature: 350° C.; capillary temperature: 350° C.; S-lens RF: 50; resolution of primary full scan: 70000, scanning range: 70-1050 m / z; secondary data-dependent scan: resolution: 17500, AGC target: 1e5, MaximunTT: 50 ms, NCE: 20, 40, 60.

[0029] Preferably, the biological sample is pretreated before detection as follows: 20 μL of plasma is added with 180 μL of a precipitant containing an internal standard, the internal standard is dissolved in equal proportions of methanol and acetonitrile, vortexed for 30 seconds, centrifuged at 12000 rpm for 10 minutes, and the supernatant is aspirated to obtain a test solution for quantitative analysis.

[0030] Also provided is a preparation of a metabolic marker in the preparation of a product for monitoring PB neurotoxicity, wherein the preparation is a composite preparation that reduces the probability of a metabolic marker diagnostic model in plasma, and the product is a food, a probiotic preparation or a pharmaceutical preparation.

[0031] Preferably, the probability of the metabolic marker diagnostic model in the plasma of the candidate food, probiotic preparation or drug preparation before and after intervention is detected, and screening is performed based on whether the probability is reduced.

[0032] The embodiments of the present invention are described in detail below.

[0033] Example 1

[0034] Screening of common metabolic markers in plasma and brain of PB neurotoxicity rats and healthy controls (I) Animal studies and sample sources:

[0035] Male Sprague–Dawley rats (n = 49, 6–8 weeks old) were purchased from Sprague Biotech (Beijing) Co., Ltd. Animal housing conditions: temperature 24 ± 2 °C, relative humidity 50 ± 5%, 12-hour day and night cycle, water and food were available ad libitum. After the animals were adapted to the laboratory conditions for one week, the rats were randomly divided into four experimental groups, 24 in each group. Polymyxin B sulfate for injection (Shanghai First Biochemical Pharmaceutical Co., Ltd.) was prepared into a solution using physiological saline. According to the commonly used clinical dose, rats were subcutaneously injected with PB (0, 3, 6, 12 mg / kg / time) twice a day. The drug was administered for 10 consecutive days and the rats were killed on the 11th day. All blood was collected using anticoagulant (EDTA anticoagulant tube), and plasma was collected by centrifugation at 5000 × g for 5 min at 4 °C and frozen at -80 °C; the brain was quickly removed in an ice bath and frozen at -80 °C.

[0036] (II) Main reagents:

[0037] LC / MS grade acetonitrile was purchased from Merck, HPLC grade methanol was purchased from Merck, and formic acid was purchased from CNW. Other reagents were commercially available and of analytical grade. Deionized water was prepared using the Milli-Q ultrapure water system from Millipore.

[0038] (III) High-resolution mass spectrometry screening of differential metabolites in plasma and brain:

[0039] 3.1 Sample preparation:

[0040] Sample pretreatment: 20 μL of plasma (healthy control group and PB administration group) was drawn and added with 180 μL of precipitant containing internal standard (methanol: acetonitrile = 1:1), vortexed for 60 s, centrifuged at 12000 rpm for 10 min, and 100 μL was drawn for metabolomics analysis. Brain tissue (0 mg / kg group and PB administration group) was weighed and homogenized with ultrapure water (containing 50% methanol) at a mass volume ratio of 1:10 (w / v) to obtain brain tissue fragmentation liquid, centrifuged at 5000 × g at 4 ° C for 5 min, 20 μL of supernatant was drawn and added with 180 μL of precipitant containing internal standard (methanol: acetonitrile = 1:1), vortexed for 60 s, centrifuged at 13000 rpm for 10 min, and 100 μL was drawn for metabolomics analysis.

[0041] 3.2 Chromatographic / mass spectrometry conditions:

[0042] High-resolution mass spectrometer QE-Orbitrap was used for detection. Chromatographic mobile phase: Mobile phase A was an aqueous solution containing 0.1% formic acid and 2.5 mmol / L ammonium formate, and mobile phase D was acetonitrile. Gradient elution program for sample determination: 0-1.0 min, 95% A; 1.0-5.0 min, 95%-40% A; 5.0-8.0 min, 40%-0% A; 8.0-11.0 min, 0% A; 11.0-14.0 min, 0%-40% A; 14.0-15.0 min, 40%-95% A; 15.0-18.0 min, 95% A, analysis time 0-18 min, 5 μL each injection, flow rate 0.25 mL / min, chromatographic column: ACQUITY BEH C18 1.7 μm, 2.1×50 mm, column temperature 30°C, and the temperature of the autosampler was maintained at 4°C. Data were collected in positive and negative ion modes of electrospray ion source (ESI), spray voltage: 3000V; evaporation temperature: 350℃; capillary temperature: 350℃; S-lens RF: 50; resolution of full scan: 70000, scanning range: 70-1050m / z. Secondary data dependent scan (Full MS / dd-MS2): resolution: 17500; AGC target: 1e5; MaximunTT: 50ms; NCE: 20, 40, 60.

[0043] High-resolution mass spectrometry results Figure 2 shown.

[0044] (IV) Data processing and statistical analysis:

[0045] Endogenous metabolites were identified by using high-resolution mass spectrometry mzCloud to obtain the 5-decimal accurate mass of each endogenous metabolite, and then identified by the molecular formula of each endogenous metabolite, and then Compound Discover software was used to automatically search the self-built library and the online public database to annotate the metabolite name. Subsequently, the metabolite matrix was analyzed using the MetaboAnalyst5.0 website, and PCA and PLS-DA model diagrams were drawn to find the differences in metabolic patterns and obvious classification trends between healthy controls and PB neurotoxic rats, and finally, the metabolic markers with differences were selected according to the standard of VIP>1.

[0046] (V) Results:

[0047] 5.1 Establishment of PB neurotoxicity rat model

[0048] About 10 minutes after subcutaneous injection of PB sulfate, rats developed redness and swelling all over the body, with the head, ears, limbs, and tail being the most obvious. After administration, they developed ataxia, lethargy, and drinking a lot of water. The higher the dose, the more obvious the symptoms. One rat in the PB high-dose (12 mg / kg) group died after the first administration, possibly due to respiratory failure caused by PB. The above symptoms are consistent with PB neurotoxicity.

[0049] 5.2 Screening of common markers in rat plasma and brain

[0050] Metabolomics total ion chromatograms of rat plasma and brain are shown in Figure 2 MetaboAnalyst5.0 website was used to calculate PCA and PLS-DA. PCA( Figure 3 A) Results show the difference between the plasma of the 0 mg / kg group and the plasma of the PB neurotoxicity group, PLS-DA ( Figure 3 B) The results showed that the plasma metabolites of the 0 mg / kg group and the PB neurotoxicity group could be completely separated. PCA ( Figure 3 C) Results show the difference between the brain of the 0 mg / kg group and the brain of the PB neurotoxicity group, PLS-DA ( Figure 3 D) The results showed that the brain metabolites of the 0 mg / kg group and the PB neurotoxicity group could be completely separated. At the same time, VIP>1 endogenous substances were selected as the main differential metabolites, and 56 differential metabolites were found in rat plasma and 86 differential metabolites in the brain. Plasma and brain common markers were screened, and a total of 23 common markers were obtained (see Figure 4 ).

[0051] Table 1 shows the change rate of common differential metabolites in plasma obtained by using the PLS-DA model. The results in Table 1 show that in rat plasma, compared with the 0 mg / kg group, the proportions of 11 endogenous substances in the PB neurotoxicity group mainly increased, and the other 12 mainly showed a downward trend. Table 2 shows the change rate of differential metabolites in the brain obtained by using the PLS-DA model. The results in Table 2 show that in the rat brain, compared with the 0 mg / kg group, the proportions of 4 endogenous substances in the PB neurotoxicity group mainly increased, and the other 19 mainly showed a downward trend. The results show that after the occurrence of PB neurotoxicity, the metabolic secretion of endogenous substances in plasma and brain is affected, causing a significant change in its content. The main components that have changed are lipids, organic acids, and carnitine substances.

[0052] Table 1

[0053]

[0054]

[0055] Table 2

[0056]

[0057]

[0058] Example 2

[0059] Confirmation of shared metabolic markers of PB neurotoxicity in plasma and brain

[0060] 1. Data Statistics

[0061] For the healthy control group and the 12 mg / kg (high dose) PB administration group, the receiver operating characteristic (ROC) curve was further used to determine whether the 23 common metabolite markers could be used as early diagnostic biomarkers for PB neurotoxicity. SPSS software was used to set the group as a state variable, and the target plasma metabolite measurement intensity as a test variable. After calculating the ROC curve, SPSS generated an output report containing the ROC curve graph and the area under the curve (AUC) value. If the AUC is greater than 0.6, it can be considered that the indicator has a certain diagnostic ability.

[0062] (II) Results Analysis

[0063] The ROC curve analysis results of the healthy control group and the high-dose PB administration group showed that the AUC values ​​of the 22 metabolites were all greater than 0.6, as shown in Table 3, Figure 5 , indicating that these 22 metabolites classified the data of healthy control and high-dose PB administration groups well, and could accurately distinguish healthy and PB neurotoxic rats, and could serve as potential metabolic markers.

[0064] Table 3 ROC parameters of common differential metabolites

[0065]

[0066]

[0067] Example 3

[0068] Establishment of PB neurotoxicity diagnostic model

[0069] 1. Data Statistics

[0070] Logistic regression analysis in SPSS was used to construct the diagnostic model, and the Hosmer-Lemeshow test was used to evaluate the goodness of model fit. When P < 0.05, it indicated that the model fit was poor, otherwise the model fit was good.

[0071] (II) Results Analysis

[0072] The common metabolic markers confirmed in Example 2 were subjected to Logistic regression analysis to obtain a fitted regression curve, i.e., the diagnostic model: Y = Logit (p) = 20.15a-14.21b + 76.60c-51.09d-

[0073] 0.99e-4.34 (Y is the diagnosis probability, Logit(p) is the logistic regression function, ae is LysoPE 20:4 / 0:0, creatine, SM d18:1 / 24:0, SM d18:0 / 24:1, LysoPC 18:0. After fitting, the area under the ROC curve AUC in the comparison between the normal neurological function group and the PB neurotoxicity group was 1.00 ( Figure 6 ), with a 95% confidence interval of 0.86-1.00, which significantly improved the diagnostic efficacy of a single marker.

[0074] The Hosmer-Lemeshow test showed P = 0.60, indicating that the model fit was good.

[0075] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. Application of metabolic markers in the preparation of products for monitoring PB neurotoxicity, characterized in that: The metabolic markers include LysoPE 20:4 / 0:0, creatine, SMd18:1 / 24:0, SM d18:0 / 24:1, LysoPC 18:0, palmitoyl sphingomyelin, phosphocholine, PC 14:0 / 22:1, creatinine, PC 18:3 / 16:0, SMd18:1 / 16:0, sphingosine, choline, LysoPC 18:1, L-carnitine, palmitic acid, PC 18:0 / 20:4, 3-hydroxy-2-octylglutaric acid, L-acetylcarnitine, LysoPC 20:4, LysoPC 16:0, PC 20:1 / 14:

1.

2. The use of the metabolic marker according to claim 1 in the preparation of a product for monitoring PB neurotoxicity, characterized in that: The analysis method of metabolic markers was performed by Logistic regression analysis to obtain the fitted regression curve: Y=Logit(p)=20.15a-14.21b+76.60c-51.09d-0.99e-4.34 Where Y is the diagnosis probability, Logit(p) is the logistic regression function, and ae are LysoPE 20:4 / 0:0, creatine, SMd18:1 / 24:0, SMd18:0 / 24:1, and LysoPC 18:0, respectively.

3. The use of the metabolic marker according to claim 2 in the preparation of a product for monitoring PB neurotoxicity, characterized in that: The biological sample is plasma, and the level of the metabolic marker in the biological sample is detected by one or more of the following methods: chromatography, spectroscopy, mass spectrometry, chemical analysis, and immunoassay.

4. The use of the metabolic marker according to claim 3 in the preparation of a product for monitoring PB neurotoxicity, characterized in that: The chromatography includes high performance liquid chromatography, thin layer chromatography, and gas chromatography; the spectroscopy includes nuclear magnetic resonance spectroscopy, refractive index spectroscopy, ultraviolet spectroscopy, and near infrared spectroscopy; the chemical analysis includes electrochemical analysis and radiochemical analysis.

5. The use of the metabolic marker according to claim 4 in the preparation of a product for monitoring PB neurotoxicity, characterized in that: The mobile phases in the chromatography method are: mobile phase A is an aqueous solution containing 0.1% formic acid and 2.5 mmol / L ammonium formate, and mobile phase D is acetonitrile; the gradient elution program for sample determination is: 0-1.0 min, 95% A; 1.0-5.0min, 95%-40%A; 5.0-8.0min, 40％-0％A； 8.0-11.0min, 0%A; 11.0-14.0min, 0%-40%A; 14.0-15.0min, 40%-95%A; 15.0-18.0min, 95%A, analysis time 0-18min, 5μL injection each time, flow rate 0.25mL / min, chromatographic column: ACQUITY BEH C18 1.7μm, 2.1×50mm, column temperature 30℃.

6. Use of the metabolic marker according to claim 3 in the preparation of a product for monitoring PB neurotoxicity, characterized in that: The mass spectrometry is a high-resolution mass spectrometry method, which first uses a chromatographic column for gradient elution, and then collects data in an electrospray ion source ESI positive and negative ion Full scan-ddMS2 mode.

7. Use of the metabolic marker according to claim 6 in the preparation of a product for monitoring PB neurotoxicity, characterized in that: In the mass spectrometry, the spray voltage is 3000 V; the evaporation temperature is 350° C.; the capillary temperature is 350° C.; the S-lens RF is 50; and the resolution of the first-stage full scan is: 70000, scan range: 70-1050m / z; secondary data-dependent scan: resolution: 17500, AGC target: 1e5, Maximun TT: 50ms, NCE: 20, 40, 60.

8. The use of the metabolic marker according to claim 2 in the preparation of a product for monitoring PB neurotoxicity, characterized in that: The biological sample was pretreated before detection as follows: 20 μL of plasma was added with 180 μL of precipitant containing internal standard, the internal standard was dissolved by mixing methanol and acetonitrile in equal proportions, vortexed for 30 seconds, centrifuged at 12000 rpm for 10 minutes, and the supernatant was aspirated to obtain the test solution for quantitative analysis.

9. The preparation of a metabolic marker for monitoring PB neurotoxicity according to any one of claims 1 to 8, characterized in that: The product is a composite product that reduces the probability of a metabolic marker diagnostic model in plasma, and the product is a food, a probiotic preparation or a pharmaceutical preparation.

10. The preparation of the metabolic marker for the preparation of a product for monitoring PB neurotoxicity according to claim 9, characterized in that: The probability of the diagnostic model of metabolic markers in plasma before and after the intervention of candidate foods, probiotic preparations or drug preparations is tested, and screening is carried out based on whether the probability is reduced.

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