Application of Inosine as biomarker in preparation of product for evaluating progression and prognosis of paraquat poisoning

Abstract

The invention relates to a biomarker for evaluating progression and prognosis of paraquat poisoning and application of the biomarker. The biomarker is a urine metabolite Inosine. Urine non-target metabonomics analysis shows that the level of the Inosine is low in urine of a patient suffering from early paraquat poisoning, is gradually reduced along with aggravation of the illness state and is remarkably increased in follow-up visit of five years; the prediction efficiency is judged according to the area (AUC) below the working characteristic (ROC) curve of a subject, the Anosine has good efficiency (AUC = 0.806) in judging the progression of paraquat poisoning, has good efficiency (AUC = 0.816) in judging the prognosis aspect in 1-4 days at the early stage of poisoning, and has excellent efficiency (AUC = 0.961) in judging the prognosis aspect in 7-14 days at the progression stage of poisoning. Moreover, the efficiency of the Inosine in predicting the progress and prognosis of paraquat poisoning is better than that of blood routine indexes (such as the number of leukocytes, the percentage of neutrophils and the percentage of lymphocytes) reported in the literature. In conclusion, as the biomarker for prognosis and prognosis of paraquat poisoning, the Inosine shows a good clinical application prospect.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of pesticide poisoning detection, and particularly relates to application of Inosine as a biomarker in preparation of a product for paraquat poisoning condition progression and prognosis evaluation. BACKGROUND

[0002] A large number of epidemiological studies have shown that long-term low-dose exposure to paraquat can significantly increase the risk of sporadic Parkinson's disease. Therefore, paraquat has posed a serious threat to human health and the ecological environment.

[0003] Paraquat poisoning is a toxic disease mainly with lung damage, accompanied by liver, kidney and other multiple organ damage. The mortality rate of patients with oral poisoning is high, and can be as high as 50%-70% in severe cases, and most of them die of respiratory failure. The patient's condition progresses rapidly, and the long-term prognosis is unclear. Blood and urine paraquat concentration determination can make a definite diagnosis and help judge the prognosis, but with the passage of time, the blood and urine paraquat concentration gradually decreases or even cannot be measured. At present, it has been reported in the literature that blood routine indexes such as white blood cell count, neutrophil percentage and lymphocyte percentage can better judge the severity of the disease and predict the prognosis of patients, but the specificity is poor. Therefore, it is urgent to develop specific and sensitive biomarkers for monitoring the condition and prognosis evaluation of paraquat poisoning patients.

[0004] Metabolites are the end products of the action of genes, proteins and environmental factors, and directly reflect the pathophysiological state of the organism, which is closer to the phenotype than gene or protein markers. In recent years, the research on metabolites as biomarkers has made remarkable progress in the fields of disease diagnosis, treatment monitoring and health evaluation. Its advantages are that it can dynamically reflect the pathophysiological state, and most of them can be detected through non-invasive samples (blood, urine), and the change of metabolite concentration can be earlier than clinical symptoms or imaging changes (such as early tumor), which is suitable for early diagnosis and real-time monitoring, such as prediction of drugs and toxicity. SUMMARY

[0005] In view of the deficiencies in the prior art, the application provides application of Inosine as a biomarker in preparation of a product for paraquat poisoning condition progression and prognosis evaluation.

[0006] Application of Inosine as a biomarker in preparation of a product for paraquat poisoning condition progression.

[0007] Further, the expression levels of the biomarker Inosine in samples of patients with different paraquat poisoning conditions are different.

[0008] Further, the condition progression is paraquat poisoning for 1-4 days and paraquat poisoning for 7-14 days.

[0009] Further, the expression level of the biomarker Inosine in paraquat poisoning 7-14 day patient sample is lower than that in paraquat poisoning 1-4 day patient sample.

[0010] Further, the sample includes a urine sample from a subject.

[0011] Application of Inosine as a biomarker in the preparation of a product for paraquat poisoning prognosis evaluation.

[0012] Further, the expression level of the biomarker Inosine in paraquat poisoning 7-14 day patient sample is lower than that in paraquat poisoning 1-4 day patient sample.

[0013] Compared with the prior art, the present application has at least the following advantages: The present application relates to the application of Inosine as a biomarker in the preparation of a product for paraquat poisoning disease progression and prognosis evaluation, and the biomarker Inosine is a urine metabolite. Through urine non-targeted metabolomics analysis, it is found that the level of Inosine is lower in the urine of patients with early paraquat poisoning, and gradually decreases as the disease worsens, and significantly increases in 5-year follow-up; the present application judges the prediction performance by the area under the curve (AUC) of the receiver operating characteristic (ROC) curve, and the performance of Inosine in judging the progression of paraquat poisoning is good (AUC=0.806), the performance in judging the prognosis in the early stage of poisoning (1-4 days) is good (AUC=0.816), and the performance in judging the prognosis in the progress stage (7-14 days) is excellent (AUC=0.961). Moreover, the performance of Inosine in predicting the progression and prognosis of paraquat poisoning is better than that of the blood routine indexes (such as white blood cell count, neutrophil percentage, and lymphocyte percentage) reported in the literature. In summary, Inosine as a biomarker for the progression and prognosis of paraquat poisoning shows good clinical application prospects, including monitoring the condition of paraquat poisoning patients and evaluating long-term prognosis. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to more clearly illustrate the specific embodiments of the present application, the drawings required to be used in the specific embodiments or prior art description will be briefly introduced below.

[0015] Figure 1 The urine non-targeted metabolomics analysis of the first embodiment of the present application screened metabolites with statistical differences; Figure 2 The expression level analysis of Inosine in the urine of paraquat poisoning patients at different stages in the second embodiment of the present application; Figure 3To evaluate the efficacy of Inosine in assessing the progression and long-term prognosis of paraquat poisoning in Example Three of the Invention; Figure 4 To evaluate the predictive efficacy of prior art white blood cell count, lymphocyte percentage, and neutrophil percentage as markers in Example Four of the Invention. DETAILED DESCRIPTION

[0016] It is necessary to point out here that the following detailed description is only used to further illustrate the Invention and cannot be understood as limiting the scope of protection of the Invention. Those skilled in the art can make some non-essential improvements and adjustments to the Invention based on the above application content.

[0017] The materials used in the experiments and the experimental methods are generally and / or specifically described in the Invention. Unless otherwise specified, the experimental methods or test methods involved are conventional methods; the reagents or instruments used, unless specified by the manufacturer, are conventional products available on the market, prepared or used by conventional methods.

[0018] Example One: Discovery of biomarkers for paraquat poisoning patient samples The Invention includes 56 cases of paraquat poisoning patients, including 20-day-old infants to 87-year-old adults, with males accounting for 55.9%. The poisoning was mainly through the digestive tract, and a few through skin contact. The median concentration of paraquat in urine was 6.0 ng / ml, and the highest concentration was 329 ng / ml. Urine samples were collected from 56 patients (n=56) 1-4 days after poisoning, 35 patients (n=35) 7-14 days after poisoning, and 38 patients (n=38) 5 years after follow-up. The study involved in the Invention was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical College, and the research subjects signed the informed consent form. LC-MS / MS method (liquid chromatography-tandem mass spectrometry) was used for urine non-target metabolomics analysis, and the analysis steps were as follows.

[0019] S1. Sample extraction: After slow thawing of the urine at 4℃, an appropriate amount of sample was added to a pre-cooled methanol / acetonitrile / water solution (2:2:1, v / v), vortex mixed, ultrasonicated at low temperature for 30 min, -20℃ for 10 min, 14000 g 4℃ centrifuged for 20 min, the supernatant was vacuum dried, 100 μL acetonitrile water solution (acetonitrile: water = 1:1, v / v) was added for reconstitution before mass spectrometry analysis, vortexed, 14000 g 4℃ centrifuged for 15 min, and the supernatant was injected for analysis.

[0020] S2. Chromatography-mass spectrometry analysis: UHPLC-Q-TOF MS.

[0021] S21. Chromatographic conditions: The sample was separated by Agilent 1290 Infinity LC ultra-high performance liquid chromatography (UHPLC) HILIC column; column temperature 25 °C; flow rate 0.5 mL / min; injection volume 2 μL; mobile phase composition A: water + 25 mM ammonium acetate + 25 mM ammonia water, B: acetonitrile; gradient elution program as follows: 0-0.5 min, 95% B; 0.5-7 min, B from 95% linearly changed to 65%; 7-8 min, B from 65% linearly changed to 40%; 8-9 min, B maintained at 40%; 9-9.1 min, B from 40% linearly changed to 95%; 9.1-12 min, B maintained at 95%; the sample was placed in the 4 °C autosampler during the whole analysis process. In order to avoid the influence of instrument detection signal fluctuation, random order was used for continuous analysis of samples. QC samples were inserted in the sample queue for monitoring and evaluating the stability of the system and the reliability of the experimental data.

[0022] S22. Q-TOF mass spectrometry conditions: AB Triple TOF 6600 mass spectrometer was used to collect the first and second spectra of the sample. After the sample was separated by Agilent 1290 Infinity LC ultra-high performance liquid chromatography (UHPLC), Triple TOF 6600 mass spectrometer (AB SCIEX) was used for mass spectrometry analysis, and electrospray ionization (ESI) positive and negative ion modes were used for detection respectively. The ESI source setting parameters are as follows: auxiliary heating gas 1 (Gas1): 60, auxiliary heating gas 2 (Gas2): 60, curtain gas (CUR): 30 psi, ion source temperature: 600 °C, spray voltage (ISVF): ±5500 V (positive and negative modes); first mass-to-charge ratio detection range: 60-1000 Da, second sub-ion mass-to-charge ratio detection range: 25-1000 Da, first mass spectrum scan accumulation time: 0.20 s / spectra, second mass spectrum scan accumulation time 0.05 s / spectra; second mass spectrum uses data-dependent acquisition mode (IDA) to obtain, and uses peak intensity value screening mode, cluster voltage (DP): ±60 V (positive and negative modes), collision energy: 35±15 eV, IDA settings as follows: dynamic exclusion of isotope ions range: 4 Da, 10 fragment spectra are collected for each scan.

[0023] S3. Data analysis process: The raw data is converted into.mzXML format by ProteoWizard, then peak alignment, retention time correction and peak area extraction are performed by XCMS software. The data extracted by XCMS is first subjected to metabolite structure identification, data preprocessing, then experimental data quality evaluation, and finally data analysis.

[0024] A total of 1717 metabolites were identified by the above method, of which 12 metabolites were statistically different at different time points after paraquat poisoning, as shown in Figure 1 According to whether these metabolites have a clear metabolic pathway and physicochemical properties, biological function is screened, and finally Inosine is selected to study its value in predicting the progress and prognosis of paraquat poisoning.

[0025] Example Two Expression level of Inosine in urine of patients with paraquat poisoning at different stages The expression level of Inosine in urine samples of patients with paraquat poisoning at different stages is shown in Figure 2 As can be seen from the figure, the expression of Inosine significantly decreases from the early stage of paraquat poisoning (1-4 days) to the progress stage (7-14 days) with the progress of the disease, and significantly increases in the follow-up of 5 years with good prognosis.

[0026] Example Three Predictive efficiency of Inosine as a biomarker Figure 3 To predict the efficiency of Inosine on the progress and long-term prognosis of paraquat poisoning, the efficiency is judged according to the AUC value: excellent (>0.9), good (0.8-0.9), better (0.7-0.8), poor (0.6-0.7), or failure (0.5-0.6). Among them Figure 3 A is the ROC curve of patients with paraquat poisoning for 1-4 days and 7-14 days, and the AUC value of Inosine is 0.806; Figure 3 B is the ROC curve of patients with paraquat poisoning for 1-4 days and 5 years, and the AUC value of Inosine is 0.816; Figure 3 C is the ROC curve of patients with paraquat poisoning for 5 years and 7-14 days, and the AUC value of Inosine is 0.961.

[0027] The above results show that Inosine has good efficiency (AUC>0.8) in monitoring the progress of patients with paraquat poisoning and judging the long-term prognosis in the early stage of poisoning (1-4 days), and excellent efficiency (AUC=0.961) in judging the long-term prognosis in the progress stage of poisoning (7-14 days).

[0028] Example Four Predictive efficiency of white blood cell count, lymphocyte percentage, and neutrophil percentage as biomarkers Figure 4The efficiency of the literature reported blood routine indexes, white blood cell count, neutrophil percentage and lymphocyte percentage, in predicting the progression of PQ poisoning and long-term prognosis was shown. The three indexes had poor efficiency in predicting the progression of the disease (AUC < 0.7), failed in judging the long-term prognosis in the early stage of poisoning (1-4 days), had good efficiency in judging the long-term prognosis in the progress stage of poisoning (7-14 days) (AUC > 0.7), but were lower than the prediction efficiency of Inosine.

[0029] The above results show that Inosine has better efficiency than the reported blood routine indexes in the progression of paraquat poisoning and the evaluation of paraquat poisoning prognosis.

[0030] The above examples are only used to illustrate the technical solutions of the present application, but not limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application, and they should be covered in the scope of the claims and the specification of the present application.

Claims

1. Application of Inosine as a biomarker in the preparation of products for the progression of paraquat poisoning.

2. The application according to claim 1, characterized in that, The expression levels of the biomarker Inosine varied in patient samples with different disease progressions in paraquat poisoning.

3. The application according to claim 2, characterized in that, The disease progression was described as paraquat poisoning for 1-4 days and paraquat poisoning for 7-14 days.

4. The application according to claim 3, characterized in that, The expression level of the biomarker Inosine in patients 7-14 days after paraquat poisoning was lower than that in patients 1-4 days after paraquat poisoning.

5. The application according to claim 1, characterized in that, The samples included urine samples from the subjects.

6. Application of Inosine as a biomarker in the preparation of products for prognostic assessment of paraquat poisoning.

7. The application according to claim 6, characterized in that, The expression level of the biomarker inosine in patients followed up for 5 years after paraquat poisoning was significantly higher than that on days 1-4 and 7-14 after poisoning.

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