CN114527209A - Marker combination for prognosis evaluation of liver cirrhosis - Google Patents

Marker combination for prognosis evaluation of liver cirrhosis Download PDF

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CN114527209A
CN114527209A CN202210108028.1A CN202210108028A CN114527209A CN 114527209 A CN114527209 A CN 114527209A CN 202210108028 A CN202210108028 A CN 202210108028A CN 114527209 A CN114527209 A CN 114527209A
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王睿林
牛明
王仲霞
景婧
何婷婷
荣文雅
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Fifth Medical Center of PLA General Hospital
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Abstract

The present application provides a marker combination for prognosis evaluation of cirrhosis, comprising: (±) -3,5, 5-trimethyl-1-hexanol, dichloromethane, pyridine, γ -butyrolactone, citronellal, ethyl lactate, guaiacol, methyl linoleate, oxoglutarate, (±) -erythro-isoleucine, 3-phenylpropanal and N-acetyl aromatic amine, preferably, the N-acetyl aromatic amine is N-acetanilide. Methods of determining the marker combinations are also provided.

Description

Marker combination for prognosis evaluation of liver cirrhosis
Technical Field
The application relates to clinical medicine, and specifically provides a marker combination for prognosis evaluation of cirrhosis.
Background
It is well known that the human body releases a large amount of Volatile Organic Compounds (VOCs), mainly from exhaled air, sweat, skin secretions, urine, faeces and saliva, all of which are produced by metabolic pathways. Traditional Chinese medicine and ancient Western doctors, including Hiboclata base, consider exhaled breath important both diagnostically and therapeutically, and use odor as a diagnostic tool. The olfactory skills of doctors have not been routinely used in modern medicine and there is a large body of literature documentations that many diseases, such as cancer, diabetes, respiratory diseases, liver diseases, etc., are associated with unique odors. Recently, "Super-smelter", a female who smells the Parkinson's disease before clinical symptoms appear, helped scientists develop a new diagnostic test that identifies the earliest stages of Parkinson's disease based on volatile organic compounds. Several studies have shown that volatile organic compounds in exhaled breath can provide valuable information for the pathophysiological condition of a patient.
In addition, patients with severe liver disease have long complained of an odor known as "liver odor", most of which comes from breathing. Some respiratory odorant compounds have been detected and used to identify healthy volunteers and patients with liver complications. For example, dimethyl sulfide has previously been identified as an important metabolite that plays a role in liver malodor caused by liver disease.
Decompensated Cirrhosis (DC) is a common consequence and the last stage of chronic liver disease of various etiologies, mainly due to chronic viral hepatitis b and c, harmful alcohol drinking and autoimmune liver diseases such as primary biliary cholangitis, with fatal complications including ascites, gastrointestinal bleeding and hepatic encephalopathy. The relevance of expired breath in decompensated liver cirrhosis patients has not been determined.
Disclosure of Invention
In this study, the inventors studied volatile organic compounds in exhaled breath of DC patients, including patients with hepatitis b cirrhosis (HLC), alcohol-related cirrhosis (ALC), and Primary Biliary Cirrhosis (PBC). Through detection of the expiratory metabonomics, the inventor determines the optimal predictor of the DC patient prognosis, explores the internal connection between DC and different causes from a new perspective, and provides reference for further early intervention and innovative treatment methods in the future.
The application provides a group of marker combinations for prognosis evaluation of cirrhosis, comprising: (±) -3,5, 5-trimethyl-1-hexanol, dichloromethane, pyridine, γ -butyrolactone, citronellal, ethyl lactate, guaiacol, methyl linoleate, oxoglutarate, (±) -erythro-isoleucine, 3-phenylpropanal, and N-acetoacetylamines. Preferably, the N-acetoacetylamide is an acetanilide.
The present application also provides a method of determining a marker combination for the prognostic assessment of cirrhosis, the marker combination comprising (±) -3,5, 5-trimethyl-1-hexanol, dichloromethane, pyridine, γ -butyrolactone, citronellal, ethyl lactate, guaiacol, methyl linoleate, oxoglutarate, (±) -erythro-isoleucine, 3-phenylpropanal and an N-acetoacetylamide, preferably the N-acetoacetylamide is N-acetanilide, the method comprising the steps of:
step 1. collecting Exhaled Breath Condensate (EBC);
step 2, analyzing the exhaled breath condensate by using a two-dimensional gas chromatography-tandem time-of-flight mass spectrum, and collecting mass spectrum data; and
and 3, analyzing the mass spectrum data by using principal component analysis and orthogonal partial least square discriminant analysis to obtain a marker combination for prognosis evaluation of cirrhosis.
The application also provides application of a marker combination for liver cirrhosis prognosis evaluation to preparation of a liver cirrhosis prognosis evaluation kit, wherein the marker combination comprises (+/-) -3,5, 5-trimethyl-1-hexanol, dichloromethane, pyridine, gamma-butyrolactone, citronellal, ethyl lactate, guaiacol, methyl linoleate, oxoglutarate, (+/-) -erythro-isoleucine, 3-phenylpropionaldehyde and N-acetyl arylamine, and preferably, the N-acetyl arylamine is N-acetanilide.
Has the advantages that:
the study of exhaled breath metabolites from subjects using comprehensive, two-dimensional gas chromatography tandem time-of-flight mass spectrometry (GC × GC-TOF-MS), Principal Component Analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) identified 12 metabolites, including (+/-) -3,5, 5-trimethyl-1-hexanol, dichloromethane, pyridine, gamma-butyrolactone, citronellal, ethyl lactate, guaiacol, methyl linoleate, oxoglutarate, (±) -erythro-isoleucine, 3-phenylpropanal and N-acetanilide, as biomarker combinations for identifying different outcomes of DCs.
Additional features and advantages of the application will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the application. Other advantages of the present application may be realized and attained by the instrumentalities and combinations particularly pointed out in the specification and the drawings.
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The accompanying drawings are included to provide an understanding of the present disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the examples serve to explain the principles of the disclosure and not limit the disclosure.
FIG. 1: graph comparing OPLS-DA scores between healthy subjects (group C), HLC, ALC and PBC groups. C: a healthy control group; HLC: hepatitis b cirrhosis group; ALC: alcohol-related liver cirrhosis group; PBC: primary biliary cirrhosis group; group of liver cirrhosis: all patients with cirrhosis, including patients in the HLC, ALC and PBC groups.
FIG. 2: is a comparative plot of OPLS-DA scores between group C and HLC (a), between group C and ALC (C), and between group C and PBC (E); VIP-p (corr) plots of OPLS-DA models for group C and HLC (B), group C and ALC (D), and group C and PBC (F).
FIG. 3: comparison of ROC curve analysis of predictive power in DC patients. (A) ROC curves for exhaled breath metabolites, MELD score, Child-pugh score, TBIL and INR for 66 DC patients. (B) ROC curves for exhaled breath metabolites, MELD score, Child-pugh score, TBIL and INR for 52 DC patients without liver transplantation.
Detailed Description
Hereinafter, embodiments of the present invention will be described in detail in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be noted that, in the present application, the embodiments and features of the embodiments may be arbitrarily combined with each other without conflict.
A first aspect of the present application provides a marker combination for prognosis of cirrhosis, comprising: (±) -3,5, 5-trimethyl-1-hexanol, dichloromethane, pyridine, γ -butyrolactone, citronellal, ethyl lactate, guaiacol, methyl linoleate, oxoglutarate, (±) -erythro-isoleucine, 3-phenylpropanal and N-acetyl aromatic amine, preferably said N-acetyl aromatic amine is N-acetanilide.
The (+ -) -3,5, 5-trimethyl-1-hexanols described herein include (+) -3,5, 5-trimethyl-1-hexanol, (-) -3,5, 5-trimethyl-1-hexanol, and mixtures of (+) -3,5, 5-trimethyl-1-hexanol and (-) -3,5, 5-trimethyl-1-hexanol.
Similarly, (+ -) -erythro-isoleucine described herein includes (+) -erythro-isoleucine, (-) -erythro-isoleucine, and mixtures of (+) -erythro-isoleucine and (-) -erythro-isoleucine.
A second aspect of the present application provides a method of determining a marker combination for the prognostic assessment of cirrhosis, said marker combination comprising (±) -3,5, 5-trimethyl-1-hexanol, dichloromethane, pyridine, γ -butyrolactone, citronellal, ethyl lactate, guaiacol, methyl linoleate, oxoglutarate, (±) -erythro-isoleucine, 3-phenylpropanal and an N-arylamine, preferably said N-arylamine is N-acetanilide, said method comprising the steps of:
step 1. collecting Exhaled Breath Condensate (EBC);
step 2, analyzing the exhaled breath condensate by using a two-dimensional gas chromatography-tandem time-of-flight mass spectrum, and collecting mass spectrum data; and
and 3, analyzing the mass spectrum data by using principal component analysis and orthogonal partial least square discriminant analysis to obtain a marker combination for prognosis evaluation of cirrhosis.
Exhaled gas condensation is a non-invasive method of collecting exhaled gas samples. Exhaled air is directed through a unique one-way valve into a cooled collection chamber where vapors, aerosols, and moisture in the breath condense along the protected RTube walls. The one-way valve is then used as a plunger to collect the droplets that adhere to the inner wall and hold the sample near the top of the RTube. For adults with normal tidal breathing efforts, the typical condensate production is 100-. All patients and volunteers were asked to exhale for at least 2 minutes under normal breathing conditions, which provided sufficient fluid volume for most laboratory tests.
In some preferred embodiments, further comprising performing sample extraction after collecting the exhaled breath condensate, preferably comprising: the incubation time and temperature were set at 15 minutes and 50 ℃ respectively, the stirrer speed was fixed at 500 rpm for 5-10 minutes, then at 100 rpm for 5-10 minutes for sample extraction, and the desorption time was 10 minutes. More preferably, the incubation time and temperature are set at 15 minutes and 50 ℃ respectively, the stirrer speed is fixed at 500 rpm for 10 minutes, then at 100 rpm for 10 minutes for sample extraction, and the sample extraction time is 35 minutes.
In some preferred embodiments, the gas chromatography conditions are: the sample introduction amount is 10 mul, helium is used as carrier gas, and the front inlet purge flow is 3ml min-1The flow rate of gas passing through the column was 1 ml/min-1(ii) a The initial temperature was maintained at 50 ℃ for 1 minute, then ramped up to 150 ℃ at a rate of 4 ℃/minute, then ramped up to 210 ℃ at a rate of 8 ℃/minute, and maintained at 210 ℃ for 5.5 minutes.
In some preferred embodiments, the mass spectrometry conditions are: the injection and transfer line and ion source temperatures were 240 deg.C, 280 deg.C and 220 deg.C, respectively; the energy in the electron collision mode is 70 eV; mass spectral data were acquired at a rate of 50 ± 5 spectra per second in a full scan mode with m/z ranging from 33 to 500, after a solvent delay of 0.3 minutes.
In a third aspect of the present application, there is provided use of a marker combination for prognosis evaluation of liver cirrhosis, the marker combination comprising (+/-) -3,5, 5-trimethyl-1-hexanol, dichloromethane, pyridine, gamma-butyrolactone, citronellal, ethyl lactate, guaiacol, methyl linoleate, oxoglutarate, (+/-) -erythro-isoleucine, 3-phenylpropanal and an N-arylamine amine, preferably the N-arylamine amine is acetanilide, for the preparation of a prognosis evaluation kit for liver cirrhosis.
In some preferred embodiments, the sample for prognostic evaluation is exhaled breath condensate.
Examples
1. Study participants
The research conforms to the ethical principle of the declaration of Helsinki of the world medical society, the experimental scheme is approved by the ethical committee of the fifth medical center of the general Hospital of the Chinese liberation force, and the national laboratory is registered in the Chinese clinical test registration center with the registration number of ChiCTR-ROC-17010345. From 4 months in 2017 to 10 months in 2017, 96 participants including 66 liver cirrhosis (DC) patients (33 male and 33 female) and 30 healthy volunteers (13 male and 17 female) were recruited from the fifth medical center of the central national release military hospital, and all participants signed informed consent. Healthy volunteers (group C) were confirmed to be healthy by normal routine examination within half a year. All diagnoses of 66 patients with cirrhosis, including 24 patients with hepatitis b cirrhosis (HLC), 22 patients with alcohol-related cirrhosis (ALC) and 20 patients with Primary Biliary Cirrhosis (PBC), were confirmed by hospital doctors according to the respective guidelines.
2. Exhaled breath condensate sample collection
Both volunteers and patients were asked to fast for at least two hours, stop taking any medication for 8 hours, and abstain from alcohol and/or quit smoking within 24 hours prior to sampling. All participants rinsed with water twice before collecting samples of Exhaled Breath Condensate (EBC). EBC samples were collected in a RTube exhaled breath condensate collector (respiratory research, RRI) with a cooling jacket and then frozen in a laboratory refrigerator at-80 ℃.
3. Analytical method
All target analytes in the samples were collected by separation on a DB-5MS column (30 m x 250 m x 0.25 m, usa) using an agilent 7890 gas chromatography system (agilent technologies, santa clara, ca, usa). Exhaled breath condensate samples were incubated at 50 ℃ for 15 minutes. However, the device is not suitable for use in a kitchenThen 10 minutes in a stirrer with a speed fixed at 500 rpm and then 10 minutes in a stirrer with 100 rpm for sample extraction. The sample extraction time was 35 minutes and the desorption time was 10 minutes. Sample injection amount is 10 mul, helium is used as carrier gas, and front inlet purge flow is 3ml min-1. The gas flow rate through the column was 1ml min-1. The initial temperature was maintained at 50 ℃ for 1 minute, then ramped up to 150 ℃ at a rate of 4 ℃/minute, then ramped up to 210 ℃ at a rate of 8 ℃/minute, and maintained at 210 ℃ for 5.5 minutes. Injection, transfer line and ion source temperatures were set at 240, 280 and 220 c, respectively. The energy in electron impact mode is 70 eV. Mass spectral data were acquired at a rate of 50 ± 5 spectra per second in a full scan mode with m/z ranging from 33 to 500, after a solvent delay of 0.3 minutes. A total of 10. mu.l of each sample was mixed as a Quality Control (QC) sample, and one QC sample was analyzed after each 10 test samples.
4. Statistical analysis
Statistical analysis was performed using IBM SPSS 22.0 software. Continuous variation of subject biological characteristics is expressed as mean standard deviation and clinical data is expressed as median [ 25, 75 percentile]. Kruskal-Wallis test or analysis of variance (ANOVA) was used to assess the difference in continuous variables, while Pearson's χ2The test is used for the classification factor. Spearman or Pearson correlation coefficients are used to assess the correlation between exhaled compounds and clinical characteristics of patients. Difference is in p<Significant at 0.05, p<The height is significant at 0.01. Metabolomics analysis and metabolite identification mass spectral data were analyzed using Chroma TOF software (v4.3x, LECO): and sequentially performing peak extraction, baseline correction, deconvolution, peak integration and peak alignment. Detection rate in removing QC samples is less than 50% or relative standard deviation>30% of the peaks. MetabioAnalyst on-line database for analyzing data and screening for display Fold Changes (FC)>2 (p) of a different compound<0.05). The normalized data were subjected to principal component analysis and OPLS-DA analysis using SIMCA-P13.0 software. The screening results were used as potential biomarkers and identified by comparison to known compounds in the National Institute of Standards and Technology (NIST) database.
5. Results
5.1 clinical characteristics of the subjects
96 subjects were recruited in the study, including 30 healthy control subjects, 24 patients with HLC, 22 patients with ALC and 20 patients with PBC. Clinical information and biochemical parameters of different subgroups, such as gender, age and complications including ascites, Hepatic Encephalopathy (HE) and upper gastrointestinal bleeding (UGIB) and Child-Pugh score, are shown in table 1. Recent research reports show that PBC male and female morbidity is 1: 10. The incidence of the disease of the male and the female in the research is 1:9.5, and the clinical characteristics of PBC are met. The gender used to assess the prognosis of cirrhosis was not significantly different in all three groups, and the baseline data were consistent in this study.
TABLE 1 clinical characteristics of patients with cirrhosis
Figure BDA0003494538390000071
Biological characteristics are expressed as mean ± sd, and clinical data as median [ P [ ]25,P75]。
5.2 multivariate analysis of exhaled breath volatile organic Compounds
Overall, the voc data obtained from 96 exhaled breath condensates were analyzed by multivariate analysis after pretreatment. The quality control panel presented a good, uniform trend based on the generated principal component analysis model and showed satisfactory stability of the process (as shown in figure 1). In addition, OPLS-DA was performed to show trends in the HLC, ALC, PBC and C groups, which highlighted significant metabolic differences (fig. 1). In addition, OPLS-DA and VIP-p (corr) profiles compared between healthy control and three DC groups further demonstrate that volatile organic compounds can distinguish between healthy individuals and DC patients of different etiology (fig. 2, a-F). Compounds with projection importance variables >1, | p (corr) | ≧ 0.5 and p <0.05 are defined as significant markers.
5.3 prognostic analysis of VOC in exhaled gas from patients with decompensated liver cirrhosis
In this study, we obtained different metabolites for the HLC, ALC and PBC groups by OPLS-DA analysis. And (3) adopting ROC curve analysis to predict prognosis, and screening 12 exhaled breath metabolites with highest diagnostic value in the HLC group, the ALC group and the PBC group. As shown in Table 2, (. + -.) -3,5, 5-trimethyl-1-hexanol, dichloromethane, pyridine, gamma-butyrolactone, citronellal, ethyl lactate, guaiacol, methyl linoleate, oxoglutarate, (. + -.) -erythro-isoleucine, 3-phenylpropanal and N-acetylphenylamine were screened to predict worse prognosis in DC patients.
Top 12 exhaled breath metabolites of the ROC curve in table 23 set
Figure BDA0003494538390000081
3.4 prognosis analysis of clinical indicators of decompensated liver cirrhosis
66 DC patients were divided into two groups according to prognosis. Patients with death and liver transplantation were classified as having a poorer prognosis in a 2 year follow-up. 45 patients were divided into good prognosis groups (GP groups) and 21 patients were divided into poor prognosis groups (BP groups). Clinical information and biochemical parameters for the different subgroups are shown in table 3. As for the EBC-based metabolite profiles of DC patients, we observed significant changes in clinical markers of ALB, INR, PT, HDL, TBIL, DBIL, ALT, CHE, TBA, PTA, WBC, RBC between the GP and BP groups. Spearman correlation analysis and Logistic regression analysis were then performed to determine indices related to prognosis. The results show that INR and TBIL can effectively predict the prognosis of DC patients (tables 4 and 5).
TABLE 3 clinical information and Biochemical parameters of DC patients
Figure BDA0003494538390000091
Figure BDA0003494538390000101
TABLE 4 clinical indicators and correlation analysis of prognosis of patients with DCs
Figure BDA0003494538390000102
Figure BDA0003494538390000111
TABLE 5 clinical indicators and correlation analysis of prognosis of patients with DCs
Figure BDA0003494538390000112
5.5 comparison of clinical diagnostic value of exhaled breath metabolites with clinical biochemical parameters
To validate the diagnostic value of exhaled breath metabolites, ROC curves were constructed to assess the potential utility of EBC metabolite biomarkers as non-invasive biomarkers for predicting adverse outcome of DC patients. The results in fig. 3(a) and table 6 show the diagnosis of exhaled metabolites ([ AUC ] ═ 0.891, 95% [ CI ] ═ 0.815-0.967), MELD score ([ AUC ] ═ 0.925, 95% [ CI ] ═ 0.851-0.999), Child-pugh score ([ AUC ] ═ 0.781, 95% [ CI ] ═ 0.664-0.898), TBIL ([ AUC ] ═ 0.898). At the same time, i observed that the model established with 12 compounds was superior to the other 8 models in assessing the prognosis of DC patients with poorer prognosis compared to survivors without liver transplantation. This ROC analysis indicated that EBC metabolite biomarkers could be used to effectively predict a worse prognosis for DC patients without liver transplant survival with AUC values of 0.906 (95% [ CI ] ═ 0.806-1.000) (fig. 3 (B)). Furthermore, the Child-pugh score ([ AUC ] 0.822, 95% [ CI ] 0.679-0.966), TBIL ([ AUC ] 0.766, 95% [ CI ] 0.595-0.936), INR ([ AUC ] 0.639, 95% [ CI ]), was compared to the MELD score ([ AUC ] 0.831, 95% [ CI ] 0.671-0.991).
TABLE 6 comparison of ROC curves for 66 patients with DC
Figure BDA0003494538390000113
Figure BDA0003494538390000121
6. Discussion of the related Art
In this study, the inventors have analyzed the metabolite profile of EBC to distinguish between DC patients with poorer prognosis and surviving patients without liver transplantation. The EBC-based metabonomics method provides a novel noninvasive method for evaluating the prognosis of the DC patient, and is beneficial to further research on clinical management of the DC patient. The ROC curve analysis of the study compared the respiratory metabolites of DC disease caused by different causes. 12 expiratory metabolites were selected as different metabolites to differentiate among DC patients with poorer prognosis (table 2). The results indicate that the best predictors of prognosis for patients with DC disease include (+ -) -3,5, 5-trimethyl-1-hexanol, dichloromethane, pyridine, gamma-butyrolactone, citronellal, ethyl lactate, guaiacol, methyl linoleate, oxoglutarate, (+ -) -erythro isoleucine, 3-phenylpropanal and acetanilide.
To accurately predict the poor prognosis of DCs, a prognostic model was established with AUC values of 0.891 (table 6) from 12 candidate metabolite biomarkers of EBC. Prognostic models based on EBC metabolite features have high sensitivity (77.8%) and specificity (85.7%), which can strongly distinguish among DC patients with poor prognosis. This ability of the EBC-associated metabolite signaling model has been shown to be superior to common clinical predictive models, including the serum markers TBIL (AUC 0.878), INR (AUC 0.769), and Child-pugh score (AUC 0.871) (fig. 3 (a)).
Meanwhile, the prognostic model of EBC 12 candidate metabolite biomarkers can strongly distinguish DC patients with poor prognosis from surviving patients without liver transplantation, and AUC value is 0.906. This diagnostic value of the EBC-associated metabolite signaling model has been shown to be superior to common clinical predictive models, including the serum markers TBIL (AUC 0.766), INR (AUC 0.639), Child-pugh score (AUC 0.822), and MELD score (AUC 0.831) (fig. 3 (B)).
The present application describes embodiments, but the description is illustrative rather than limiting and it will be apparent to those of ordinary skill in the art that many more embodiments and implementations are possible within the scope of the embodiments described herein. Although many possible combinations of features are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are possible. Any feature or element of any embodiment may be used in combination with, or instead of, any other feature or element in any other embodiment, unless expressly limited otherwise.
The present application includes and contemplates combinations of features and elements known to those of ordinary skill in the art. The embodiments, features and elements that have been disclosed in this application may also be combined with any conventional features or elements to form unique inventive aspects as defined by the claims. Any feature or element of any embodiment may also be combined with features or elements from other inventive aspects to form yet another unique inventive aspect, as defined by the claims. Thus, it should be understood that any of the features shown and/or discussed in this application may be implemented alone or in any suitable combination. Accordingly, the embodiments are not to be restricted except in light of the attached claims and their equivalents. Further, various modifications and changes may be made within the scope of the appended claims.

Claims (7)

1. A marker combination for prognosis evaluation of cirrhosis of the liver comprising: (±) -3,5, 5-trimethyl-1-hexanol, dichloromethane, pyridine, γ -butyrolactone, citronellal, ethyl lactate, guaiacol, methyl linoleate, oxoglutarate, (±) -erythro-isoleucine, 3-phenylpropanal and N-acetyl aromatic amine, preferably, the N-acetyl aromatic amine is N-acetanilide.
2. A method of determining a marker combination for the prognostic assessment of cirrhosis of the liver, said marker combination comprising (±) -3,5, 5-trimethyl-1-hexanol, dichloromethane, pyridine, γ -butyrolactone, citronellal, ethyl lactate, guaiacol, methyl linoleate, oxoglutarate, (±) -erythro-isoleucine, 3-phenylpropanal and an N-acetoacetarylamine, preferably said N-acetoacetylamide is N-acetanilide, said method comprising the steps of:
step 1. collecting Exhaled Breath Condensate (EBC);
step 2, analyzing the exhaled air condensate by using a two-dimensional gas chromatography-tandem time-of-flight mass spectrum, and collecting mass spectrum data; and
and 3, analyzing the mass spectrum data by using principal component analysis and orthogonal partial least square discriminant analysis to obtain a marker combination for prognosis evaluation of cirrhosis.
3. The method of claim 2, wherein the gas chromatography conditions are: the sample introduction amount is 10 mul, helium is used as carrier gas, and the front inlet purge flow is 3ml min-1The flow rate of gas passing through the column was 1 ml/min-1The initial temperature was maintained at 50 ℃ for 1 minute, then ramped up to 150 ℃ at a rate of 4 ℃/minute, then ramped up to 210 ℃ at a rate of 8 ℃/minute, and maintained at 210 ℃ for 5.5 minutes.
4. The method of claim 3, wherein the mass spectrometry conditions are: the injection and transfer line and ion source temperatures were 240 ℃, 280 ℃ and 220 ℃, respectively; the energy in the electron collision mode is 70 eV; mass spectral data were acquired at a rate of 50 ± 5 spectra per second in a full scan mode with m/z ranging from 33 to 500, after a solvent delay of 0.3 minutes.
5. The method of claim 4, further comprising performing sample extraction after collecting exhaled breath condensate, comprising: the culture time and temperature are respectively set to 15 minutes and 50 ℃, the speed of a stirrer is fixed at 500 revolutions per minute for 5-10 minutes, then the stirrer is used for sample extraction after 100 revolutions per minute for 5-10 minutes, and the desorption time is 10 minutes; preferably, the incubation time and temperature are set at 15 minutes and 50 ℃ respectively, the stirrer speed is fixed at 500 rpm for 10 minutes and then used for sample extraction after 100 rpm for 10 minutes.
6. Use of the marker combination for prognosis evaluation of liver cirrhosis according to claim 1 for preparing a kit for prognosis evaluation of liver cirrhosis.
7. Use according to claim 6, wherein the sample for prognosis of cirrhosis is an exhaled breath condensate.
CN202210108028.1A 2022-01-28 2022-01-28 Marker combination for prognosis evaluation of liver cirrhosis Pending CN114527209A (en)

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