MODEL FOR EVALUATING DEGREE OF LIVER FIBROSIS CONSTRUCTED BASED ON BILE ACIDS

Abstract

Provided is a model for evaluating the degree of liver fibrosis constructed based on bile acids. A plurality of bile acids are simultaneously detected by a liquid chromatography-tandem mass spectrometer to further improve the accuracy in combination with other liver indicators; moreover, a multiple regression analysis method is applied to establish a grading diagnosis model for the degree of liver fibrosis caused by a chronic liver disease, which can significantly improve the sensitivity and specificity of the existing non-invasive diagnosis of liver fibrosis. When the model is used to evaluate the degree of liver fibrosis of a patient, the highest AUC is up to 0.9278; the sensitivity is up to 86.79%; and the specificity is up to 89.01%. The detection results are completely consistent with the pathological results of clinical liver biopsy. Therefore, patients need not receive a liver biopsy.

Claims

1. A method for evaluating degree of liver fibrosis, wherein, a degree or a level of liver fibrosis is evaluated by detecting a concentration of a bile acid in a sample.

2. The method of claim 1, wherein, the bile acid is selected from any one or more of GCA, TCA, CDCA, GCDCA, TCDCA, DCA, GDCA, TDCA, LCA, GLCA, TLCA, UDCA, GUDCA, TUDCA and CA.

3. The method of claim 1, wherein, the bile acid is selected from any one or more of UDCA, TUDCA, LCA, GLCA, GCA and TCDCA.

4. The method of claim 3, wherein, the bile acid is selected from any one or more of LCA, TUDCA and GLCA.

5. The method of claim 4, wherein, the bile acid is selected from any one or more of LCA and TUDCA.

6. The method of claim 5, wherein, the bile acid is selected from LCA.

7. The method of claim 6, wherein, a regression equation is constructed to calculate a predicted value of liver fibrosis, thus evaluating the degree of liver fibrosis, and the regression equation is as follows: BAS=43.18×LCA+16.74×TUDCA+13.91×GLCA+5.57×UDCA+0.89×TCDCA+0.77×GCA+0.21×TCA+0.1×CDCA+0×TDCA−0.08×GDCA−0.16×GCDCA−0.43×CA−3.29×GUDCA−4.39×DCA−35.19×TLCA, in which, BAS represents a predicted value of liver fibrosis, and wherein, the predicted value is correlated to the degree of liver fibrosis.

8. The method of claim 7, wherein, a regression equation is constructed to calculate a predicted value of liver fibrosis, thus evaluating the degree of liver fibrosis, and the regression equation is as follows: BASA=61.92×LCA+38.53×TUDCA+4.66×UDCA+2.14×TCA+0.9×GDCA+0.42×DBIL+0.27×RBC+0.26×GCDCA+0.24×CDCA+0.22×GLU+0.19×WBC+0.03×GGT+0.02×ALT+0.02×TBIL+0.01×AST−0.04×IBIL−0.05×TDCA−0.16×CA−0.67×GCA−0.8×GLCA−1.51×TCDCA−3.77×GUDCA−5.96×DCA−16.07×TLCA, in which, BASA represents a predicted value of liver fibrosis.

9. The method of claim 8, wherein, the regression equation has a value of BAS or BASA; when BAS is greater than 0.63, the liver fibrosis is a stage 3-4 liver fibrosis; when BAS is lower than 0.63, the liver fibrosis is a stage 1-2 liver fibrosis; when BASA is greater than 0.63, the liver fibrosis is a stage 3-4 liver fibrosis; and when BASA is lower than 0.63, the liver fibrosis is a stage 1-2 liver fibrosis.

10. A system for evaluating degree of liver fibrosis, comprising a data input/output interface and a data analysis unit, wherein the data input/output interface needs to input a concentration of at least one bile acid, after the concentration is analyzed by the data analysis unit, the data input/output interface outputs an evaluation result of the degree of liver fibrosis.

11. The system of claim 10, wherein, the bile acid comprises any one or more of GCA, TCA, CDCA, GCDCA, TCDCA, DCA, GDCA, TDCA, LCA, GLCA, TLCA, UDCA, GUDCA, TUDCA and CA.

12. The system of claim 11, wherein, the bile acid comprises any one or more of UDCA, TUDCA, LCA, GLCA, GCA and TCDCA.

13. The system of claim 12, wherein, the bile acid comprises any one or more of LCA, TUDCA and GLCA.

14. The system of claim 13, wherein, the bile acid comprises any one or more of LCA and TUDCA.

15. The system of claim 14, wherein, the bile acid comprises LCA.

16. The system of claim 15, wherein, the data analysis unit calculates a predicted value of liver fibrosis based on the regression equation, thus evaluating a degree of liver fibrosis.

17. The system of claim 16, wherein, the regression equation is as follows: BAS=43.18×LCA+16.74×TUDCA+13.91×GLCA+5.57×UDCA+0.89×TCDCA+0.77×GCA+0.21×TCA+0.1×CDCA+0×TDCA−0.08×GDCA−0.16×GCDCA−0.43×CA−3.29×GUDCA−4.39×DCA−35.19×TLCA, in which, BAS represents a predicted value of liver fibrosis.

18. The system of claim 17, wherein, the data input/output interface further needs to input a concentration/concentrations of one or more of DBIL, RBC, GLU, WBC, GGT, ALT, TBIL, AST and IBIL.

19. The system of claim 18, wherein, the regression equation is as follows: BASA=61.92×LCA+38.53×TUDCA+4.66×UDCA+2.14×TCA+0.9×GDCA+0.42×DBIL+0.27×RBC+0.26×GCDCA+0.24×CDCA+0.22×GLU+0.19×WBC+0.03×GGT+0.02×ALT+0.02×TBIL+0.01×AST−0.04×IBIL−0.05×TDCA−0.16×CA−0.67×GCA−0.8×GLCA−1.51×TCDCA−3.77×GUDCA−5.96×DCA−16.07×TLCA, in which, BASA represents a predicted value of liver fibrosis.

20. The system of claim 19, wherein, the regression equation has a value of BAS or BASA; when BAS is greater than 0.63, the liver fibrosis is a stage 3-4 liver fibrosis; when BAS is lower than 0.63, the liver fibrosis is a stage 1-2 liver fibrosis; when BASA is greater than 0.63, the liver fibrosis is a stage 3-4 liver fibrosis; and when BASA is lower than 0.63, the liver fibrosis is a stage 1-2 liver fibrosis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0084] FIG. 1 shows a detection diagram of a plurality of bile acids simultaneously detected by liquid chromatography-tandem mass spectrometry in Example 1;

[0085] FIG. 2 shows a LASSO regression analysis diagram in Example 1;

[0086] FIG. 3 shows a PLS-DA analysis diagram of an evaluation model 1 (BAS) for degree of liver fibrosis in Example 1;

[0087] FIG. 4 shows a PLS-DA analysis diagram of an evaluation model 2 (BASA) for degree of liver fibrosis in Example 1;

[0088] FIG. 5 shows a comparison diagram of the evaluation model 1 (BAS) for degree of liver fibrosis, the evaluation model 2 (BASA) for degree of liver fibrosis in Example 1, as well as ROC curves of the existing APRI and FIB.4;

[0089] FIG. 6 shows a ROC curve graph for the evaluation of clinical application of an evaluation model 1 (BAS) for degree of liver fibrosis in Example 2;

[0090] FIG. 7 shows a ROC curve graph for the evaluation of clinical application of an evaluation model 2 (BASA) for degree of liver fibrosis in Example 2.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0091] The present invention will be further described in detail with reference to the accompanying drawings and examples hereafter. It should be indicated that the following examples are aimed at facilitating the understanding to the present invention, but not constructed as limiting the present invention. The reagents used in the examples are products known in the art and are commercially available.

Example 1 Construction of an Evaluation Model for Degree of Liver Fibrosis

[0092] 1. Data of Cases

[0093] 152 cases (case group) of chronic hepatitis B patients receiving liver biopsy from Zhejiang Provincial People's Hospital from 2017 to 2019 were brought into this example. All the patients had no clinical manifestation of compensatory liver diseases and laboratory basis, nephrotic syndrome complicating pregnancy, and hematological system diseases.

[0094] 2. Establishment of a Model

[0095] 1) Liver biopsy and liver fibrosis staging of the cases:

[0096] All the selected patients received liver biopsy under abdomen ultrasound guidance. Liver biopsy was performed by percutaneous liver puncture for 1 s; a specimen was immediately put to a plastic specimen tube and frozen after being collected, then sent for examination. Liver tissue was put to a plastic embedding box and immobilized with neutral formalin, and dehydrated by gradient ethanol, subjected to transparency treatment by xylene, immersed and embedded by paraffin wax, then sliced, and stained by hematoxylin-eosin and subjected to reticular fiber staining. The quality evaluation of the liver tissue specimen and pathological diagnosis of the liver tissue were completed by an experienced pathologist independently. Liver histopathologic diagnosis is reference to the consensus on the evaluation of diagnosis and therapeutic effect of liver fibrosis in 2002; and the liver histopathologic fibrosis includes five stages, namely, S0, S1, S2, S3 and S4. The patients were divided into two groups according to the grading results, namely, the stage 1-2 (S0-S2) fibrosis and the stage 3-4 (S3-S4) fibrosis. There were 98 cases in the stage 1-2 fibrosis group, and 53 cases in the stage 3-4 fibrosis group.

[0097] 2) Biochemical Detection of Blood, Liver Functions, 15 Bile Acids, and PLT:

[0098] 4 ml whole blood was collected after fasting for 8 h, and centrifuged for 10 mins at 3500 r/min to collect blood serum; ALB, ALT, AST, GGT, ALP, TBIL, DBIL, IBIL, GLU, RBC, WBC and HGB were analyzed by a Roche cobas c702 fully automatic biochemical analyzer and the matching reagents; and APRI and FIB-4 were calculated; a plurality of bile acids were simultaneously analyzed by an AB SCIEX Triple Quad4500MD liquid chromatography-tandem mass spectrometer and the matching reagents; and LSM was collected; PLT was analyzed by a SYSME XE-2100 automatic blood cell analyzer and the matching reagents; three levels of quality control materials were detected every batch for indoor quality control; the (Out Of Control) rules are subjected to 13S, 22S and R4S.

[0099] 3) Statistical Method:

[0100] R language software was used to process the data. Based on the fibrosis grouping of the patients, a multiple analysis method (PCA, ANOVA and PLS-DA) was used to judge the patient's degree of liver fibrosis, and LASSO regression analysis was performed on the all detection results to establish a same grading mathematical model for liver fibrosis, and inclusion and exclusion criteria of the independent variables were respectively P<0.05 and P>0.10; and the regression model efficiency was evaluated by a calibration curve and a ROC curve method.

[0101] 3. Result Analysis:

[0102] 1) The detection method provided in CN2020101870483 was taken as a method for simultaneously detecting 15 bile acids by liquid chromatography-tandem mass spectrometry. The detection diagram is shown in FIG. 1

[0103] 2) Based on LASSO, it is prompted that 15 bile acids have a distinct correlation to the occurrence of obvious liver fibrosis; and ALB, ALT, AST, GGT, ALP, TBIL, DBIL, IBIL, GLU, RBC, WBC, HGB and PLT also have a correlation to the occurrence of obvious liver fibrosis. The analysis result is shown in Table 1.

TABLE-US-00001 TABLE 1 Comparison of the detection results between the stage 1-2 (S0-S2) liver fibrosis and the stage 3-4 (S3-S4) liver fibrosis 95% CI Indicator β OR p-value Lower Upper ALB −0.110 0.896 0.001 1.166 4.626 ALT −0.017 0.983 0.566 −12.998 7.142 AST 0.032 1.032 0.060 −30.683 0.675 GGT 0.020 1.020 0.018 −61.066 −6.083 ALP 0.002 1.002 0.005 −37.182 −7.067 TBIL −0.141 0.868 0.003 −8.823 −1.852 DBIL 0.487 1.627 0.001 −3.667 −1.004 IBIL 0.137 1.147 0.010 −5.760 −0.807 GLU 0.577 1.781 0.001 −0.943 −0.239 RBC 0.137 1.146 0.196 −0.130 0.619 WBC 0.190 1.209 0.641 −0.542 0.875 HGB −0.013 0.987 0.076 −0.940 18.552 PLT −0.010 0.990 0.000 27.841 68.440 UDCA 5.568 261.887 0.135 −0.391 0.054 GUDCA −3.289 0.037 0.126 −0.866 0.109 TUDCA 16.741 1.864 × 10.sup.7  0.025 −0.168 −0.012 CA −0.432 0.649 0.870 −0.466 0.394 GCA 0.767 2.153 0.002 −2.262 −0.514 TCA 0.207 1.230 0.002 −1.081 −0.259 CDCA 0.103 1.108 0.333 −0.723 0.247 GCDCA −0.155 0.856 0.014 −6.194 −0.723 TCDCA 0.894 2.445 0.006 −2.508 −0.452 DCA −4.394 0.012 0.047 0.003 0.398 GDCA −0.075 0.927 0.348 −0.498 0.177 TDCA 0.003 1.003 0.434 −1.859 0.806 LCA 43.177 5.643 × 10.sup.16 0.022 −0.050 −0.004 GLCA 13.907 1.0958 × 10.sup.6   0.192 −0.024 0.005 TLCA −35.188 0.000 0.036 −0.005 0.000

[0104] The higher the OR value is, the greater the impact of the people with stage 3-4 (S3-S4) on the indicator relative to the people with stage 1-2 (S0-S2), and the more obvious the indicator exposure is.

[0105] It can be seen from Table 1 that the bile acids, UDCA, TUDCA, LCA, GLCA, GCA and TCDCA have a particularly high correlation to the occurrence of obvious liver fibrosis; of which, LCA has the highest correlation with an OR value being up to 5.643×10.sup.16 and a very high weight; the second one is TUDCA with an OR value being up to 1.864×10.sup.7; the third one is GLCA with an OR value being up to 1.0958×10.sup.6; then followed by UDCA, TCDCA, GCA and TCA; weights of the above indicators are greater than other liver index values. By comparison, the correlation of the 15 bile acids to the occurrence of obvious liver fibrosis is apparently higher than other liver index values. As can be seen, it is of obvious advantage to establish an evaluation model for the degree of liver fibrosis by bile acids.

[0106] The degree of the concentration of bile acid to the degree of liver fibrosis can by represented by an OR value in Table 1. Certainly, the correlation may be distinguished by a β value, a p-value, or the like in Table 1; and OR value is the most visualized and distinct. For example, LCA has an OR value being up to 5.643×10.sup.16 which is 10.sup.16 folds of other bile acids; with such a high correlation, it can be understood that LCA concentration can be detected independently to judge the degree of liver fibrosis; for another example, TUDCA has an OR value being up to 1.864×10.sup.7 which is 10.sup.7 folds of other bile acids; GLCA has an OR value being up to 1.0958×10.sup.6, followed by UDCA, TCDCA, GCA, and TCA. The single detection of the concentration of a bile acid with a high correlation can be completely used to judge the degree of liver fibrosis, thus distinguishing the people with stage 1-2 liver fibrosis (S0-S2) and the people with stage 3-4 liver fibrosis (S3-S4).

[0107] Multi-factor regression analysis was performed on the correlation of the measured concentrations of the patient's 15 bile acids to the occurrence of obvious liver fibrosis to establish an evaluation model 1 (BAS) for the degree of liver fibrosis: BAS=43.18×LCA+16.74×TUDCA+13.91×GLCA+5.57×UDCA+0.89×TCDCA+0.77×GCA+0.21×TCA+0.1×CDCA+0×TDCA−0.08×GDCA−0.16×GCDCA−0.43×CA−3.29×GUDCA−4.39×DCA−35.19×TLCA.

[0108] Moreover, to further improve the detection sensitivity and specificity, DBIL, RBC, GLU, WBC, GGT, ALT, TBIL, AST and IBIL were picked according to the degree of correlation to the occurrence of obvious liver fibrosis and subjected to multi-factor regression analysis together with the measured concentrations of the patient's 15 bile acids, thus establishing an evaluation model 2 (BASA) for the degree of liver fibrosis: BASA=61.92×LCA+38.53×TUDCA+4.66×UDCA+2.14×TCA+0.9×GDCA+0.42×DBIL+0.27×RBC+0.26×GCDCA+0.24×CDCA+0.22×GLU+0.19×WBC+0.03×GGT+0.02×ALT+0.02×TBIL+0.01×AST−0.04×IBIL−0.05×TDCA−0.16×CA−0.67×GCA−0.8×GLCA−1.51×TCDCA−3.77×GUDCA−5.96×DCA−16.07×TLCA.

[0109] 2) PLS-DA analysis diagram of the evaluation model 1 (BAS) for the degree of liver fibrosis is shown in FIG. 3. As can be seen, the people with stage 1-2 (S0-S2) liver fibrosis and the people with stage 3-4 (S3-S4) liver fibrosis may be completely separated by the model, namely, the concentrations of the 15 bile acids.

[0110] 2) PLS-DA analysis diagram of the evaluation model 2 (BASA) for the degree of liver fibrosis is shown in FIG. 4. As can be seen, the people with stage 1-2 (S0-S2) liver fibrosis and the people with stage 3-4 (S3-S4) liver fibrosis may be completely separated by the model; and the separation effect is better than that of the evaluation model 1 for the degree of liver fibrosis.

[0111] 3) ROC curves of the new and previous models were compared by means of the evaluation model 1 (BAS) for degree of liver fibrosis, the evaluation model 2 (BASA) for degree of liver fibrosis newly established herein, as well as APRI and FIB.4. The results are shown in FIG. 5, Tables 2 and 3.

TABLE-US-00002 TABLE 2 Comparison results of the ROC curves among BAS, BASA, FIB.4 and APRI Indicator BAS BASA FIB-4 APRI Areas Under The 0.834 0.928 0.802 0.757 ROC Curve (AUC) Standard error a 0.0349 0.0231 0.0403 0.0419 95% confidence 0.763-0.891 0.873-0.964 0.727-0.865 0.679-0.825 interval b Z statistics 9.578 18.506 7.498 6.146 Significance level <0.0001 <0.0001 <0.0001 <0.0001 P (area = 0.5) Youden index 0.5239 0.7580 0.5127 0.4038 Relevant standard <0.63 <0.63 >1.72 >0.70 Sensibility 75.47 86.79 66.04 60.38 Specificity 76.92 89.01 85.23 80.00

TABLE-US-00003 TABLE 3 Comparison result of sensitivity and specificity among BAS, BASA, FIB-4 and APRI Indicator Standard Sensibility 95% CI Specificity 95% CI +LR −LR BAS ≤0.63 75.47 61.7-86.2 76.92 66.9-85.1 3.27 0.32 BASA ≤0.63 86.79 74.7-94.5 89.01 80.7-94.6 7.90 0.15 FIB-4 >1.72 66.04 51.7-78.5 85.23 76.1-91.9 4.47 0.40 APRI >0.70 60.38 46.0-73.5 80.00 70.2-87.7 3.02 0.50

[0112] It can be seen from Tables 2-3 and FIG. 5 that the evaluation model 1 (BAS) for the degree of liver fibrosis has an AUC value of 0.834, a sensitivity of 75.47% and a specificity of 76.92; the evaluation model 2 (BASA) for the degree of liver fibrosis has an AUC value of 0.928, a sensitivity of 86.79% and a specificity of 89.01%; the above results are much higher than the detection efficiency of the existing model APRI (AUC value: 0.757), and FIB.4 (AUC value: 0.802).

[0113] It can be further seen from Tables 2-3 that according to the calculation results of the BAS and BASA of the people with stage 1-2 (S0-S2) liver fibrosis and the people with stage 3-4 (S3-S4) liver fibrosis, the standard of BAS and BASA is ≤0.63; when BAS is greater than 0.63, the liver fibrosis is a stage 3-4 liver fibrosis; when BAS is lower than 0.63, the liver fibrosis is a stage 1-2 liver fibrosis; when BASA is greater than 0.63, the liver fibrosis is a stage 3-4 liver fibrosis; and when BASA is lower than 0.63, the liver fibrosis is a stage 1-2 liver fibrosis.

Example 2 Clinical Application of BAS and BASA of the Evaluation Model for the Degree of Liver Fibrosis

[0114] 32 cases of chronic hepatitis B patients from Zhejiang Provincial People's Hospital in October 2021 were selected in this example, and divided into two groups, 16 cases in each group. The degree of liver fibrosis was respectively evaluated by the evaluation model 1 (BAS) for the degree of liver fibrosis and the evaluation model 2 (BASA) for the degree of liver fibrosis provided in Example 1. The evaluation results were compared with the final results of the liver biopsy, and AUC values of the BAS and BASA were verified and calculated. The evaluation results of the degree of liver fibrosis are shown in Tables 1-2.

TABLE-US-00004 TABLE 1 Comparison of BAS evaluation results Specimen Liver biopsy result BAS evaluation result Specimen 1 S0-S2 S0-S2 Specimen 2 S0-S2 S0-S2 Specimen 3 S0-S2 S0-S2 Specimen 4 S0-S2 S0-S2 Specimen 5 S0-S2 S0-S2 Specimen 6 S0-S2 S0-S2 Specimen 7 S0-S2 S0-S2 Specimen 8 S0-S2 S0-S2 Specimen 9 S0-S2 S0-S2 Specimen 10 S0-S2 S0-S2 Specimen 11 S0-S2 S0-S2 Specimen 12 S0-S2 S0-S2 Specimen 13 S0-S2 S0-S2 Specimen 14 S3-S4 S3-S4 Specimen 15 S0-S2 S3-S4 Specimen 16 S0-S2 S3-S4

TABLE-US-00005 TABLE 2 Comparison of BASA evaluation results Specimen Liver biopsy result BAS evaluation result Specimen 1 S0-S2 S0-S2 Specimen 2 S0-S2 S0-S2 Specimen 3 S0-S2 S0-S2 Specimen 4 S0-S2 S0-S2 Specimen 5 S0-S2 S0-S2 Specimen 6 S0-S2 S0-S2 Specimen 7 S0-S2 S0-S2 Specimen 8 S0-S2 S0-S2 Specimen 9 S0-S2 S0-S2 Specimen 10 S0-S2 S0-S2 Specimen 11 S0-S2 S0-S2 Specimen 12 S0-S2 S0-S2 Specimen 13 S0-S2 S0-S2 Specimen 14 S0-S2 S0-S2 Specimen 15 S0-S2 S0-S2 Specimen 16 S3-S4 S3-S4

[0115] It can be seen from Table 1 that when BAS is used for evaluation, the evaluation results of the degree of fibrosis of the 15th and the 16th specimens in the 16 specimens are slightly higher; the evaluation results of other specimens are consistent with the results of the liver biopsy; the accuracy rate is 87.5% and the verification result of the AUC value is up to 0.924 (FIG. 6).

[0116] It can be seen from Table 2 that when BASA is used for evaluation, the evaluation results of the degree of fibrosis of the 16 specimens are completely consistent with the results of the liver biopsy; the accuracy rate is 100% and the verification result of the AUC value is up to 1.000 (FIG. 7).

[0117] The present invention is disclosed above, but the present invention is not limited thereto. Any person skilled in the art can make various alterations and modifications within the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be subjected to the scope defined in the claims.