Application of Pseudouridine as biomarker in preparation of products for paraquat poisoning diagnosis, disease course analysis and prognosis evaluation

Abstract

The invention relates to a biomarker for diagnosis, disease course analysis and prognosis of paraquat poisoning and application of the biomarker. The biomarker is a urine metabolite Pseudouridine. Urine non-target metabonomics analysis shows that the relative amount of Pseudouridine is relatively low in urine of early-stage poisoning patients, is gradually reduced along with aggravation of illness, is increased in follow-up visit of five years, and is recovered to the level of a healthy control group; according to the invention, the prediction efficiency is judged through the area under the line (AUC) of a subject's working characteristic (ROC) curve, the Pseudouridine has good efficiency (AUC = 0.732) in the aspect of diagnosing whether paraquat is poisoned or not, has good efficiency (AUC = 0.855) in the aspect of judging the progression of paraquat poisoning, has good efficiency (AUC = 0.807) in the aspect of judging long-term prognosis in the early stage of poisoning (1-4 days), and has excellent efficiency (AUC = 0.979) in the aspect of judging long-term prognosis in the progression stage of poisoning (7-14 days). And the prediction efficiency of the Pseudouridine is better than the blood routine examination index reported in the literature. In conclusion, the Pseudouridine, as a biomarker for paraquat poisoning diagnosis, disease course analysis and prognosis, shows a good clinical application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pesticide poisoning detection, and particularly relates to application of Pseudouridine as a biomarker in preparation of products for paraquat poisoning diagnosis, disease course analysis and prognosis evaluation. BACKGROUND

[0002] Paraquat (1,1'-dimethyl-4,4'-bipyridinium salt, paraquat, PQ) is a highly efficient, broad-spectrum, inexpensive but highly toxic organic heterocyclic herbicide. Paraquat poisoning can cause systemic toxic reactions, leading to multiple system organ dysfunction or failure such as pulmonary fibrosis, gastrointestinal erosion, liver damage, kidney damage, etc. Due to the lack of specific antidote, the mortality rate of paraquat poisoning is as high as 60% or more. Moreover, the patient's condition progresses rapidly, and the long-term prognosis is unclear.

[0003] The diagnostic criteria for paraquat poisoning mainly depend on clear paraquat exposure history, detection of paraquat in blood / urine and typical clinical manifestations. Detection of blood, urine, gastric contents and residual poison is an important basis for clinical diagnosis of paraquat poisoning. Blood and urine paraquat concentration determination can confirm the diagnosis and help judge the prognosis, but with the passage of time, blood and urine paraquat concentration gradually decreases or even cannot be measured. Paraquat is mainly quantitatively detected by high performance liquid chromatography, high performance liquid chromatography-mass spectrometry and other methods. These methods have good precision, high sensitivity and accuracy, but require professional technical personnel and rely on expensive instruments and equipment, and are difficult to detect in medical institutions without physical and chemical detection departments. At present, there are some literatures reporting that blood routine indicators such as white blood cell count, neutrophil percentage and lymphocyte percentage can better reflect the severity of the patient's condition and have certain predictive value for the prognosis of the patient, but the specificity is poor. Therefore, it is urgent to develop specific and sensitive biomarkers to assist in the diagnosis of paraquat poisoning and judge the progression and prognosis of patients.

[0004] Metabolomics is a science that studies the types, quantities and change rules of metabolic products (endogenous metabolites) with molecular mass less than 1500 Da caused by external stimulation, pathophysiological changes and gene mutations of living beings, and is the closest omics to phenotype. Metabolites are the end products of gene, protein and environmental factors, and directly reflect the pathophysiological state of the organism, which is closer to the phenotype than gene or protein markers. Metabolite concentration changes can be earlier than clinical symptoms or imaging changes (such as early tumor), which is suitable for early diagnosis and real-time monitoring. Many metabolites can be detected through blood, urine, saliva and other body fluids, reducing the need for invasive procedures such as biopsy. The development of high-throughput metabolomics technology enables the simultaneous detection of thousands of metabolites, significantly improving the efficiency of biomarker discovery. Metabolites as biomarkers show important value in the diagnosis, disease monitoring and prognosis evaluation of various diseases. SUMMARY

[0005] In view of the deficiencies of the prior art, the application proposes the use of Pseudouridine as a biomarker in the preparation of paraquat poisoning diagnosis, disease course analysis and prognosis evaluation products.

[0006] The technical scheme of the application is: The use of Pseudouridine as a biomarker in the preparation of paraquat poisoning diagnosis products.

[0007] Further, the biomarker Pseudouridine is lowly expressed in paraquat poisoning patient samples.

[0008] Further, the sample includes a urine sample from a subject.

[0009] The use of Pseudouridine as a biomarker in the preparation of paraquat poisoning disease course analysis products.

[0010] Further, the biomarker Pseudouridine has different expression levels in paraquat poisoning patient samples at different disease courses.

[0011] Further, the disease course is paraquat poisoning for 1-4 days and paraquat poisoning for 7-14 days.

[0012] Further, the biomarker Pseudouridine has a lower expression level in paraquat poisoning patient samples for 7-14 days than in paraquat poisoning patient samples for 1-4 days.

[0013] The use of Pseudouridine as a biomarker in the preparation of paraquat poisoning prognosis evaluation products.

[0014] Further, the biomarker Pseudouridine has no significant difference in expression level between paraquat poisoning patient samples followed up for 5 years and healthy control samples.

[0015] Compared with the prior art, the application has at least the following advantages: The present application relates to the application of Pseudouridine as a biomarker in the preparation of paraquat poisoning diagnosis, course analysis and prognosis evaluation products, the biomarker Pseudouridine is a urine metabolite. Through urine non-target metabolomics analysis, it is found that the relative amount of Pseudouridine is lower in the urine of patients with early paraquat poisoning, and gradually decreases with the aggravation of the disease, and increases in the 5-year follow-up, and returns to the level of the healthy control group; 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 Pseudouridine in diagnosing whether paraquat is poisoned is better (AUC=0.732), the performance in judging the progress of paraquat poisoning is good (AUC=0.855), the performance in judging long-term prognosis in the early stage (1-4 days) of poisoning is good (AUC=0.807), and the performance in judging long-term prognosis in the progress stage (7-14 days) of poisoning is excellent (AUC=0.979). Moreover, the performance of Pseudouridine in predicting the progress and prognosis of paraquat poisoning is better than that of the blood routine indexes (such as white blood cell count, neutrophil percentage, lymphocyte percentage) reported in the literature. In summary, Pseudouridine as a biomarker for paraquat poisoning diagnosis, course analysis and prognosis shows good clinical application prospect, including paraquat poisoning diagnosis, disease monitoring and prognosis evaluation. BRIEF DESCRIPTION OF DRAWINGS

[0016] 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.

[0017] Figure 1 The urine non-target metabolomics analysis of the present application example one is used to screen metabolites with statistical difference; Figure 2 The present application example two Pseudouridine is used for analyzing the expression level of Pseudouridine in the urine of healthy control group and paraquat poisoning patients at different stages; Figure 3 The present application example three Pseudouridine is used for the efficiency of Pseudouridine in paraquat poisoning diagnosis, course analysis and long-term prognosis evaluation; Figure 4 The present application example four is used for the prediction efficiency of white blood cell count, lymphocyte percentage and neutrophil percentage as markers in the prior art. DETAILED DESCRIPTION

[0018] Further details of the present application are described below. It is necessary to point out that the following specific embodiments are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application according to the above application content.

[0019] The materials used in the experiment and the test method are generally and / or specifically described in the present application. The test method or test method involved, if not specially stated, is a conventional method; the reagent or instrument used, if not specified by the manufacturer, is a conventional product available on the market, prepared or used by a conventional method.

[0020] Example 1: Discovery of biomarkers in paraquat poisoning patient samples The present application includes 56 cases of paraquat poisoning patients, including 20-day infants to 87-year-old old people, with males accounting for 55.9%. The poisoning is mainly through the digestive tract, and a few through skin contact. The median concentration of paraquat in urine is 6.0 ng / ml, and the highest concentration is 329 ng / ml. 56 urine samples (n=56) were collected 1-4 days after poisoning, 35 urine samples (n=35) were collected 7-14 days after poisoning, and 38 urine samples (n=38) were collected 5 years after follow-up. According to the age and gender of the patients, 56 healthy controls were matched 1:1, and 56 urine samples (n=56) were collected. The research involved in the present application was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical College, and the research objects 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.

[0021] S1. Sample extraction: After the urine was slowly thawed 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, vacuum dried, 100 μL acetonitrile water solution (acetonitrile: water = 1:1, v / v) was added for reconstitution, vortexed, 14000 g 4℃ centrifuged for 15 min, and the supernatant was taken for analysis.

[0022] S2. Chromatography-mass spectrometry analysis: UHPLC-Q-TOF MS.

[0023] 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.

[0024] 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 separation by Agilent 1290 Infinity LC ultra-high performance liquid chromatography (UHPLC), the sample was analyzed by Triple TOF 6600 mass spectrometer (AB SCIEX) for mass spectrometry, and was detected by electrospray ionization (ESI) positive and negative ion modes respectively. The ESI source setting parameters are as follows: nebulizing gas 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 was obtained by data-dependent acquisition mode (IDA), and peak intensity value screening mode was used, 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 were collected for each scan.

[0025] S3. Data analysis process: The raw data was converted into.mzXML format by ProteoWizard, and then XCMS software was used for peak alignment, retention time correction and peak area extraction. The data extracted by XCMS was first subjected to metabolite structure identification, data preprocessing, then experimental data quality evaluation, and finally data analysis.

[0026] 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, physicochemical properties, and biological function, Pseudouridine (Pseudouridine) was finally selected to study its value in paraquat poisoning diagnosis, prediction of disease progression, and prognosis.

[0027] Example Two Pseudouridine expression levels in urine of healthy controls and paraquat poisoning patients at different stages The expression levels of Pseudouridine in urine samples of paraquat poisoning patients at different stages and healthy controls are shown in Figure 2 As can be seen from Figure 2 , with the progression of the disease, the expression level of Pseudouridine in the early stage of paraquat poisoning (1-4 days) was significantly lower than that of the healthy control group, and further decreased in the progress stage (7-14 days). The expression level of 5 years was significantly increased and had no statistical difference compared with the healthy control group.

[0028] Example Three Prediction performance of Pseudouridine as a biomarker Figure 3 To predict the prediction performance of Pseudouridine for paraquat poisoning diagnosis, disease analysis and long-term prognosis, the performance 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 the healthy control group and the paraquat poisoning 1-4 day patient, and the AUC value of Pseudouridine is 0.732; Figure 3 B is the ROC curve of paraquat poisoning patients 1-4 days and 7-14 days, and the AUC value of Pseudouridine is 0.855; Figure 3 C is the ROC curve of paraquat poisoning patients 1-4 days and 5 years, and the AUC value of Pseudouridine is 0.807; Figure 3 D is the ROC curve of paraquat poisoning patients 5 years and 7-14 days, and the AUC value of Pseudouridine is 0.979.

[0029] The above results show that Pseudouridine has better performance in judging whether PQ is poisoned (AUC = 0.732), better performance in predicting disease progression and early poisoning (1-4 days) to judge long-term prognosis (AUC = 0.855, AUC = 0.807), and excellent performance in judging long-term prognosis in the progress of poisoning (7-14 days) (AUC = 0.979).

[0030] Example Four Prediction performance of white blood cell count, lymphocyte percentage, and neutrophil percentage as biomarkers Figure 4 The performance of the literature reported blood routine indexes, white blood cell count, neutrophil percentage and lymphocyte percentage in predicting paraquat poisoning disease progression and long-term prognosis is shown. The three have poor performance in predicting disease progression (AUC < 0.7), fail to judge long-term prognosis in the early stage of poisoning (1-4 days), have better performance in judging long-term prognosis in the progress of poisoning (7-14 days) (AUC > 0.7), but all are lower than the performance of Pseudouridine.

[0031] The above results show that Pseudouridine has better performance in judging whether PQ is poisoned (AUC = 0.732), better performance in predicting disease progression and early poisoning (1-4 days) to judge long-term prognosis (AUC = 0.855, AUC = 0.807), and excellent performance in judging long-term prognosis in the progress of poisoning (7-14 days) (AUC = 0.979).

[0032] The above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions 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 Pseudouridine as a biomarker in the preparation of products for the diagnosis of paraquat poisoning.

2. The application according to claim 1, characterized in that, The biomarker Pseudouridine was expressed at low levels in paraquat poisoning patient samples.

3. The application according to claim 2, characterized in that, The samples included urine samples from the subjects.

4. Application of Pseudouridine as a biomarker in the preparation of products for analysis of paraquat poisoning course.

5. The application according to claim 4, characterized in that, The expression levels of the biomarker Pseudouridine varied in samples from patients at different stages of paraquat poisoning.

6. The application according to claim 5, characterized in that, The course of the disease is described as 1-4 days and 7-14 days after paraquat poisoning.

7. The application according to claim 6, characterized in that, The expression level of the biomarker Pseudouridine in patients 7-14 days after paraquat poisoning was lower than that in patients 1-4 days after paraquat poisoning.

8. Application of Pseudouridine as a biomarker in the preparation of products for prognostic assessment of paraquat poisoning.

9. The application according to claim 8, characterized in that, The expression level of the biomarker Pseudouridine in patients followed up for 5 years after paraquat poisoning was not significantly different from that in healthy control samples.

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