Application of uridine as serum marker in prognosis of hepatitis B related chronic-acute liver failure

Abstract

The invention discloses application of uridine as a serum marker in prognosis of hepatitis B related chronic-acute hepatic failure, the uridine content is an independent predictive factor of 28-day mortality of patients with hepatitis B related chronic-acute hepatic failure, and when the mortality of the patients is increased, the uridine expression quantity is reduced, so that the content of the uridine in the prognosis of hepatitis B related chronic-acute hepatic failure is reduced. Uridine can be used for constructing a hepatitis B related chronic-acute liver failure prognosis prediction model. The serum marker uridine provided by the invention can be used for identifying high-death-risk patients in the early stage of hepatitis B-related chronic-acute hepatic failure, provides important reference for clinical doctors to accurately judge the severity of illness, and can identify the high-death-risk patients in the early stage and take corresponding positive treatment measures.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of medicine, and relates to application of uridine as a serum marker in prognosis of hepatitis B virus (HBV)-associated acute-on-chronic liver failure (HBV-ACLF), in particular to a serum uridine detection kit as a marker for predicting prognosis of an HBV-ACLF patient, to provide a reference for clinical treatment. BACKGROUND

[0002] Hepatitis B virus (HBV) infection is a major problem threatening human health and safety, which not only leads to liver cancer and acute exacerbation of cirrhosis, but also causes hepatitis B virus (HBV)-associated acute-on-chronic liver failure (HBV-ACLF), which is also an important cause of death in patients with HBV infection. Although progress has been made in organ support, liver transplantation and new drug therapy in recent years, the mortality rate of acute-on-chronic liver failure (ACLF) is still high, with a 28-day mortality rate of up to 30%, and the mortality rate of patients who develop organ failure in the first week after the occurrence of ACLF can be as high as 40%-50%.

[0003] The pathophysiological process of ACLF is very complex, which can recover, worsen or remain stable within the first few days of hospitalization, thus having a major impact on the overall prognosis of ACLF. The first week after the onset of ACLF is very important, and early identification of patients with high mortality risk of ACLF can provide an important reference for clinical treatment, thus improving the survival rate of patients. However, the current diagnostic techniques still have limitations in the early identification of ACLF, and lack of specific and sensitive biomarkers, resulting in that some patients cannot be diagnosed and intervened in time before the disease worsens. SUMMARY

[0004] The first object of the present application is to provide the application of uridine as a serum marker in the preparation of a product for evaluating the prognosis of patients with hepatitis B virus (HBV)-associated acute-on-chronic liver failure (HBV-ACLF), in view of the deficiencies of the prior art.

[0005] As a preferred embodiment, the content of uridine is an independent predictor of the 28-day mortality rate of patients with hepatitis B virus (HBV)-associated acute-on-chronic liver failure (HBV-ACLF).

[0006] As a preferred embodiment, the expression level of uridine decreases when the mortality rate of the patient increases.

[0007] As a preferred embodiment, the product is used for detecting serum samples of patients.

[0008] As a preferred embodiment, the product comprises a kit for detecting the content of the serum marker, and the kit contains reagents for detecting the content of the serum marker.

[0009] A second object of the present application is to provide a detection product for evaluating or assisting in evaluating the prognosis of hepatitis B-related acute-on-chronic liver failure; the detection product contains reagents for detecting the content of a serum marker, which is uridine.

[0010] Preferably, the detection product comprises a kit, a chip, and a test paper.

[0011] A third object of the present application is to provide an application of uridine in constructing a prognosis prediction model for hepatitis B-related acute-on-chronic liver failure.

[0012] Compared with the prior art, the present application has the following beneficial effects:

[0013] The present application provides a new serum marker, uridine, which can be used to predict the 28-day prognosis of HBV-ACLF patients. Through serum metabolomics analysis, the present application reveals the serum metabolite changes in patients with HBV-ACLF who died within 28 days and survived patients in the early stage of the disease. It is found that uridine is an independent predictor of 28-day death in patients with HBV-ACLF. Subsequently, a prediction model for patient prognosis is constructed using serum uridine, and it is found that the constructed model can effectively predict the short-term mortality of patients. This provides an important reference for the treatment of HBV-ACLF. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 is the metabolomics detection result of serum of HBV-ACLF death and survival patients at admission. A is the serum metabolomics OPLS-DA score plot of HBV-ACLF 28-day death and survival patients in the discovery cohort; B is the volcano plot of serum metabolite expression of HBV-ACLF death and survival patients; C is the quantitative enrichment analysis plot of serum metabolite metabolic pathways of HBV-ACLF death and survival patients.

[0015] Figure 2 is the screening of serum differential metabolites. A is the LASSO regression analysis of 103 differential metabolites to screen 10 metabolites independently related to 28-day death; B is the differential metabolite regression coefficient trajectory plot (collinearity plot).

[0016] Figure 3 is the relative expression amount of metabolite uridine and ROC curve analysis. A is the relative expression amount of uridine in HBV-ACLF death and survival patients in the discovery cohort; B is the relative expression amount of uridine in HBV-ACLF death and survival patients in the verification cohort; C is the ROC curve plot of uridine predicting 28-day death of patients in the discovery cohort; D is the ROC curve plot of uridine predicting 28-day death of patients in the verification cohort. DETAILED DESCRIPTION

[0017] The technical solutions of the present application are further described in detail below with reference to the drawings. It should be noted that the specific embodiments are only detailed descriptions of the present application and should not be regarded as limitations of the present application.

[0018] Example 1

[0019] 1. Serum sample collection

[0020] 3-5 mL of venous blood was collected within 3 days of hospitalization using a non-anticoagulant tube, and was labeled and registered in detail. The blood collection tube was placed in a centrifuge and balanced, 3000 r / min, 10 min. The centrifuged blood sample was placed on a test tube rack, and the blood sample was visibly divided into two layers, with the upper layer being light yellow, which was the desired serum, and the lower layer being the blood cell component. The upper layer of serum was evenly divided into two EP tubes using a pipette, and the EP tube cap was tightly closed. The EP tube was placed in a specimen box in a -80°C refrigerator, and was placed in order and in groups for standby, and the sample name and time were labeled.

[0021] 2. Clinical data collection

[0022] Patients with chronic HBV infection were screened using the International Classification of Diseases, 10th edition (ICD-10) code B18.1. At the same time, the screened patients were further checked for the presence of hepatitis B surface antigen (HBsAg) positive, hepatitis B core antigen (HBeAg) positive or HBV DNA detection positive, and one of them was considered to have HBV infection. Patients with HBV infection for more than 6 months were diagnosed as chronic HBV infection. Subsequently, standardized data collection was used to collect patient clinical data. The diagnosis of ACLF was determined according to the APASL guidelines.

[0023] Patients diagnosed as HBV-ACLF at the time of admission were finally included in the study, and were divided into 28-day death group (48 people) and 28-day survival group (40 people) according to the follow-up results. This part of the patients was randomly divided into a discovery cohort (30 dead patients and 20 surviving patients) and a validation cohort (18 dead patients and 20 surviving patients), wherein the discovery cohort was used for screening and modeling of differential metabolites, and the validation cohort was used to evaluate the prognostic prediction performance of the identified markers in an independent population, and the two were independent of each other.

[0024] The following experiments were performed in the discovery cohort.

[0025] 3. Labeling of serum samples

[0026] (1) Serum sample pretreatment: Take 20 μL of serum sample, add 60 μL of ice-cold methanol, vortex to mix, and let stand for 15 minutes. Then centrifuge at 14,000 rpm for 30 minutes. Collect the supernatant and concentrate it by centrifugation and evaporation to obtain a powder containing serum metabolites. At the same time, 10 μL of each sample is precipitated as a mixed sample. The metabolite powder of the mixed sample is obtained by the same method and stored in a freezer at -80°C.

[0027] (2) Danylation labeling: using 12 C-dansyl chloride labeling of individual samples, using 13 C-Dansulfonyl chloride / labeled mixed sample. Redissolve the metabolite powder in 25 μL of water / acetonitrile mixture (5:1 v / v). Then add: 125 μL of ABC buffer (Na₂CO₃ / NaHCO₃, 500 mM, pH 9.4), 125 μL of acetonitrile, vortex to mix, and then add 25 μL of... 12 C-dansyl chloride solution (20 mg / mL) or 13 The reaction mixture was incubated with C-dansyl chloride solution (20 mg / mL) in a 60°C water bath for 60 minutes. After the reaction was complete, 5 μL of NaOH (250 mM) was added to terminate the reaction. Finally, the reaction mixture was diluted with 425 mM 50% formic acid solution and analyzed by LC-MS.

[0028] 4. LC-MS Analysis

[0029] Labeled serum samples were analyzed using an Impact II quadrupole time-of-flight mass spectrometer (QTOF, Bruker, USA) and an UltiMate 3000U HPLC system (Thermo Scientific). The mobile phase settings were as follows: Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% formic acid acetonitrile solution; The chromatographic gradient was as follows: t = 0 min: Phase B at 25%; t = 10 min: Phase B increased to 99% and held until t = 13 min; t = 13.1 min: Phase B decreased back to 25% and held until t = 16 min. The flow rate was 400 μL / min, and the column temperature was set to 45 °C. Before formal sample analysis, quality control (QC) samples were tested at least five times until the signal stabilized.

[0030] 5. Characteristic analysis of metabolomics

[0031] (1) Multivariate statistics: OPLS-DA analysis of serum metabolomics was performed using SMICA 14.1. OPLS-DA analysis was used to distinguish differences between groups.

[0032] (2) Differential metabolites preliminary screening: Excel was used to screen the differential metabolite pairs of serum metabolomics between death and survival HBV-ACLF patients within 28 days with |FC|>1.2, P<0.05 as the threshold. The differential metabolite pairs were compared with the Danacy standard library and HMDB database according to mass-to-charge ratio and retention time, etc. to identify the obtained differential metabolites.

[0033] (3) Metabolic pathway quantitative enrichment analysis: The preliminary screening differential substances were subjected to quantitative enrichment analysis, and the metabolic pathways with greater impact were inferred according to P value and Pathway Impact.

[0034] (4) Correlation analysis: Spearman correlation analysis was performed between the preliminary screening differential metabolites obtained above and the 28-day death of patients, and the differential substances were screened with a correlation coefficient |p|>0.5, P<0.05 as the standard to obtain potential biomarker candidate list.

[0035] (5) Screening of prognostic markers: The independent predictors obtained by LASSO regression analysis were listed as prognostic markers, which met the correlation coefficient |p|>0.5, P<0.05. The best prognostic marker was found by ROC curve analysis.

[0036] 6. Statistical data analysis

[0037] SPSS software package (IBM Corp., Armonk, NY, USA), R language software (version 4.2.2) and GraphPad Prism software (GraphPad Software Inc., CA, USA) were used for data analysis. Normality test was performed on continuous variables. In single factor analysis, T test and non-parametric test were used for analysis. Chi-square test and Fisher's exact test were used for analysis of categorical variables. LASSO regression analysis was performed using the "glmnet" package in R language software, and a regression model was constructed. In SPSS, a logistic regression analysis was used to construct a model, and ROC curves were drawn using GraphPad Prism. P<0.05 was considered to be statistically different.

[0038] Results

[0039] In the discovery cohort, by analyzing the differential metabolites, it was found that the serum metabolomics of death patients was significantly different from that of survival patients. In order to identify potential metabolites related to patient death, Student's t test and fold change (FC) analysis were used to compare the differences between peak pairs. The volcano plot showed that a total of 432 pairs of peaks had statistically significant differences between the HBV-ACLF death group (ACLF-D) and the HBV-ACLF survival group (ACLF-S) (Figure 1 Among A) in the serum samples, the differential metabolites were classified into three categories according to the confidence level of identification of 2,493 pairs of peaks: confirmed identification, high-confidence putative identification and putative identification. 730 metabolites were included in the subsequent analysis (including metabolites with confirmed identification and high-confidence putative identification). Finally, 103 metabolites were identified as differential expression metabolites. Among them, 48 metabolites were down-regulated in the 28-day death group of HBV-ACLF patients, and 55 metabolites were up-regulated in the 28-day death group of HBV-ACLF patients. Figure 2 Quantitative enrichment analysis showed that purine metabolism, glyoxylate and dicarboxylate metabolism, nitrogen biosynthesis, arginine biosynthesis, alanine, aspartate and glutamate metabolism were significantly changed. Figure 1 C) in the serum samples.

[0040] After 103 differential metabolites were included in LASSO regression analysis, it was found that 10 differential metabolites were independently related to the death of patients within 28 days. Figure 2 The differential metabolites with correlation coefficient |p|>0.5 and P<0.05 were screened, and the metabolites obtained by LASSO analysis were intersected, and uridine, 3-hydroxy-4-aminopyridine isomer, 2,3,4-trihydroxybenzoic acid isomer and 4-fluorocatechol were listed as potential biomarkers (Table 1).

[0041] Table 1 Correlation coefficient between 28-day death event of patients in the discovery cohort and differential metabolites (absolute value of correlation coefficient >0.5)

[0042]

[0043] To evaluate the prediction ability of each metabolite for 28-day death event, receiver operating characteristic curve (ROC) analysis was performed on uridine, 3-hydroxy-4-aminopyridine isomer, 2,3,4-trihydroxybenzoic acid isomer and 4-fluorocatechol, respectively. It was found that the area under the curve (AUC) of uridine was the largest, indicating that its ability to predict 28-day death event in the discovery cohort was the strongest (Table 1, Figure 3 C) in the serum samples.

[0044] To further verify the effectiveness of the above markers, the serum of the validation cohort patients was used to perform steps 3-6, and the validation results are shown in Figure 3 In the serum samples of the validation cohort, the level of uridine was significantly decreased, and the AUC value for predicting 28-day death event was as high as 0.913 Figure 3 D), which indicates that uridine still shows strong prognostic prediction ability in the validation set, supporting its potential as a prognostic marker for HBV-ACLF patients.

[0045] The experimental data show that the serum marker uridine provided by the application has excellent diagnostic sensitivity and specificity in predicting the short-term mortality risk of HBV-ACLF patients.

[0046] The above examples are not limited to the embodiments, and for those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Any modification and variation of the application still falls within the protection scope of the application.

Claims

1. Use of uridine as a serum marker in the preparation of a product for evaluating the prognosis of a patient with hepatitis B-related acute-on-chronic liver failure.

2. Use according to claim 1, characterized in that, The content of uridine is an independent predictor of 28-day mortality in patients with hepatitis B-related acute-on-chronic liver failure.

3. Use according to claim 2, characterized in that, The expression of uridine decreases when the mortality of the patient increases.

4. Use according to claim 1, characterized in that, The product is used for detecting serum samples of patients.

5. The use according to claim 1, characterized in that, The product comprises a kit for detecting the content of the serum marker, and the kit contains reagents for detecting the content of the serum marker uridine.

6. A detection product for prognosis evaluation or auxiliary prognosis evaluation of hepatitis B related acute-on-chronic liver failure, wherein, The detection product contains reagents for detecting the content of the serum marker, and the serum marker is uridine.

7. The test product of claim 6, wherein, The detection product comprises a kit, a chip, and a test paper.

8. Use of uridine in constructing a prognosis prediction model for hepatitis B-related acute-on-chronic liver failure.

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