Method and system for extracting and quantifying bile acids obtained from extracellular vesicles

Abstract

The present invention provides methods and systems for the extraction and quantification in vitro of bile acids contained in extracellular vesicles (EVs) for use in the area of protocols, diagnostics or other applications.

Description

[0001] METHOD AND SYSTEM FOR THE EXTRACTION AND QUANTIFICATION OF BILE ACIDS OBTAINED FROM EXTRACELLULAR VESICLES

[0002] DESCRIPTIVE MEMORY

[0003] Technical Field

[0004] The present invention falls within the field of biomedicine and chemical analysis. Specifically, it focuses on methods and systems for the in vitro extraction and quantification of bile acids present in extracellular vesicles (EVs) obtained from biological samples (blood, urine, etc.) for use in protocols, diagnostics, or other applications.

[0005] BACKGROUND OF THE INVENTION

[0006] Bile acids are unsympathetic molecules (they are both soluble and insoluble in water) with a steroidal skeleton that are produced by liver cells from cholesterol.

[0007] Along with water, cholesterol, bilirubin, salts, and other compounds, bile acids are part of bile and act as detergents in the small intestine by decreasing the surface tension of fats, causing them to emulsify, which are subsequently broken down by the action of lipases. They are necessary for the absorption of fat-soluble vitamins. They have a mild cathartic action, improve biliary drainage, and prevent infections, as bile is an excellent breeding ground.

[0008] Bile acids are frequently found conjugated with glycine and taurine. Thus, cholic acid forms glycocholic and taurocholic acids, respectively, giving rise to the group of secondary bile acids.

[0009] They have recently been considered to be relevant molecules in cell signaling, acting both in the liver and in other organs and tissues. In fact, through the activation of receptors such as vitamin D or other nuclear receptors (FXR, NR1H4), they regulate lipid and carbohydrate metabolic pathways, and their effects are important for the body's energy homeostasis.

[0010] Regarding the use of bile acids as biomarkers, only analyses of their presence in blood outside the gallbladder are known, primarily for the diagnosis and monitoring of some gastroenterological pathologies, but their usefulness is limited because they are not absolute indicators of pathology. When their concentrations are measured directly from this biological matrix, the complexity of managing this matrix is ​​compounded by the relatively low concentration of bile acids.

[0011] For example, alcoholic liver disease (ALD) is liver damage that occurs after years of excessive alcohol consumption, which is also influenced by environmental factors. Some people may have an inherited risk for the disease, and while it doesn't occur in all drinkers, the chances of developing the disease increase depending on how long you've been drinking and how much alcohol you consume. You may not even experience any symptoms, or they may develop slowly.

[0012] Currently, when a patient is clinically evaluated, they 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 performed. Biopsies are associated with high risks (which increase in critically ill patients), low availability, and high costs (associated with the intervention 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 the diagnosis or management of this disease.

[0013] Consequently, this type of bile acid-associated disease would benefit from a sensitive and specific diagnosis without the need for biopsy or other invasive procedures; all that is needed is a biological sample, for example, peripheral blood.

[0014] State of the Art

[0015] To date, there are some approaches to problems or techniques close to those addressed in the present invention, however, they do not aim at the extraction and quantification of bile acids present in extracellular vesicles.

[0016] In WO2021183879, from one of the inventors of the present invention, methods are disclosed for detecting a liver disease, such as alcohol-associated liver disease (ALD) or alcoholic hepatitis (AH), which are determined by the elevated concentration of EVs or cargoes within those EVs. This document establishes that AH can exhibit circulating EVs at a concentration greater than 1.5 x 10 11EVs per milliliter (EVs / mL) of plasma, which can be considered a biomarker of high mortality risk. The document 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, for example: 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.

[0017] On the other hand, in the publication by Livshits, M. et al. (Sci Rep 5, 17319 (2015) a theoretical analysis is presented where the centrifugation conditions are reviewed, including the selection of the rotor, which corresponds to an aspect that generates disparate results despite selecting identical speeds. The centrifugation sequence includes four stages of 1,290g, 2,000g, 10,000g and 100,000g. Regarding the quantification and concentration of exosomes, it is carried out by NTA in order to corroborate the expected theoretical value versus the obtained value.

[0018] On the other hand, the company Creative Biolabs Exosome, on its website www.creative-biolabs.com / exosome / exosome-isolation.htm, presents the exosome-related services it provides. It shows separation using five-stage centrifugations, at speeds of 300g, 2,000g, 10,000g, 100,000-200,000g, and again at 100,000-200,000g. Regarding the NTA technique, it is one of the five technologies available for quantifying exosomes provided by this company. Consequently, although it describes the production of exosomes, it does not describe the extraction of bile acids from extracellular vesicles.

[0019] Finally, the publication of patent application CN115698265A describes a method for obtaining microvesicles, such as exosomes or others, which differs completely from the method according to the invention, although in Example 10 they mention the analysis by NTA to determine the concentration and size of the nanoparticles. In one section of the document they refer to a comparison with the standard ultracentrifugation method, where the speeds selected after separating the supernatant and filtering are 12,000g and 120,000g, however, the lower centrifugation speed is not mentioned.

[0020] However, none of the protocols described teach a methodology and / or system intended to obtain extracellular vesicles for use in detection or measurement in the field of protocols, diagnostics or other applications.

[0021] Given that chronic diseases are reversible or their management improves the patient's quality of life if diagnosed early and then monitored and treated appropriately, this makes it necessary and relevant to have methodologies or systems that facilitate the extraction and quantification of biomarkers of good diagnostic quality.

[0022] BRIEF DESCRIPTION OF THE FIGURES

[0023] Figure 1. (A) Total bile acid concentration (ng / mL). (B) Total bile acid concentration (ng / mL). Two-way and one-way ANOVA with Bonferroni post-test. ***P < 0.001. Bile acids (BA), cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA).

[0024] Figure 2. Comparison of serum bile acid samples from healthy controls, alcohol-associated hepatitis, alcohol-associated cirrhosis, and alcohol use disorder. (A) Total bile acid concentration (ng / mL). (B) Total conjugated bile acid concentration. (C) Total unconjugated bile acid concentration (ng / mL).

[0025] Figure 3. Comparison of bile acid samples in extracellular vesicles from healthy controls, alcohol-associated hepatitis, alcohol-associated cirrhosis, and alcohol use disorder. (A) Total bile acid concentration (ng / mL). (B) Total conjugated bile acid concentration. (C) Total unconjugated bile acid concentration (ng / mL).

[0026] Figure 4. A. Determination of exosome size and concentration by NTA. B. Characterization of EVs by Western Blot and TEM.

[0027] Figure 5. Graphical representation of the LC-MS analysis showing the bile acid profile of the sample. Nomenclatures

[0028] • BAs = bile acids

[0029] • EVs = extracellular vesicles

[0030] • CA = cholic acid - cholic acid

[0031] • DCA = deoxycholic acid

[0032] • CDCA = chenodeoxycholic acid - chenodeoxycholic acid

[0033] • LCA = lithocholic acid

[0034] • UDCA = ursodeoxycholic acid - ursodeoxycholic acid

[0035] • T-BA = taurine-conjugated bile acid

[0036] • G-BA = glycine-conjugated bile acid

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] The invention corresponds to a method and system for isolating and quantifying bile acids (BA) present inside the extracellular vesicle (EVs) or exosomes.

[0039] Currently, measurements of bile acid content or other biomarkers in serum, other than EVs, do not allow for the prediction of bile acid-associated diseases. In some cases, detectable values ​​are not obtained. In contrast, in the present invention, the method for isolating and measuring bile acids contained in EVs shows different correlations than those that could be established from bile acids detected in serum, other than EVs, plasma, or urine.

[0040] Bile acids can be extracted from EVs present in any type of biological sample, such as blood or urine, among others.

[0041] The method according to the invention involves a series of operations beginning with a sequence of centrifugations, where instead of searching for large, high-molecular-weight molecules like those regularly obtained in procedures involving ultracentrifugation (proteins, peptides, among others), the present invention seeks to collect bile acids, which are small molecules compared to those mentioned above, but which are "encapsulated in EVs" and have a very high predictive power in certain pathologies.

[0042] In the sequence of centrifugations selected to obtain bile acids, optionally, a sample preparation is first performed, where proteins and other components are precipitated and removed by weight, for example, in the case of blood, the samples are centrifuged to obtain serum or plasma. Sample preparation will include a clarification or conditioning operation of the sample prior to centrifugation, or filtering to remove larger components present in blood (clot, proteins, etc.), urine (sediment, proteins, etc.), or other biological samples.

[0043] Ultracentrifugation is then performed to separate the vesicles. The bile acids found outside the EVs remain in the supernatant after centrifugation and ultracentrifugation and are discarded. Only the bile acids present inside the vesicles contained in the pellet are recovered.

[0044] After executing the centrifugation sequence, the extracellular vesicles are quantified and characterized by Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscope (TEM) and Western Blot. These three technologies are a complementary tool to confirm the presence of vesicles in the obtained pellet, following international standards for EV analysis (only NTA and TEM correspond to the standard, Western blot is an alternative method). However, these techniques alone or together do not detect or quantify bile acids present in EVs. Although quantification is not a fundamental part of the steps required by the methodology according to the invention, it is carried out as a verification control for the presence of EVs in the pellet.

[0045] Figure 4A shows an EV characterization curve using NTA, which shows the dispersion of exosome size and concentration. Figure 4B shows images of EV characterization using Western blotting and TEM.

[0046] In parallel, bile acids (BA) are extracted from the interior of the vesicle using any feasible technique existing in the state of the art, for example, by liquid-liquid extraction, precipitation or solid phase, in order to precipitate the high molecular weight components of the vesicle (proteins, nucleic acids and others) and thus release the BAs contained therein, to subsequently quantify them. It is not trivial to search for bile acids inside the vesicles, since ultracentrifugation is usually used to search for proteins or extract some lipids from the cell wall, but there is no history of the presence of bile acids in them. In a preferred embodiment, the extraction of BAs is carried out by liquid-liquid extraction, with solvents related to them and precipitation, in order to precipitate the proteins and nucleic acids that make up the EV and release the BAs from it.

[0047] LC-MS profile of bile acids obtained from EVs

[0048] Subsequently, the analytical part is performed using LC-MS (Liquid Chromatography - Mass Spectrometry) or LC-MS / MS or another technique that allows the quantification of BAs simultaneously and at low concentrations, with high analytical sensitivity and specificity. It is not evident whether the identification and quantification method used by LC-MS allows the analysis of bile acids obtained from the vesicles, nor whether the sensitivity of the equipment would allow them to be quantified, because they are found at very low concentrations and could be bound or conjugated to other EV components, not allowing their separation by liquid chromatography (LC) or their quantification by mass spectrometry (MS).

[0049] Therefore, thanks to this methodology it is possible to rescue and quantify the bile acids present inside the extracellular vesicles present in the pellet, at very low concentrations (nanograms and picograms).

[0050] Differences and advantages

[0051] It should be noted that a person skilled in the art would recognize that in a simple centrifugation or ultracentrifugation of a biological sample, free bile acids (BAs) remain in the supernatant and not in the pellet.

[0052] The present invention differs from the prior art in that, instead of working with the BAs present in the supernatants, it looks for the BAs present in the extracellular vesicles (EVs) isolated from the pellet. That is, it is not logical, and there is no suggestion in the prior art to motivate the use of the BAs present within EVs as a specific biomarker.

[0053] The method according to the present invention allows simultaneous quantification of bile acids by LC-MS, which exclusively accounts for their concentration within the EVs. There is no interference from free (unconjugated) or conjugated BAs present outside the vesicle in the serum or plasma directly from the biological sample, since this set of BAs is previously eliminated in the supernatant.

[0054] EVs carry a variety of biological information; in the case of BAs in EVs, the information is more valuable than that provided by BAs outside EVs for better diagnosing and / or prognosticating liver diseases or other pathologies involving bile acids, where it is feasible to identify BA profiles of clinical value. BAs in EVs provide more and better information than BAs outside EVs in biological samples.

[0055] The free or non-conjugated bile acids analyzed correspond to:

[0056] • CA = Cholic Acid - cholic acid

[0057] • DCA = Deoxycholic Acid - deoxycholic acid

[0058] • CDCA = Chenodeoxycholic Acid - chenodeoxycholic acid

[0059] • LCA = Lithocholic Acid - lithocholic acid

[0060] • UDCA = Ursodeoxycholic acid - ursodeoxycholic acid

[0061] In addition to their respective Taurine-conjugated bile acid (T-BA) or Glycine-conjugated bile acid (G-BA) or any other conjugated or non-conjugated bile acid present in the extracellular vesicles.

[0062] Method

[0063] The method for the in vitro extraction and quantification of bile acids obtained from extracellular vesicles according to the invention comprises the following steps: a) Performing a centrifugation gradient of 8,000g or greater in at least two steps, with increasing speeds until a pellet containing EVs is obtained. In a preferred embodiment of the invention, the centrifugation gradient comprises the following steps:

[0064] - centrifuge the biological sample at a speed greater than or equal to 8,000g, preferably at a speed between 10,000 and 15,000g, discarding the pellet, to eliminate elements in the precipitate that could subsequently interfere with the identification and characterization of the EVs, which is subsequently obtained by ultracentrifugation;

[0065] - ultracentrifuging the clarified medium (supernatant) at a high speed greater than or equal to 20,000g, preferably at a speed between 100,000 and 150,000g, and extracting the pellet containing the extracellular vesicles, where the ultracentrifugation can last from 1 to 24 hours for at least 100,000g; b) optionally, quantifying and characterizing the extracellular vesicles (EVs) present in the sample; c) processing the pellet containing the extracellular vesicles to extract the bile acids (BA) present inside, preferably by liquid-liquid extraction, precipitation or solid phase extraction or any other technique feasible in the state of the art; d) determining the concentration of the bile acids (BA) extracted from the extracellular vesicles, for example, by a technique with high sensitivity and specificity such as LC-MS

[0066] The method provides data for analysis in the field of diagnosis and monitoring the progress of diseases related to bile acid metabolism.

[0067] Bile acids (BA) are preferably 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).

[0068] BA levels are measured using LC-MS, such as HPLC-MS / MS or UPLC-MS / MS, among others.

[0069] The quantification and characterization of extracellular vesicles (EVs) was performed by NTA, TEM and Western Blot analysis.

[0070] Preferably, the sequence of centrifugation steps for EV isolation corresponds to high speeds between 8,000 and 20,000 g for the centrifugation steps. Optionally, a pre-preparation step is performed, such as a low-speed centrifugation (between 0 and 13,000 g, preferably between 2,000 and 8,000 g).

[0071] System

[0072] A system for the extraction and quantification of bile acids obtained from extracellular vesicles is also provided, comprising the following components: a. Means for performing an ultracentrifugation gradient of at least two increasing stages until obtaining a pellet with EVs. Preferably, the system comprises the following elements: i. Means for centrifuging at a speed greater than or equal to 8,000 g, preferably at a speed between 10,000 and 15,000 g; ii. Means for ultracentrifuging at a speed greater than or equal to 20,000 g, preferably at a speed between 100,000 and 150,000 g; b. Optionally, means for quantifying and characterizing extracellular vesicles (EVs); c. Means for extracting bile acids (BA) from extracellular vesicles; and d. Means for determining the concentration of bile acids (BA) in EVs.

[0073] EXAMPLES

[0074] The examples shown below serve to illustrate the invention, however, they should not be considered to limit its scope.

[0075] Example 1: Extraction of bile acids present in serum and Evs.

[0076] Preliminary analysis of serum BA composition and profile was observed in samples from Alcohol-Associated Liver Disease (ALD) groups (Alcohol-Associated Hepatitis, Alcohol-Associated Cirrhosis, and Alcohol Use Disorder) compared to healthy controls.

[0077] The same type of analysis was performed using the method of the invention, to extract BA from extracellular vesicles, but adapted for smaller sample volumes and lower analytical sensitivity.

[0078] Figure 1A shows the results in serum, while Figure 1B shows the unconjugated or free BA present in EVs extracted according to the method of the present invention. The content of cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA) was analyzed. The conditions analyzed correspond to values ​​in healthy individuals (control), alcoholic hepatitis (AH), cirrhosis, and alcohol use disorder (AUD).

[0079] From these results, it is possible to confirm the presence of bile acids contained in extracellular vesicles isolated from patient samples and to appreciate that there is a characteristic and distinct pattern of bile acids in serum and in EVs. Example 2: Analysis of the concentration of total, conjugated and unconjugated bile acids

[0080] The results of the extraction of total, conjugated and unconjugated bile acids present in serum were compared (Fig. 2A, B and C respectively).

[0081] Similarly, total bile acids, both conjugated and unconjugated, present in extracellular vesicles were extracted (Fig. 3A, B, and C, respectively). BAs were extracted using a liquid-liquid system with acetonitrile, stirring the mixture, and centrifugation was performed, precipitating the proteins and nucleic acids that make up the EV and releasing the BAs. In this specific case, we used gradient microcentrifugation and ultracentrifugation.

[0082] EVs were quantified using nanotracking analysis (NTA) (see Figure 4A), and BAs in extracellular vesicles were characterized using liquid chromatography (LC)-mass spectrometry (MS). A representative LC-MS profile is shown in Figure 5, where:

[0083] • AC: 0.12; LCA: < 0; DCA: 0.54; CDCA: < 0; UDCA: < 0

[0084] • GCA: 28.45; GLCA: < 0; GDCA: 0.74; GCDCA: 205.08; GUDCA: 2.11

[0085] • TCA: 21.2; TLCA: 0.51; TDCA: 0.73; TCDCA: 81.94; TUDCA: < 0

[0086] The TCA is appreciated at minute 3.

[0087] Example 3: Extraction step of bile acids from EVs

[0088] An extraction was performed according to the conditions shown in the diagram in Figure 6.

[0089] The pellet obtained in the centrifugation flow (containing the EVs) was reconstituted with 1% saline buffer (PBS 1%) and mixed well for 1 minute in vortex.

[0090] Then, 10 p.1 of deuterated internal standard of all bile acids to be quantified were added, then an organic precipitating agent friendly to the LC-MS equipment and to the BAs, in this case acetonitrile, was added.

[0091] It was vortexed for 1 minute and subsequently centrifuged at 10,000g to precipitate proteins, nucleic acids and other components of the EVs. Example 4: Preliminary centrifugation tests for the isolation of BAs present within

[0092] EVs

[0093] During the development of the invention, conditions were evaluated that did not provide optimal results for the analytical objective. Some of these conditions include the following:

[0094] 1.- When centrifuging the serum sample directly between 100,000 and 150,000g, the precipitate obtained contains EVs and other elements that did not allow its correct characterization and quantification, and also interfered with the BAs extraction and quantification process.

[0095] 2.- When centrifuging at speeds lower than 100,000g, in the second step, the EVs could not be isolated correctly.

[0096] 3.- When centrifuging only at 10,000g and looking for EVs in the precipitate, they were not found and therefore BAs could not be found when quantifying, since they all remained in the supernatant.

[0097] Therefore, it was found that it is imperative to carry out the series of centrifugations indicated in the method according to the invention.

[0098] Example 5: Preliminary quantification tests of BAs present within EVs

[0099] Some of the methodologies include the following:

[0100] 1.- When measuring BAs directly using LC-MS or another analytical method in serum, only BAs outside the EVs were measured. Therefore, the objective of measuring BAs within EVs is not met.

[0101] 2. By adding a chemical or biological agent to release or extract BAs from EVs prior to EV isolation by ultracentrifugation, the sum of BAs in serum outside the EVs, combined with the BAs within the EVs, was measured. The use of the extraction agent allowed EVs to be broken down and BAs released from within. The analytical data show a different behavior than that of the BAs present within EVs.

[0102] The above shows the relevance of the steps of the method according to the invention.

Claims

CLAIMS A method for the extraction and quantification of bile acids obtained from extracellular vesicles, CHARACTERIZED in that it comprises the steps of: a. Performing a centrifugation gradient greater than or equal to 8,000g in at least 2 stages, with increasing speeds to obtain a pellet with extracellular vesicles (EVs); b. subjecting the pellet containing the extracellular vesicles to an extraction to obtain the bile acids (BAs) present inside; and c. determining the content and concentration of the BAs extracted from the EVs. The method according to claim 1, CHARACTERIZED in that the bile acids (BAs) are selected from the group consisting of cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA) or ursodeoxycholic acid (UDCA), in addition to the conjugated bile acids T-CA, T-CDCA, T-DCA, T-LCA, T-UDCA, G-CA, G-CDCA, G-DCA, G-LCA or G-UDCA, among others.The method according to claim 1 or 2, CHARACTERIZED in that the measurement of the BA levels is carried out by LC-MS or an equivalent technique. The method according to claim 1, CHARACTERIZED in that it comprises a parallel step of quantifying and characterizing the extracellular vesicles (EVs) present in the sample. The method according to claim 5, CHARACTERIZED in that the quantification of the extracellular vesicles (EVs) is carried out by Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM) and Western Blot. The method according to claim 1, CHARACTERIZED in that a preliminary centrifugation step is carried out at a speed between 0 to 8,000g. The method according to claim 1, CHARACTERIZED in that the centrifugation gradient of step (a) comprises the following steps: i) centrifuging the biological sample at a speed greater than or equal to 8,000g, discarding the pellet; and. (i) ultracentrifuging the supernatant from the previous step at a speed greater than or equal to 20,000g, and extracting the pellet containing the extracellular vesicles. The diagnostic method according to claim 8, CHARACTERIZED in that the centrifugation of step (i) is carried out at a speed between 10,000 and 15,000g. The method according to claim 1, CHARACTERIZED in that the ultracentrifugation of step (i) is carried out at a speed between 100,000 and 150,000g. A system for the extraction and quantification of bile acids obtained from extracellular vesicles, CHARACTERIZED in that it comprises: a. Means for carrying out a centrifugation gradient greater than or equal to 8,000g of at least 2 stages with increasing speeds until obtaining a pellet with EVs; b. Means for extracting bile acids (BA) from extracellular vesicles; c. Means for determining the concentration of bile acids (BA). A system according to claim 11, CHARACTERIZED in that the means for carrying out the centrifugation gradient comprise: i. Means for centrifuging at a speed of between 10,000 and 15,000g; and i. Means for ultracentrifuging at a speed of between 100,000 and 150,000g. A system according to claim 11, characterized in that the extraction means are selected from liquid-liquid extraction media and precipitation media. A system according to claim 11, characterized in that it comprises means for quantifying and characterizing extracellular vesicles (EVs). A system according to claim 11, characterized in that the concentration of bile acids is performed by LC-MS.

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