In vitro method for the diagnosis and prognosis of alcohol-related liver disease

Abstract

The present invention discloses an in vitro method for the detection or diagnosis of alcohol-related liver disease using biomarkers. The method is based on measuring bile acids (BAs) contained in extracellular vesicles (EVs).

Description

[0001] IN VITRO METHOD FOR THE DIAGNOSIS AND PROGNOSIS OF LIVER DISEASE

[0002] ASSOCIATED WITH ALCOHOL

[0003] DESCRIPTIVE MEMORY

[0004] Technical Field

[0005] The present invention falls within the field of biomedicine and gastroenterology. Specifically, it focuses on in vitro analysis using biomarkers present in blood samples for the diagnosis and monitoring of alcohol-associated liver disease.

[0006] BACKGROUND OF THE INVENTION

[0007] Excessive alcohol consumption is a serious and silent problem that causes 3.3 million deaths annually worldwide.

[0008] Alcohol use disorder can increase the risk of many health problems, including bleeding in the digestive tract, malnutrition, neurological and brain damage, dementia and memory loss, heart damage, high blood pressure, cancer of the mouth, throat, esophagus, stomach, liver, colon, or breast, inflammation of the pancreas (pancreatitis), liver disease, including cirrhosis, and others.

[0009] Alcohol-induced liver disease (ALD) is damage to the liver and its function that occurs after years of excessive alcohol consumption. Environmental factors also play a role, and some people may have an inherited risk for the disease. ALD includes a wide range of stages, from simple fatty liver disease, alcohol-associated steatohepatitis with or without fibrosis, cirrhosis, and hepatocellular carcinoma (HCC).

[0010] Alcohol-associated liver disease does not occur in all people who consume alcohol, although the risk of developing the disease increases depending on the length of time the person has been drinking and the amount of alcohol consumed. Alternatively, the patient may not experience symptoms or may develop slowly. During medical care for the diagnosis, management, and prognosis of ALD, patients undergo a physical examination to look for an enlarged liver or spleen or a swollen abdomen, resulting from excess fluid, in addition to evaluating other clinical parameters. Blood tests, liver function tests, coagulation studies, and a liver biopsy are also performed.

[0011] ALD has limited therapeutic options. Biopsies carry high risks (which increase in severely ill patients), low availability, and high costs (associated with the involvement of medical personnel to obtain and analyze the biopsy and the requirement to perform it in a specialized procedure unit). There are no other specific predictive biomarkers for diagnosing or managing the disease.

[0012] State of the Art

[0013] To date, there are some approaches to problems or techniques close to those addressed in the present invention, however, they would not be able to predict liver disease in alcohol consumers.

[0014] In document WO2021183879, from one of the inventors of the present invention, methods are disclosed for detecting liver disease, such as alcohol-associated liver disease (ALD) or alcohol-associated hepatitis (AH), which are determined by the elevated concentration of extracellular vesicles (EVs) or cargoes within those EVs. This document establishes that in HA, circulating EVs can be exhibited at a concentration greater than 1.5 x 1011 EVs per milliliter (EVs / mL) of plasma, which can be considered a biomarker of high mortality risk.This paper teaches that, compared to healthy controls and heavy drinkers, humans with AH may exhibit the presence of enriched cargoes in circulating EVs, specifically increased sphingolipids, such as, but not limited to: sphingosine (SPH), sphinganine (SPA), sphingosine 1-phosphate (SIP), C14:0 ceramides (C14-cer), C16:0 ceramides (C16-cer), C18:0 ceramides (C18-cer), C20:0 ceramides (C20-cer), C22:0 ceramides (C22-cer), C24:0 ceramides (C24-cer), and / or C24:l ceramides (C24-cer). Although this document suggests that determining an increase in EVs and / or their charges can be used to diagnose HA, this development does not identify or suspect that said EVs may contain bile acids, and therefore does not predict that these can be quantified by any particular analytical technology, that is, it did not focus on the study of concentration and / or composition of bile acids in EVs.

[0015] On the other hand, in the publication by Tejasav S. Sehrawat, et al. (Hepatology, vol. 73, No. 2, 571-584. 2021) -where one of the authors is the inventor of the present invention- it is described that the concentration of circulating EVs can be used in the diagnosis and differentiation of HA from heavy drinkers, decompensated AC and other etiologies of End-stage Liver Disease (ESLD) and also predict 90-day survival. In this article, in addition, an analysis of sphingolipids as cargos in EVs isolated from plasma samples of healthy controls, heavy drinkers, people with ESLD, decompensated AC and HA is performed. The sphingolipids analyzed are: sphingosine, sphinganine, sphingosine 1-phosphate, C14:0 ceramide, C16:0 ceramide, C18:0 ceramide, C18:l ceramide, C20:0 ceramide, C22:0 ceramide, C24:0 ceramide and C24:l ceramide.This publication does not identify or consider evaluating changes in the concentration and / or composition of bile acids in EVs and, although it uses mass spectrometry to measure sphingolipids, it does not use it coupled with liquid chromatography or nanoparticle tracking analysis.

[0016] In the thesis by Francesc Ibáñez Cabanes (PhD Thesis, Universitat Politécnica de Valencia. 2021), using EVs from primary astrocyte cultures, it was shown that ethanol induces increased EV secretion and alters the levels of certain proteins and microRNAs (miRNAs) associated with neuroinflammation. It is indicated that during the process of exosomal biogenesis, the participation of a family of enzymes, called sphingomyelinases, which would be related to the biogenesis and secretion of EVs, has been demonstrated.According to the results of this document, it is suggested that ethanol promotes an increased release of EVs by activating sphingomyelinase enzymes through MAMs (mitochondria-associated membranes) and that ethanol is able to alter the miRNA profiles related to inflammation present in circulating EVs from young people with acute alcohol intoxication (AEI), suggesting that circulating EVs and their miRNA profiles are potential candidates for biomarkers of neuroinflammation associated with alcohol abuse. However, this publication does not consider or mention the alternative of analyzing bile acids in circulating EVs or determining the circulating concentration of EVs.

[0017] In the scientific article by Katharina Brandl et al. (Journal of Hepatology. Vol. 69 (2), p396-405; 2018) it is reported that modulation of bile acid metabolism or signaling could represent a promising target for the treatment of alcohol-associated hepatitis in humans, since they observed that, in serum samples, total and conjugated bile acids were significantly elevated in patients with alcohol-associated hepatitis compared to controls and patients with alcohol use disorder. In contrast, there was no significant difference in unconjugated bile acids comparing the three groups in terms of absolute concentrations.Analysis of bile acid composition in different patient populations revealed significantly elevated levels of glycocholic acid (G-CA), taurocholic acid (T-CA), and taurochenodeoxycholic acid (T-CDCA) in patients with alcohol-associated hepatitis compared with alcohol-dependent patients and controls. In contrast, patients with alcohol-associated hepatitis showed significantly lower levels of chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), glycodeoxycholic acid (G-DCA), and cholic acid (CA), as well as altered ratios between the components of total unconjugated bile acids, compared with control patients and patients with alcohol use disorders. In this publication, bile acid levels were measured by mass spectrometry, without considering coupling it to liquid chromatography, and also without considering the use of nanoparticle tracking analysis.Furthermore, this article does not consider measuring EV levels or bile acids present as cargo in EVs.

[0018] However, none of the available tests are specific and sufficient for the in vitro diagnosis of ALD in alcohol users. The available tests are late and have low specificity, as is the case, for example, with bilirubin tests.

[0019] Since chronic liver diseases are reversible if diagnosed early and then properly monitored and treated, the development of an early diagnosis and management methodology specific to alcoholic liver disease is necessary and relevant.

[0020] Early diagnosis methodologies are applied to specific risk groups, such as patients who abuse alcohol, relatives of patients with a genetic liver disease, or people at risk of viral hepatitis infection.

[0021] BRIEF DESCRIPTION OF THE FIGURES

[0022] Figure 1. Bile acids present in serum. (A) Total bile acid composition. (B) Total bile acid concentration (ng / mL). Two-way and one-way ANOVA with Bonferroni posttest. ***P < 0.001. Bile acids (BA), cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA). Figure 2. Bile acids present in extracellular vesicles. (A) Total bile acid composition. (B) Total bile acid concentration (ng / mL). Two-way and one-way ANOVA with Bonferroni posttest. ***P < 0.001.

[0023] Figure 3. Relative proportion of 15 bile acid content, including glycine- and taurine-conjugated forms, in samples from healthy controls, alcohol use disorder, alcohol-associated cirrhosis, and alcohol-associated hepatitis. A. Analysis of total bile acid composition in serum samples. B. Analysis of total bile acid composition in extracellular vesicles.

[0024] Nomenclatures

[0025] • BA = bile acids

[0026] • EVs = Extracellular vesicles

[0027] • CA = Cholic Acid - cholic acid

[0028] • DCA = Deoxycholic Acid - deoxycholic acid

[0029] • CDCA = Chenodeoxycholic Acid - chenodeoxycholic acid

[0030] • LCA = Lithocholic Acid - lithocholic acid

[0031] • UDCA = Ursodeoxycholic acid - ursodeoxycholic acid

[0032] • T-BA = Taurine-conjugated bile acid

[0033] • G-BA = Glycine conjugated bile acid

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] The basis of the invention is based on the selection of a group of biomarkers that allow the in vitro diagnosis of alcohol-associated liver disease, even in early stages, before symptoms exist.

[0036] During liver disease, liver cells release extracellular vesicles (EVs) containing specific markers, where a specific selection of such markers is used in the diagnosis and prognosis of ALD with sufficient sensitivity and specificity for the diagnosis of said disease, to ensure therapeutic efficacy.

[0037] Specific biomarkers correspond to bile acids (BAs), whose presence and specific profile in extracellular vesicles (EVs) are not evident from the state of the art. Thus, the results obtained allowed us to identify a characteristic signature given by certain BAs in EVs. This, combined with the determination of extracellular vesicle concentrations, allows us to relate a clinical condition or pathology to a particular pattern of bile acids (BAs) present in extracellular vesicles (EVs) and their individual charge (absolute concentration of each quantified analyte).

[0038] While the invention is primarily directed at diagnosing alcohol-related liver disease, the described methodology would allow diagnosing any other pathology where bile acids are involved.

[0039] In the method according to the invention, the bile acid content in extracellular vesicles is analyzed. Biomarkers with a pattern that differs from control values ​​in healthy individuals are selected.

[0040] Development of the invention

[0041] A preliminary analysis of serum BA composition revealed a different profile in the groups with ALD (alcohol-associated liver disease, alcohol-associated cirrhosis, and alcohol use disorder) compared to healthy controls. For example, an increase in CDCA in the form conjugated with glycine and taurine, as well as an increase in T-CA and G-CA, consistent with previous reports in the serum literature, was observed.

[0042] However, during the development of the present invention it was possible to confirm the presence of bile acids contained in extracellular vesicles isolated from samples of control patients and those with ALD spectrum and to analyze them effectively.

[0043] In EVs from individuals with alcohol-associated hepatitis, a significant increase in total BA concentration is observed compared to that obtained with respect to controls (healthy individuals).

[0044] It was found that there is a BA profile in EVs in the Alcohol Liver Disease groups that differs from the profile of healthy controls.

[0045] Furthermore, it is possible to determine a BA signature in extracellular vesicles different from that found in serum.

[0046] In the BAs present in EVs, it is possible to observe an increase in UDCA in its form conjugated with Glycine, the levels of which are different between healthy controls and alcohol-associated hepatitis, in terms of the proportion of each BA with respect to the total.

[0047] UDCA is a hydrophilic bile acid that makes up a small fraction of the total bile acid pool in humans (3-4%). ​​Its hydrophilic characteristic is relevant because it exerts a protective effect against cytotoxicity associated with the accumulation of hydrophobic BAs, in addition to mediating effects on apoptosis and mitochondrial dysfunction.

[0048] On the other hand, a decrease in UDCA levels was determined in EVs, so this variable is a predictor of the presence of disease in susceptible individuals.

[0049] The bile acids analyzed correspond to:

[0050] • CA = Cholic Acid - cholic acid

[0051] • DCA = Deoxycholic Acid - deoxycholic acid

[0052] • CDCA = Chenodeoxycholic Acid - chenodeoxycholic acid

[0053] • LCA = Lithocholic Acid - lithocholic acid

[0054] • UDCA = Ursodeoxycholic acid - ursodeoxycholic acid

[0055] In addition to its respective bile acid conjugated with Taurine (T-BA) or Glycine (G-BA).

[0056] The relevant values ​​for each BA correspond to GCA, G-CDCA, G-UDCA, T-CA and T-CDCA, the most relevant variations in EVs in terms of the invention being:

[0057] • Increase: G-CDCA: the ratio with respect to control values ​​is greater than or equal to 2.

[0058] • Decrease: T-UDCA and G-UDCA: the ratio with respect to the control values ​​is greater than or equal to 1.1.

[0059] In addition to analyzing the proportion of each bile acid present in the EVs, various ratios between them were evaluated, preferably using glycine- or taurine-conjugated bile acids, which yielded the most significant results. This allowed for the selection of ratios among bile acids that can identify alcohol-associated pathologies with greater sensitivity and specificity than serum or other liver tests. The relevant ratios, normalized to control values, indicative of alcohol-associated disease are as follows:

[0060] G-CDCA / T-UDCA greater than 1, preferably greater than 5

[0061] G-CDCA / G-UDCA greater than 5, preferably greater than 10 T-CDCA / T-UDCA greater than 10, preferably greater than 20

[0062] In one embodiment of the invention, before performing the diagnostic method described below, the sample is processed to extract bile acids from the EVs according to the following steps:

[0063] - Centrifuge a peripheral blood sample and obtain the clarified Plasma / Serum medium, depending on whether the sample is collected in a tube with anticoagulant or without anticoagulant respectively (both tubes are feasible to use);

[0064] - ultracentrifuge the clarified medium at different speeds to discard material less heavy than the vesicles (e.g. BAs outside the vesicle), and extract the pellet containing the extracellular vesicles, that is, repeating the centrifugation and pellet extraction sequence at least once with the same ultracentrifugation speed or a different speed;

[0065] - optionally, quantify and characterize the extracellular vesicles (EVs) present in the sample by NTA (Nanoparticle Tracking Analysis);

[0066] - subjecting the pellet containing the extracellular vesicles to a liquid-liquid extraction to obtain the bicarbonate acids (BA) present inside;

[0067] - optionally, determine the concentration of 7a-hydroxy-4-cholesten-3-one (C4) and Fibroblast Growth Factor 19 (FGF19) present inside the EVs.

[0068] Diagnostic method

[0069] The in vitro diagnostic method for alcohol-associated liver disease according to the invention comprises the steps of:

[0070] - determining the concentration and / or ratios of one or more biomarkers selected from bile acids (BA) extracted from extracellular vesicles present in plasma or serum of a peripheral blood sample, where the bile acids (BA) are selected from cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA) ), and the respective bile acids conjugated with Taurine (T-CA, T-CDCA, T-DCA, T-LCA, T-UDCA) or with Glycine (G-CA, G-CDCA, G-DCA, G-LCA, G-UDCA); - compare the levels of the selected biomarkers with a set of previously established control values ​​to determine the association with alcohol-associated liver disease and establish the diagnosis when at least one of the following ratios, normalized with respect to the control values, is met: o G-CDCA / T-UDCA greater than 1, o G-CDCA / G-UDCA greater than 5, o T-CDCA / T-UDCA greater than 10;where the control values ​​correspond to the values ​​obtained from equivalent markers and ratios in healthy individuals free of alcohol-associated liver disease.

[0071] The bile acids (BA) are selected from cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA) or ursodeoxycholic acid (UDCA), in addition to their respective bile acid conjugated with Taurine (T-CA, T-CDCA, T-DCA, T-LCA or T-UDCA) or with Glycine (G-CA, G-CDCA, G-DCA, G-LCA or G-UDCA). Preferably, the bile acids (BA) correspond to T-CA, T-CDCA, T-DCA, T-LCA, T-UDCA, G-CA, G-CDCA, G-DCA, G-LCA, G-UDCA.

[0072] BA and C4 levels are measured using HPLC (LC / MS). FGF19 levels are measured using ELISA.

[0073] Extracellular vesicles (EVs) were quantified using Nanoparticle Tracking Analysis (NTA). Preferably, the concentration of extracellular vesicles is greater than 1.2 E 11 particles / mL.

[0074] In one embodiment of the invention, the ratio of G-CDCA in the EVs relative to the control values ​​is greater than or equal to 2.

[0075] In one embodiment of the invention, the ratio of T-UDCA and G-UDCA in the EVs relative to the control values ​​is greater than or equal to 1.1.

[0076] The in vitro diagnostic method according to the invention makes it possible to determine that a subject has been diagnosed with cirrhosis when the ratio of CDCA to control values ​​is greater than or equal to 5.5.

[0077] In turn, the diagnosis corresponds to alcohol-associated hepatitis (AH) when the ratio of DCA and UDCA with respect to the control values ​​is greater than or equal to 4 and the DCA ratio is greater than or equal to 15. In one embodiment of the invention, the ratio of G-CDCA / T-UDCA is greater than 5. In one embodiment of the invention, the ratio of G-CDCA / G-UDCA is greater than 10. In one embodiment of the invention, the ratio of T-CDCA / T-UDCA is greater than 20.

[0078] Based on this information, a correlation was determined between the levels of bile acids contained in extracellular vesicles and the concentration of extracellular vesicles present in the sample. Thus, a sensitive and accurate in vitro method for the diagnosis and prognosis of alcohol-associated liver disease was developed, based on the determination of Bas in EVs.

[0079] Traditionally used blood tests or other clinical tests, such as liver function tests, coagulation studies, and liver biopsies, may be performed as a complement to confirming alcohol-associated liver disease, but they are not part of the scope of the invention. Traditionally used liver damage tests are not required or necessary to perform the in vitro method for diagnosing and prognosing alcohol-associated liver disease according to the present invention.

[0080] EXAMPLES

[0081] The examples shown below serve to illustrate the invention, however, they should not be considered to limit its scope.

[0082] Example 1: Analysis of bile acids present in serum.

[0083] The content of various bile acids (BA) was analyzed in samples from various conditions: cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA). The conditions analyzed correspond to values ​​in healthy individuals (control), alcohol-associated hepatitis (AH), cirrhosis, and alcohol use disorder (AUD).

[0084] Figure 1 shows the bile acid content in serum. The variation in total bile acid composition in proportional terms does not show a clear trend among the different bile acids (Table 1); however, when comparing the bile acid concentrations (Table 2), an increase in the concentration of each of the BAs analyzed is clearly observed, although the rate of change is greater in the case of LCA and UDCA.

[0085] Table 1: Change ratio between percentage of bile acids with respect to control values ​​shown in Fig. 1A.

[0086] Table 2: Rate of change between bile acid concentrations with respect to the control values ​​shown in Fig. IB.

[0087] Example 2: Analysis of bile acids present in extracellular vesicles (cargo).

[0088] Similar to the previous example, the extracellular vesicle loading of the bile acids CA, CDCA, DCA, LCA, and UDCA was analyzed. The conditions analyzed correspond to values ​​in healthy individuals (control), alcohol-associated hepatitis (AH), cirrhosis, and alcohol use disorder (AUD).

[0089] Figure 2 shows the bile acid content in extracellular vesicles. The variation in the total percentage composition of bile acids does not show a clear trend among the different bile acids (Table 3). On the other hand, the ratio with respect to the control value of bile acid concentration (Table 4) shows an increase in the concentration of each of the BAs analyzed, with less dispersion compared to that observed in serum. Table 3: Ratio of change between the percentage of bile acids in extracellular vesicles compared to the control values ​​shown in Fig. 2A.

[0090] Table 4: Rate of change between bile acid concentrations in extracellular vesicles with respect to the control values ​​shown in Fig. 2B.

[0091] Example 3: Analysis of extracellular vesicle and bile acid concentration in patients under various conditions.

[0092] When analyzing samples from healthy (control) patients, alcohol-associated hepatitis (AH), cirrhosis, and alcohol use disorder (AUD), the following is observed:

[0093] A higher total AB concentration in the AUD group (1366.28 ng / mL) compared to the control group (552.42 ng / mL) (p=0.003).

[0094] Chenodeoxycholic acid concentration was higher in the AH patient group (734.23 ng / mL) (p=0.04) compared to all other groups.

[0095] The average concentration of extracellular vesicles was higher in the AH group (1,292 E 11 +- 6.4 E 10 particles / mL) and in the AUD (9.9 E 10 + 4.9 E 9 particles / mL) (p=0.005) compared to the control group and cirrhosis. Example 4: Analysis of the proportion of bile acids in serum and extracellular vesicles, compared in patients with liver disease

[0096] Fifteen bile acids were analyzed in serum and extracellular vesicles, compared with patients with liver disease. The results were graphed to represent the proportion of each bile acid in the analyzed samples (see Figures 3A and 3B).

[0097] Figure A presents the serum results, where GCA - TCA - GCDCA - TCDCA - GDCA showed a statistically significant difference. The serum samples presented the following variations in the total concentration of bile acids and conjugates in the groups with alcoholic liver disease in relation to the control group: o Increase: G-CDCA, T-CDCA, G-CA, T-CA o Decrease: G-DCA, T-DCA

[0098] Figure 3B shows the composition in the case of extracellular vesicles, where GCA, G-CDCA, G-UDCA, T-CA and T-CDCA showed a statistically significant difference.

[0099] In extracellular vesicles, an increase in the proportion of ursodeoxycholic acid (UDCA) conjugated primarily with glycine (GUDCA) and taurine (T-UDCA) was found relative to the total bile acids compared to serum. Although the observed trends between groups persisted, a different BA signature was demonstrated in EVs.

[0100] TCDCA increases in both serum and EVs. However, in the case of GCDCA, the trend in serum is not observed; it is relatively constant. However, in EVs, it increases in pathologies associated with alcohol consumption.

[0101] The proportion of ursodeoxycholic acid in samples with alcohol-related pathologies decreases compared to healthy controls. This is seen in the case of TUDCA and also in GUDCA, which decreases at a much greater rate compared to serum samples.

[0102] Table 5: Average composition of bile acid samples in serum and EVs under different conditions. The bile acids with the greatest differences between the profiles for each sample type are presented.

[0103] In BA-EVs, an increase in UDCA in its form conjugated with Glycine can be observed, and the levels are different between healthy controls and alcohol-associated hepatitis, when analyzing the proportional composition with respect to the total BA.

[0104] On the other hand, when analyzing the reasons of interest, in different liver conditions, it is possible to rescue relationships between bile acids that when normalized with respect to the relationship for the control results (Table 6) present results in EVs that differ by at least one order of magnitude with respect to the values ​​obtained in serum BA samples.

[0105] Table 6: Comparison of ratios between BA-Serum VS BA-EVs biomarkers of interest

Claims

CLAIMS 1. An in vitro diagnostic method for alcohol-associated liver disease, CHARACTERIZED in that it comprises the stages of: - determining the concentration and / or ratios of one or more biomarkers selected from bile acids (BA) extracted from extracellular vesicles present in plasma or serum of a peripheral blood sample, where the bile acids are selected from cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), and the respective bile acids conjugated with Taurine (T-CA, T-CDCA, T-DCA, T-LCA, T-UDCA) or with Glycine (G-CA, G-CDCA, G-DCA, G-LCA, G-UDCA); - compare the levels of the selected biomarkers with a set of previously established control values ​​to determine the association with alcohol-associated liver disease and establish the diagnosis when at least one of the following ratios, normalized with respect to the control values, is met: o G-CDCA / T-UDCA greater than 1, o G-CDCA / G-UDCA greater than 5, o T-CDCA / T-UDCA greater than 10; where the control values ​​correspond to the values ​​obtained from bile acids (BA) extracted from extracellular vesicles and equivalent ratios in healthy individuals free of alcohol-associated liver disease.

2. The diagnostic method according to claim 1, CHARACTERIZED in that the bile acids (BA) correspond to T-CA, T-CDCA, T-DCA, T-LCA, T-UDCA, G-CA, G-CDCA, G-DCA, G-LCA, G-UDCA.

3. The diagnostic method according to claim 1 or 2, CHARACTERIZED in that the measurement of BA levels is performed by HPLC (LC / MS).

4. The diagnostic method according to claim 1, CHARACTERIZED in that the concentration of extracellular vesicles is greater than 1.2 E 11 particles / mL.

5. The diagnostic method according to claim 4, CHARACTERIZED in that the quantification of extracellular vesicles (EVs) is carried out by Nanoparticle Tracking Analysis (NTA).

6. The diagnostic method according to claim 1, CHARACTERIZED in that the ratio of G-CDCA in the EVs with respect to the control values ​​is greater than or equal to 2.

7. The diagnostic method according to claim 1, CHARACTERIZED in that the ratio of T-UDCA and G-UDCA in the EVs with respect to the control values ​​is greater than or equal to 1.

1.

8. The diagnostic method according to claim 1, CHARACTERIZED in that the diagnosis corresponds to cirrhosis when the ratio of CDCA to the control values ​​is greater than or equal to 5.

5.

9. The diagnostic method according to claim 1, CHARACTERIZED in that the diagnosis corresponds to alcohol-associated hepatitis (AH) when the ratio of DCA and UDCA with respect to the control values ​​is greater than or equal to 4 and the DCA ratio is greater than or equal to 15.

10. The diagnostic method according to any of claims 1 to 7, CHARACTERIZED in that the ratio of G-CDCA / T-UDCA is greater than 5.

11. The diagnostic method according to any of claims 1 to 7, CHARACTERIZED in that the ratio of G-CDCA / G-UDCA is greater than 10.

12. The diagnostic method according to any of claims 1 to 7, CHARACTERIZED in that the ratio of T-CDCA / T-UDCA is greater than 20.

Images