Improved methods for evaluating liver function

Abstract

Improved methods and kits are provided for non-invasively assessing a patient's liver function, including rapidly and efficiently processing, detecting, and quantifying identifiable compounds from a patient's blood or serum sample. Methods are provided for estimating the one-year risk of experiencing a clinical event for an individual patient with chronic liver disease. Methods are provided for determining hepatic reserve capacity in a subject.

Description

[Technical Field]

[0001] This application claims priority and benefits of U.S. Provisional Patent Application No. 63 / 007,810, filed on April 9, 2020, as a PCT International Application on April 9, 2020, the entirety of which is incorporated herein by reference. [Background technology]

[0002] Because chronic liver disease progresses slowly and is often asymptomatic, liver function and disease severity testing to monitor patients and predict outcomes is of great clinical value. Several such tests have been proposed and developed. The HALT-C trial was designed to investigate and directly compare the clinical utility of several experimental tests that attempted to quantify liver function. Everson et al., HALT-C Trial Group. Aliment Pharmacol Ther. 2009;29:589-601. In HALT-C, the bicholate test for assessing liver function showed particular strength in monitoring patients and predicting clinical outcomes. Everson et al. Hepatology 2012;55:1019-29.

[0003] The dual cholate clearance test relies on the hepatic spontaneous elimination of endogenous bile acid cholate (cholic acid, CA). In the dual cholate clearance test, the patient is given two distinguishable cholates, each labeled with a stable isotope to distinguish it from naturally occurring endogenous cholic acid. For example, the patient receives a 20 mg dose of cholic acid-24- in an intravenous bolus. 13 The patient receives C(13C-CA). Simultaneously, the patient drinks a 40 mg dose of cholic acid-2,2,4,4-d4(4D-CA) dissolved in NaHCO3 and mixed with juice. In the HALT-C study, peripheral blood samples were collected at two doses: before administration (time 0) and at 5, 10, 15, 20, 30, 40, 45, 60, 75, 80, 90, 105, 120, 150, and 180 minutes. The serum concentrations of labeled cholate at all these time points were used for oral D4-CA and IV. 13A C-CA clearance curve was generated. As a result, the oral cholate clearance (cholate Cl) normalized to body weight was obtained. oral ), IV cholate clearance normalized to body weight (cholate Cl IV Measurements of ), and their ratios (cholate shunt) were obtained. Each test had the ability to identify patients who would likely have a negative outcome. Among the various measurements, cholate Cl oral It was found to be an excellent predictor of future clinical outcomes and indicators of liver disease. Everson et al. Hepatology 2012;55:1019-29. A minimal model based on spline function was developed and validated. Everson et al. Aliment Pharmacol Ther. 2007;26:401-10. The minimal model was used for oral D4-CA and IV. 13 To accurately reproduce the C-CA clearance curve, serum collection is required in samples at time 0 and at 5, 20, 45, 60, and 90 minutes. This is the basis of the HepQuant-SHUNT® test.

[0004] Previously, the inventors have used a single ion monitoring LC-MS for the separation and detection of analytes, including a multi-step extraction procedure for two analytes from human serum, D4-CA and 13 We developed and partially validated a liquid chromatography-mass spectrometry (LC-MS) assay for quantifying C-CA. However, prior art sample preparation and analysis are cumbersome, and previous methods using GC-MS or LC-MS analysis suffer from certain drawbacks.

[0005] U.S. Patent No. 8,613,904, Everson et al., discloses a method for evaluating liver function in a patient, comprising administering two identifiable stable isotope-labeled cholate compounds followed by obtaining a patient serum sample, and cumbersome sample preparation and analysis of the patient serum sample using GC-MS. The cholate compounds are isolated from the serum sample by a method including isolation and derivatization of the analyte. Derivatization of the sample analyte is used because volatilization of the analyte is required for GC analysis. Sample preparation included the steps of adding an unlabeled cholic acid internal standard to 0.5 mL of patient serum sample, diluting the sample with an aqueous sodium hydroxide solution, applying the diluted sample to a solid-phase extraction (SPE) cartridge (e.g., Waters® Sep-pak C18), eluting the sample from the SPE cartridge, drying and acidifying the sample eluate with diluted HCl, extracting the acidified sample with diethyl ether, evaporating the ether layer to form an evaporated sample, treating the evaporated sample with 2,2-dimethoxypropane (DMP) in methanol and HCl in the dark for 30 minutes, derivatizing the treated sample with hexamethyldisilazane (HMDS) catalyzed with pyridine and trimethylchlorosilane (TMCS) and heating to 55-60°C for 2 hours, evaporating the solvent from the derivatized sample, and reconstituting the sample by repeated addition and evaporation of hexane to form a reconstituted sample. The reconstituted sample is injected into a capillary GC-MS system for ratio analysis. Sample derivatization can lead to problems associated with chemical decomposition and the formation of new products, which may occur under high thermal conditions. In addition, the laborious manual sample preparation methods for GC-MS require extensive preparation time, resulting in low throughput and reduced analyte recovery from the sample.

[0006] U.S. Patent No. 8,778,299, Everson, discloses a method for evaluating liver function, comprising obtaining a patient serum sample following administration of two identifiable stable isotope-labeled cholate compounds, and processing and analyzing the patient serum sample using HPLC-MS. U.S. Patent No. 8,778,299 discloses a manual method for sample extraction for HPLC-MS, comprising the steps of: adding an unlabeled cholic acid internal standard to at least 0.5 mL of a patient serum sample; diluting the sample with an aqueous sodium hydroxide solution; applying the diluted sample to a solid-phase extraction (SPE) cartridge (e.g., Waters® reverse-phase Sep-pak C18); eluting the sample from the SPE cartridge; drying and acidifying the sample eluate with diluted HCl; extracting the acidified sample with diethyl ether; separating and evaporating the ether layer; and dissolving the evaporated sample in a mobile phase buffer to form a reconstituted sample. The reconstituted sample is injected into the HPLC-MS system, for example, using multimode electrospray (MM-ES) ionization via atmospheric pressure chemical ionization (APCI). Selective ion monitoring (SIM) is performed for cholic acid, 13C-cholic acid, and 4-D-cholic acid, for example, unlabeled and stable isotope-labeled cholic acids at 407.30, 408.30, and 411.30 m / z, respectively. The HPLC-MS method eliminates the need for sample derivatization required in the GC-MS method. Unlike GC analysis, sample volatilization is not required for LC, reducing analysis time and avoiding problems associated with chemical decomposition and the formation of new products that can occur under high thermal conditions. However, several manual steps are still required for analyte extraction, and ion overlap between administered isotopes needs to be corrected using simultaneous equations to address slight ion bleed-over and contamination of ion peaks in the LC-MS method.

[0007] For example, to support the FDA's clinical assay development and pre-market device approval, improved sample handling and quantification methods are desirable to enhance the performance of analytical assays and optimize assay efficiency. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] U.S. Patent No. 8,613,904 [Patent Document 2] U.S. Patent No. 8,778,299 [Overview of the Initiative]

[0009] This specification provides an improved method for evaluating liver function in a patient, comprising rapidly and efficiently processing, detecting, and quantifying identifiable compounds from a patient's blood or serum sample. Improved methods for processing blood or serum samples, and for the detection and quantification of analytes for use in liver function tests, are provided herein. Simplified methods for processing blood or serum samples suitable for automation have been developed.

[0010] A method is provided for quantifying one or more identifiable compounds in a blood or serum sample from a subject, the method comprising: receiving a blood or serum sample obtained from a subject having, suspected of having, or currently having chronic liver disease or liver impairment, wherein the sample was collected from the subject less than 3 hours after oral and / or intravenous administration of one or more identifiable compounds to the subject; processing the blood or serum sample to form a processed sample; injecting the processed sample into a mass detection system; measuring the concentration of one or more identifiable compounds in the processed sample, wherein the measurement includes mass detection; and quantifying the concentration of one or more identifiable compounds in the blood or serum sample. The processed sample may be a supernatant or an eluate. Processing of the blood or serum sample may include forming a supernatant. Optionally, the supernatant may be injected onto a separation system including preparation components and / or analytical components to form an eluate that can be injected into a mass detection system. In some embodiments, the method does not include a separation system. The optional separation system may include a chromatography system. In some embodiments, the method does not include chromatography.

[0011] A method is provided for quantifying one or more identifiable compounds in a blood or serum sample from a subject, the method comprising: receiving a blood or serum sample obtained from a subject having, suspected of having, or currently having chronic liver disease or liver impairment, wherein the sample was collected from the subject less than 3 hours after oral and / or intravenous administration of one or more identifiable compounds to the subject; processing the blood or serum sample to form a supernatant; injecting the supernatant into a separation system comprising preparation components and analyte components to form an eluate; and measuring the concentration of one or more identifiable compounds in the eluate, the method comprising quantifying the concentration of one or more identifiable compounds in the sample using a mass assay system. Optionally, the separation system may include a mobile phase component.

[0012] An optional separation system may include a chromatography system. The chromatography system may include a liquid chromatography (LC) system, and optionally, the LC system may be selected from the group consisting of HPLC and UPLC systems.

[0013] In some embodiments, the mass detection system may include a mass spectrometer. The mass spectrometer includes an ion source system and a mass decomposition / detection system. The ion source system may be selected from the group consisting of electrospray ionization (ES), matrix-assisted laser desorption / ionization (MALDI), fast atomic bombardment (FAB), chemical ionization (CI), atmospheric pressure chemical ionization (APCI), liquid secondary ionization (LSI), laser diode thermal desorption (LDTD), and surface-enhanced laser desorption / ionization (SELDI). The mass decomposition / detection system may be selected from the group consisting of triple quadrupole mass spectrometers (MS / MS), single quadrupole mass spectrometers (MS), Fourier transform mass spectrometers (FT-MS), and time-of-flight mass spectrometers (TOF-MS). The triple quadrupole mass spectrometer (MS / MS) may be operated in multiple reaction mode (MRM), optionally in negative ion multiple reaction mode.

[0014] In some embodiments, injection includes injecting the supernatant into a preparation component and eluting the preparation component onto an analysis component. In some embodiments, the preparation component and the analysis component are inline. In some embodiments, the supernatant is injected into a preparation column, and the flow is reversed to elute the preparation column onto an analysis column inline. The preparation component may include a solid-phase resin, and the analysis component may include a solid-phase resin. The solid-phase resins of the preparation and analysis components can each be independently selected from the group consisting of normal-phase resins, reverse-phase resins, hydrophobic-interacting solid-phase resins, hydrophilic-interacting solid-phase resins, ion-exchange solid-phase resins, size-exclusionary solid-phase resins, and affinity-based solid-phase resins. In some embodiments, the preparation and analysis components each include a reverse-phase resin, and optionally, the reverse-phase resin is independently a C8 or C18 resin.

[0015] A method has been developed for the analysis of blood or serum samples using LC-MS / MS with multiple reaction monitoring (MRM). The differences between this method and previous methods include: (i) unlabeled cholic acid is now quantified in each individual sample, not just in the baseline sample; (ii) previous multi-step extraction procedures, including combinations of solid-phase extraction, liquid-liquid extraction, evaporation, and reconstitution, have been replaced by automated online extraction procedures; and (iii) analyte detection and quantification are now based on analyte ion transitions in multiple reaction modes (MS / MS vs. MS). The advantages of the improved method provided herein include improved analyte recovery from the sample, increased analyte selectivity, increased sample throughput, reduced processing time, reduced patient sample volume, and improved limit of quantification (LOQ).

[0016] In some embodiments, a method is provided for quantifying one or more identifiable compounds in a blood or serum sample from a subject, the method comprising: receiving a blood or serum sample obtained from a subject having, suspected of having, or currently having chronic liver disease or liver impairment, wherein the sample was collected from the subject less than 3 hours after oral administration of a first identifiable compound to the subject; adding a protein precipitation solution to the sample to form a precipitated sample and a supernatant; injecting the supernatant onto an analytical column; and measuring the concentration of the first identifiable compound in the analytical column eluate, the measurement comprising quantifying the concentration of the first identifiable compound in the sample by multiple reaction mode (MRM) liquid chromatography-quadrupole mass spectrometry (LC-MS / MS). Optionally, the method comprises centrifugation of the precipitated sample to form a supernatant. Optionally, the method comprises injecting the supernatant into an extraction column and eluting the extraction column onto an analytical column. In some embodiments, after injecting the supernatant into the extraction column, the method includes reversing the flow to apply the extracted supernatant onto the analysis column.

[0017] In some embodiments, a second identifiable compound was also administered to the subjects by parenteral administration, or optionally by intravenous administration, less than three hours before sample collection from the subjects. The first and second identifiable compounds may be administered to the subjects within 15 minutes, 10 minutes, 5 minutes, 2 minutes, or simultaneously.

[0018] In some embodiments, the protein precipitation solution may contain a miscible organic solvent. The protein precipitation solution may be an aqueous solution containing at least 50% by volume of a miscible organic solvent. The protein precipitation solution may contain a water-miscible organic solvent selected from the group consisting of methanol, ethanol, isopropanol, acetonitrile, and acetone. In some embodiments, the miscible organic solvent is an organic alcohol. The organic alcohol may be a C1-C6 organic alcohol. The organic alcohol may be selected from the group consisting of methanol, ethanol, and isopropanol. In another embodiment, the protein precipitation solution may contain dimethoxyacetone, which can decompose into acetone and methanol when exposed to an acidic aqueous solution.

[0019] In some embodiments, the protein precipitate solution further comprises an identifiable compound of the internal standard.

[0020] The volume of the blood or serum sample may be 10 μL or more, 20 μL or more, 30 μL or more, 40 μL or more, 50 μL or more, or preferably 50 to 500 μL. The blood sample may be whole blood. The blood sample may be venous blood or capillary blood.

[0021] In some embodiments, the analyte extraction and recovery rates of identifiable compounds from blood or serum samples are >80%, >85%, >90%, >95%, or >97%.

[0022] In some embodiments, the first and / or second identifiable compound may exhibit a high hepatic extract of at least 50%, 60%, 70%, 75%, or at least 80% in the first pass through the liver of a healthy subject after oral administration.

[0023] In some embodiments, the first and / or second identifiable compound is an identifiable bile acid, bile acid conjugate, bile acid analog, or FXR agonist. Identifiable bile acids, bile acid conjugates, or bile acid analogs include identifiable cholic acid (CA), dehydrolithocholic acid (dehydroLCA), lithocholic acid (LCA), isodeoxycholic acid (isoDCA), isolithocholic acid (isoLCA), allolithocholic acid (alloLCA), glycolithocholic acid (GLCA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), taurolithocholic acid (TLCA), apocholic acid (apoCA), 23-nordeoxycholic acid (nor-DCA), 12- Ketritocholic acid (12-ketoLCA), 7-ketritocholic acid (7-ketoLCA), 6,7-diketritocholic acid (6,7-diketoLCA), glycodeoxycholic acid (GDCA), 6-keto-litocholic acid (6-ketoLCA), glycochenodeoxycholic acid (GCDCA), hyodeoxycholic acid (HDCA), ursodeoxycholic acid (UDCA), cholic acid (CA), taurodeoxycholic acid (TDCA), allocholic acid (ACA), β-hyodeoxycholic acid (β-HDCA), Muroko Taurocholic acid (MuroCA), Hyocholic acid (HCA), 12-Dehydrocholic acid (12-DHCA), β-Mulicolic acid (β-MCA), Norcholic acid (NorCA), 7-Ketodeoxycholic acid (7-KetDCA), Glycocholic acid (GCA), α-Mulicolic acid (α-MCA), Glycohyodeoxycholic acid (GHDCA), 3β-Cholic acid (βCA), Glycorsodeoxycholic acid (GHCA), ω-Mulicolic acid (ωMCA), Taurocholic acid (TCA), Glycohyocholic acid (GHCA), Tau Rohyodeoxycholic acid (THDCA), 7,12-diketritocholic acid (7,12-diketoLCA), dehydrocholic acid (DHCA), ursocholic acid (UCA), taurohyocholic acid (THCA), taurobeta-mulicolic acid (TβMCA), tauroalpha-mulicolic acid (TαMCA), glycodehydrocholic acid (GDHCA), tauroω-mulicolic acid (TωMCA), taurohydrocholic acid (TDHCA), ursodeoxycholic acid (UDCA), hyodeoxycholic acid (HDCA), (3α,It may be selected from the group consisting of 6α-dihydroxy-5β-cholan-24-oic acid), deoxycholic acid, all β-cholic acids, lithocholic acid 3-hemisuccinate, epideoxycholic acid, methyl ester of ursodeoxycholic acid, ursodeoxycholic acid, obeticholic acid (2α-ethyl-chenodeoxycholic acid), methyl ester of cholic acid, cholalcohol, epilithocholic acid, or an isotope-labeled derivative, or an analog or epimer thereof. In some embodiments, the stable isotope-labeled bile acid, bile acid conjugate, or bile acid analog is 2,2,4,4-d4-cholic acid (D4-CA; CA-D4), 24-, 13 C-cholic acid ( 13C-CA), 2,2,3,4,4-d5 cholic acid (D5-CA), 3,6,6,7,8,11,11,12-d8 cholic acid (D8-CA), lithocholic acid-2,2,4,4-D4 (LCA-D4), ursodeoxycholic acid-2,2,4,4-D4 (UDCA-D4), ursodeoxycholic acid (24-13C-UDCA), deoxycholic acid-2,2,4,4-D4 (DCA-D4), glycochenodeoxycholic acid-2,2,4,4-D4 (GCDCA-D4), glycochenodeoxycholic acid (glycine-2,2,3,4,4,6,6,7,8-D9 -CDCA), Glycodeoxycholic acid-2,2,4,4-D4 (GDCA-D4), Glycocholic acid-2,2,4,4-D4 (GCA-D4), Glycocholic acid (Glycine-1-13C-CA), Deoxycholic acid-24-13C (DCA-24-13C), Deoxycholic acid (2,2,4,4,11,11-D6-DCA), α-Mulicoleic acid (2,2,3,4,4-D5-αMCA), β-Mulicoleic acid (2,2,3,4,4-D5-βMCA), Chenodeoxycholic acid (2,2,3,4,4,6,6,7,8-D9-CDCA), Chenodeoxy Cholic acid (2,2,3,4,4-D5-CDCA), chenodeoxycholic acid (2,2,4,4-D4-CDCA), chenodeoxycholic acid (24-13C-CDCA), γ-mulicolic acid (2,2,3,4,4-D5-γMCA), omega-mulicolic acid (2,2,3,4,4-D5-ωMCA), taurochenodeoxycholic acid, sodium salt (taurine-2,2,3,4,4,6,6,7,8-D9-CDCA), taurochenodeoxycholic acid, sodium salt (taurine-2,2,4,4-D4-CDCA), taurocholic acid, sodium salt (taurine Taurodeoxycholic acid, sodium salt (Taurine-2,2,4,4-D4-CA), Taurodeoxycholic acid, sodium salt (Taurine-2,2,4,4,11,11-D6-DCA), Taurodeoxycholic acid, sodium salt (Taurine-2,2,4,4-D4-DCA), Tauroursodeoxycholic acid, sodium salt (Taurine-2,2,4,4-D4-UDCA), Tauroursodeoxycholic acid, sodium salt (Taurine-13C2-UDCA), Glycolic acid (Glycine-2,2,4,4-D4-LCA), 11,Selected from the group consisting of 12-double hydrogenated chenodeoxycholic acid (D2-chenodeoxycholic acid, D2-CA), glycosodeoxycholic acid (glycine-2,2,4,4-D4-UDCA), and glycosodeoxycholic acid (glycine-13C2-UDCA).

[0024] In some embodiments, the identifiable compound is an identifiable bile acid, a bile acid conjugate, or a bile acid analog. In some embodiments, the identifiable bile acid is an isotope-labeled bile acid, preferably a stable isotope-labeled bile acid. In some embodiments, the stable isotope-labeled cholic acid is cholic acid-2,2,4,4-D4(D4-CA;CA-D4),24- 13 C-Cholic acid ( 13 These are C-CA and 2,2,3,4,4-d5 cholic acid (D5-CA).

[0025] A method is provided for screening or monitoring liver function, liver disease, or liver impairment in a subject, the method comprising: taking a blood or serum sample from a subject who has, is suspected of having, or is at risk of having, chronic liver disease, after orally administering a composition containing an identifiable compound to the subject, wherein the blood or serum sample is collected from the subject less than 3 hours after orally administering the identifiable compound to the subject; measuring the concentration of the orally administered identifiable compound in the blood or serum sample from the subject, wherein the measurement includes quantifying the concentration of the identifiable compound in the sample by LC-MS / MS as described in claim 1; and comparing the concentration of the identifiable compound in the blood sample over time with (i) one or more cutoff values ​​of identifiable compound concentrations established from a known patient population, and / or (ii) the concentration of the identifiable compound in one or more earlier samples from the same subject. In some embodiments, the blood or serum sample is collected from the subject within 180 minutes, 150 minutes, 120 minutes, 90 minutes, 75 minutes, 65 minutes, 60 minutes, 55 minutes, 45 minutes, 35 minutes, 30 minutes, 25 minutes, or 15 minutes after administration of an identifiable compound. In some embodiments, the blood or serum sample consists of a single blood or serum sample. In some embodiments, the blood or serum sample consists of multiple samples. In some embodiments, the blood or serum sample consists of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more samples. In some embodiments, the blood or serum sample consists of two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve samples. In some embodiments, the blood or serum sample consists of two to seven samples.

[0026] In some embodiments, the blood or serum sample consists of a single blood or serum sample collected at approximately 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 minutes after oral administration of an identifiable compound, or at one point in time selected from any point in time thereafter.

[0027] In some embodiments, a single blood or serum sample is collected at one time point selected from approximately 30–180 minutes, 45–120 minutes, 50–80 minutes, 45 minutes, 60 minutes, or 90 minutes after oral administration of an identifiable compound.

[0028] In some embodiments, the blood or serum sample consists of a single blood or serum sample collected at approximately 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 minutes after intravenous administration of an identifiable compound, or at one point in time selected from any point in time thereafter.

[0029] In some embodiments, the blood or serum sample consists of multiple blood or serum samples and is optionally collected at two or more time points selected from baseline, approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 minutes after administration of an identifiable compound, or any point in between.

[0030] In some embodiments, the concentration of a identifiable compound in a single blood or serum sample, after oral administration only, compared to one or more cutoff values ​​of identifiable compound concentrations in a known patient population, is an estimate of the portal vein hepatic filtration rate (portal HFR) in the subject.

[0031] In some embodiments, a method for estimating portal vein HFR in a subject further includes converting the concentration of a identifiable compound to an estimated portal vein HFR (mL / min / kg) in the subject by using an equation, and comparing the estimated portal vein HFR in the subject to one or more portal vein HFR (FLOW) cutoff values ​​established from a known patient population or within the subject over time.

[0032] In some embodiments, a method is provided for converting STAT values ​​in a subject to estimated portal vein HFR (FLOW) (mL / min / kg) values ​​in the subject, the method comprising the equation, The equation includes y=A(x)+C, and in the equation, x = LOG estimated portal vein HFR (FLOW) value (mL / min / kg) in the subject. y = LOG STAT value in the subject (μM adjusted for a 75kg body weight), The gradient coefficient is A = 0.9 to 1.1. C is a constant between -0.05 and 0.05.

[0033] In some embodiments, a method is provided for converting STAT values ​​in a subject to estimated portal vein HFR (FLOW) (mL / min / kg) values ​​in the subject, the method comprising the equation, y = 0.9702x + 0.0206, where x = LOG estimated portal vein HFR value (mL / min / kg) in the subject and y = LOG STAT value (μM adjusted for 75 kg body weight) in the subject.

[0034] In some embodiments, a method is provided for converting the STAT value in a subject to an estimated portal vein HFR (FLOW) (mL / min / kg) value in the subject, the method comprising the equation, Ln(x) = 1.031 × Ln(y) - 0.0212, where x = estimated portal vein HFR value in the subject (mL / min / kg) and y = STAT value in the subject (μM adjusted for a 75 kg body weight).

[0035] In some embodiments, the concentration of a discriminable compound in a single sample after oral administration only is compared to one or more cutoff values ​​of discriminable compound concentrations in a known patient population to estimate the DSI value in the subject.

[0036] In some embodiments, a method for estimating DSI values ​​in a subject further includes converting the concentration of an identifiable compound into a DSI value in the subject by using an equation, and comparing the estimated DSI value in the subject with one or more DSI value cutoff values ​​established from a known patient population or within the subject over time.

[0037] In some embodiments, an equation is provided, y = A ln(x) + C, for converting the concentration of an identifiable compound in a single specific sample (STAT value) to an estimated DSI value in the subject, where A = a slope value between 8.5 and 10.5, C = a constant between 18 and 22, x = the STAT value (μM adjusted for a 75 kg body weight), and y = the DSI value in the subject.

[0038] In some embodiments, an equation is provided for converting STAT values ​​to estimated DSI values ​​in the subject, and optionally, the equation is: y = 9.4514ln(x) + 21.12, and in the equation, x = STAT value (μM adjusted for a 75kg body weight), y = DSI value in the subject. As shown in Figure 12B, this equation is obtained in a test of n=1736, R 2 The result was 0.8499.

[0039] In another embodiment, an equation is provided for converting the concentration of an identifiable compound (STAT value) in a single specific sample to an estimated DSI value in the subject, the equation being: y = A(Ln x) 2 +B(Ln x)+C, and in the equation, y = estimated DSI value for the subject, x = STAT value in the target. The coefficient A is between 1 and 1.5. The coefficient B is between 9 and 10. C is a constant between 19.5 and 22.

[0040] In some embodiments, an equation is provided for converting the STAT value to the estimated DSI value in the subject, the equation, y = 1.3816(Ln x) 2 The formula includes +9.2339(Ln x)+20.196, y = estimated DSI value, and x = STAT value in the subject. As shown in Figure 12C, this equation is obtained in a study of n=1783, R 2 The result was 0.8684.

[0041] In some embodiments, the liver impairment or liver disease in the subject is selected from the group consisting of chronic hepatitis C, chronic hepatitis B, cytomegalovirus, Epstein-Barr virus, alcoholic liver disease, amiodarone toxicity, methotrexate toxicity, nitrofurantoin toxicity, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hemoglobinosis, Wilson's disease, autoimmune chronic hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis (PSC), and hepatocellular carcinoma (HCC).

[0042] In some embodiments, estimated portal vein HFR values ​​or estimated DSI values ​​in subjects are used to screen patients for liver function or liver disease, to monitor patients with liver disease receiving antiviral therapy, to monitor disease progression in patients with chronic liver disease, to determine disease stage in patients diagnosed with HCV or PSC, to prioritize patients with liver disease for liver transplantation, to determine the selection of patients with chronic hepatitis B who should receive antiviral therapy, to assess the risk of liver decompensation in hepatocellular carcinoma (HCC) patients undergoing evaluation for hepatectomy, to identify subgroups of patients on waiting lists with low MELD (model of end-stage liver disease score) who are at risk of dying while waiting for an organ donor, as an endpoint in clinical trials, to replace liver biopsy in pediatric populations, to track allograft function, to measure liver function recovery in living donors, to measure functional impairment in biliary liver disease in subjects, to initiate treatment or intervention in patients, or to identify HCV patients in early stage F0-F2 in combination with ALT.

[0043] In some embodiments, a method is provided for assessing hepatic shunt and / or relative liver function in subjects who have, are suspected of having, or are at risk of having, liver impairment or chronic liver disease, the method comprising: (a) obtaining a plurality of blood or serum samples, the plurality of blood or serum samples being collected from subjects at intervals of less than 3 hours after the subjects have been orally administered a first identifiable compound and simultaneously administered a second identifiable compound intravenously; (b) quantifying the first and second identifiable compounds in the samples by a method including LC-MS / MS using MRM; and (c) formula AUC oral / AUC iv ×Dose iv / Dose oral A step of calculating the hepatic shunt in a subject using ×100%, wherein the formula is AUC oral AUC is the area under the curve for the serum concentration of the first identifiable compound. iv(d) a step of calculating the area under the curve of a second identifiable compound, and a step of comparing the hepatic shunt in the subject with one or more shunt cutoff values ​​established from a known patient population, wherein the hepatic shunt in the subject compared with one or more shunt cutoff values ​​is an indicator of the subject's relative liver function.

[0044] In some embodiments, the sample comprises blood or serum samples collected from the subject at two or more, three or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, or fifteen or more time points, preferably collected at intervals of about 90 minutes or less after administration, preferably collected at about 5, 20, 45, 60, and 90 minutes after administration of an identifiable compound.

[0045] In some embodiments, a method is provided for determining the portal vein HFR value in patients who have, are suspected of having, or are at risk of having chronic liver disease, and the method is: (i) a certain dose (dose oral Receiving multiple blood or serum samples collected from patients with or at risk of chronic liver disease after orally administering an identifiable compound of ) to the patient, wherein the samples were collected from the patient at intervals of less than 3 hours after administration, (ii) Measuring the concentration of an identifiable compound in each sample, wherein the measurement includes processing the sample and analyzing the processed sample using MRM and LC-MS / MS to obtain the concentration of the identifiable compound. (iii) Generating individualized oral clearance curves from the concentrations of identifiable compounds in each sample, including using a computer algorithm curve that fits the model-identifiable compound clearance curve, (iv) Calculate the area under the individualized oral clearance curve (AUC) (mg / mL / min), divide the dose (mg) by the AUC of the orally administered identifiable compound to obtain the oral identifiable compound clearance in the patient, (v) Obtaining the patient's portal vein HFR value (mL / min / kg) by dividing the orally identifiable compound clearance by the patient's body weight in kg.

[0046] In some embodiments, determining the concentration of identifiable compounds in each sample involves analyzing the sample, including any suitable techniques known in the art. Any suitable known chromatographic techniques, spectroscopic techniques, or combinations of techniques may be used. For example, determining the concentration of identifiable compounds in a sample may involve analysis including chromatographic techniques, such as gas chromatography (GC) or liquid chromatography (LC). Any suitable known chromatographic techniques, or combinations of techniques, such as partition chromatography, normal-phase chromatography, displacement chromatography, reversed-phase chromatography, size exclusion chromatography, ion exchange chromatography, biocompatibility chromatography, or aqueous normal-phase chromatography, may be used. In certain embodiments, reversed-phase chromatography, such as C8 or C18 reversed-phase chromatography, may be used in the extraction and analysis columns. Detection and quantification of identifiable compounds in a sample may include high-performance liquid chromatography (HPLC), HPLC-diode array detection (HPLC-DAD), HPLC-fluorescence, ultra-high-performance liquid chromatography (UPLC), GC-MS, LC-MS, or LC-MS / MS. Mass spectrometry (MS) may be used alone or in combination with chromatographic techniques to quantify identifiable compounds in a sample. In some embodiments, LC-MS may be used to quantify identifiable compounds in a sample. LC-MS may use selected ion monitoring (SIM) over a specific mass range of atomic mass units (amu) that encompass the precise mass of the identifiable compounds. In other embodiments, the identifiable compounds may be radiolabeled compounds, for example, 3 H or14 It may be a compound radiolabeled using 1C. The analytical technique may include liquid scintillation counting (LSC). The identifiable compound may be a stable isotope-labeled compound, for example, 2 H or 13 It may be labeled with 1C. In some embodiments, LC-MS / MS is used. In certain embodiments, LC-MS / MS is used with multiple reaction monitoring (MRM) to quantify identifiable compounds in the sample. In certain embodiments, MS / MS is used with multiple reaction monitoring (MRM) to quantify identifiable compounds in the sample. In some embodiments, MS / MS is used without LC or GC. In some embodiments, MS / MS is used with LC or GC.

[0047] A method is provided for determining the whole-body HFR value in patients who have, are suspected of having, or are at risk of having chronic liver disease, and the method is as follows: (i) a certain dose (dose iv Receiving multiple blood or serum samples collected from patients with or at risk of chronic liver disease after intravenous administration of an identifiable compound of ) to a patient, wherein the samples were collected from the patient at intervals of less than 3 hours after administration, (ii) Measuring the concentration of identifiable compounds in each sample, including LC-MS / MS using MRM, (iii) Generating individualized intravenous clearance curves from the concentrations of identifiable compounds in each sample, including using a computer algorithm curve that fits the model-identifiable compound clearance curve, (iv) calculating the individualized area under the intravenous clearance curve (AUC) (mg / mL / min), dividing the dose (mg) by the AUC of the intravenously administered identifiable compound to obtain the intravenous identifiable compound clearance in the patient, and (v) dividing the intravenous identifiable compound clearance by the patient's body weight in kg to obtain the patient's whole-body HFR value (mL / min / kg).

[0048] In other embodiments, a method is provided for determining the disease severity index (DSI) value in a patient, and the method is: (a) Obtaining one or more liver function test values ​​in a patient having or at risk of having chronic liver disease, wherein the one or more liver function test values ​​are obtained from one or more liver function tests selected from the group consisting of SHUNT, portal vein hepatic filtration rate (portal HFR), and systemic hepatic filtration rate (systemic HFR), and the liver function test includes measuring identifiable compounds in a blood or serum sample from the subject by a method including LC-MS / MS using MRM as described in claim 1, (b) Obtaining a DSI value in a patient using a disease severity index equation (DSI equation), wherein the DSI equation includes one or more terms and a constant for obtaining the DSI value, wherein at least one term of the DSI equation independently represents the liver function test value in the patient from step (a) or the mathematically transformed liver function test value in the patient from step (a), and multiplying at least one term of the DSI equation by a coefficient specific to the liver function test.

[0049] Methods for estimating DSI values ​​in subjects may optionally include (c) comparing the patient's DSI value over time with one or more DSI cutoff values, one or more normal healthy controls, or one or more DSI values ​​within the patient.

[0050] In some embodiments, the mathematically transformed liver function test values ​​in the patient are selected from the logarithm, real number, natural logarithm, real number, or reciprocal of the liver function test values ​​in the patient.

[0051] In some embodiments, each term of the DSI equation independently represents the liver function test values ​​in the patient from step (a), or the mathematically transformed liver function test values ​​in the patient from step (a).

[0052] In some embodiments, the DSI equation is: The equation is DSI = f(shunt, portal vein HFR, whole-body HFR), where shunt is the shunt value in the subject, portal vein HFR is the portal vein HFR value in the subject, and whole-body HFR is the whole-body HFR value in the subject. The equation is DSI = A (shunt) + B (log portal vein HFR) + C (log whole-body HFR) + D, where A = a value between 5 and 6, or between 5.2 and 5.8; B = a value between 6 and 8, or between 6.5 and 7.5; C = a value between 8 and 10, or between 8.5 and 9.5; and D = a value between 40 and 60, or between 44 and 55.

number

[0053] In some embodiments, comparing a patient's DSI value to one or more DSI cutoff values ​​indicates at least one clinical outcome. In some embodiments, the clinical outcome or clinical event is selected from the group consisting of Child-Turcotte-Pugh (CTP) elevation, varicose veins, encephalopathy, ascites, and liver-related death.

[0054] In some embodiments, comparing DSI values ​​within a patient over time is used to monitor the effectiveness of treatment for chronic liver disease in the patient, and a decrease in DSI values ​​over time within the patient indicates the effectiveness of the treatment.

[0055] In some embodiments, comparing DSI values ​​in patients over time is used to monitor the need for treatment of chronic liver disease in patients, where an increase in DSI values ​​over time in a patient indicates the need for treatment in that patient.

[0056] In some embodiments, treatment of chronic liver disease in patients is selected from the group consisting of antiviral therapy, antifibrotic therapy, antibiotics, immunosuppressive therapy, anticancer therapy, FXR agonists, ursodeoxycholic acid, insulin sensitizers, interventional therapy, liver transplantation, lifestyle modifications and dietary restrictions, hypoglycemic index diet therapy, antioxidants, vitamins, transjugular intrahepatic portosystemic shunt (TIPS), catheter-guided thrombolysis, balloon dilation and stent placement, balloon dilation and drainage, weight loss, exercise, and alcohol avoidance.

[0057] In some embodiments, comparing intracellular DSI values ​​over time is used to monitor the state of chronic liver disease in patients, and changes in intracellular DSI values ​​over time are used to inform patients of the state of the disease and the risk of future clinical outcomes, with an increase in intracellular DSI values ​​over time indicating a worse prognosis, and a decrease in intracellular DSI values ​​over time indicating a better prognosis.

[0058] In some embodiments, a method is provided for estimating the clinical event rate in patients with chronic liver disease, the method comprising obtaining the patient's baseline DSI value (dsi0) and, optionally, the patient's repeated DSI value (dsi0). T This includes obtaining a DSI value where T = the number of months between baseline sample collection and repeated DSI sample collection, and calculating an estimated per capita annual event of observations and optionally repeated DSI values ​​as a function of baseline DSI values. In some embodiments, the calculation includes a Poisson regression model equation.

[0059] In some embodiments, the Poisson regression model equation is: Y = β0 + β1X1 + β2X2 + β3X3, in the equation, Y = log(ln(rate)) of event rats, X1, X2, and X3 are dsi0, dsiT, (dsi T -dsi0), and (dsi T A variable selected from the group consisting of *dsi0). β0 (intercept), β1, β2, and β3 are regression coefficients.

[0060] In some embodiments, regression coefficients are obtained from clinical studies of multiple patients with chronic liver disease and defined clinical event rates over time. In some embodiments, clinical events are selected from a group consisting of Childs-Turcotte-Pugh 2 point score progression (CTP+2), variceal bleeding, ascites, encephalopathy, or death.

[0061] In some embodiments, calculating the estimated per capita annual events of observations as a function of baseline DSI values ​​and optionally iterated DSI values ​​involves a Poisson regression model equation, which is: Y=β0+β1dsi0, Y = β0 + β1dsi0 + β2dsi T , Y = β0 + β1dsi0 + β2dsi24, Y = β0 + β1dsi0 + β2dsi T +β3(dsi0*dsi T ), Y=β0+β1dsi0+β2dsi24+β3(dsi0*dsi24), Y = β0 + β1dsi0 + β2dltaDSI, Y = β0 + β1dsi0 + β2(Δdsi), Y = β0 + β1dsi0 + β2(dsi) T -dsi0), and Selected from the group consisting of Y = β0 + β1dsi0 + β2(dsi24 - dsi0), in the formula, dsi0 is the baseline DSI value for the subject. dsi T This is the DSI value at T months in the subject, dsi24 is the DSI value over 24 months for the subject. dltaDSI=Δdsi=(dsi T -dsi0) and Y = log(ln(rate)) of event rats, β0 (intercept), β1, β2, and β3 are regression coefficients.

[0062] In some embodiments, a method is provided for estimating baseline or repeated DSI values ​​in a subject, the method comprising: obtaining a blood or serum sample from a subject after orally administering a composition containing an identifiable compound to the subject, wherein the blood or serum sample is collected from the subject less than 3 hours after orally administering the identifiable compound to the subject; and measuring the concentration of the orally administered identifiable compound in the blood or serum sample from the subject, the method comprising quantifying the concentration of the identifiable compound in the sample, including LC-MS / MS. Optionally, the blood or serum sample may consist of a single blood or serum sample.

[0063] In some embodiments, a method is provided for estimating the clinical event rate in patients with chronic liver disease, the method comprising: obtaining the patient's baseline DSI value (dsi0); and calculating an estimated per capita annual event rate as a function of the baseline DSI value. In some embodiments, the calculation includes a Poisson regression model equation Y = β0 + β1dsi0, where β0 is a coefficient in the range of -7.359 to -5.279, optionally β0 = -6.2997; β1 is a coefficient in the range of 0.107 to 0.191, optionally β1 = 0.1498; and dsi0 = the baseline DSI value from the patient.

[0064] In some embodiments, a method is provided for estimating the clinical event rate in patients with chronic liver disease, the method comprising obtaining the patient's baseline DSI value (dsi0) and the patient's repeated DSI value (dsi0). T This includes obtaining a DSI, where T = the number of months between baseline sample collection and repeated DSI sample collection, and calculating the estimated per capita per year of observation as a function of baseline DSI values. In some embodiments, the calculation is performed using a Poisson regression model equation. The formula includes Y = β0 + β1dsi0 + β2(dsi24 - dsi0), and in the equation, β0 is a coefficient in the range of -8.417 to -6.057, and is arbitrarily set to β0 = -7.2008, and β1 is a coefficient in the range of 0.127 to 0.217, and is arbitrarily set to β1 = 0.1726, and β2 is a coefficient within the range of 0.092 to 0.185, and can be arbitrarily selected. β2 = 0.1395, dsi0 = patient's baseline DSI value, and dsi24 = repeated DSI value for patients from whom patient samples were taken 24 months after baseline.

[0065] In some embodiments, a method is provided for providing a baseline or subsequent DSI value in a subject, the method comprising: obtaining a blood or serum sample from a subject after simultaneous oral and intravenous administration of first and second compositions containing an identifiable compound to the subject, wherein the blood or serum sample is collected from the subject less than 3 hours after oral administration of the identifiable compound to the subject; and measuring the concentration of the orally and intravenously administered identifiable compound in the blood or serum sample from the subject, the measurement comprising quantifying the concentration of the identifiable compound in the sample by LC-MS / MS.

[0066] A method is provided for monitoring the effectiveness of treatment for chronic liver disease in patients requiring treatment for chronic liver disease, the method comprising: determining a baseline disease severity index (DSI) value in the patient before treatment; determining at least one subsequent DSI value in the patient after initiation of treatment; and comparing the at least one subsequent DSI value to the baseline DSI value, wherein a decrease in the at least one subsequent DSI value compared to the baseline DSI value in the patient indicates treatment effectiveness in the patient. Determining the DSI value may involve measuring the concentration of one or more identifiable compounds in a blood or serum sample from the patient, including LC-MS / MS or MS / MS without LC.

[0067] In some embodiments, a decrease in at least one subsequent DSI value over time compared to the baseline DSI value indicates improved liver function, improved portal circulation, reduced portosystemic shunt, reduced hepatic fibrosis, reduced Ishak fibrosis score, reduced disease severity, and / or reduced risk of clinical outcomes in the patient. The decrease in at least one subsequent DSI value over time compared to the baseline DSI value in the patient may be at least approximately -1.5 points, at least approximately -2 points, or at least approximately -3 points. In another embodiment, an increase or no change in at least one subsequent DSI value over time compared to the baseline DSI value indicates that the patient is unresponsive to treatment.

[0068] In some embodiments, DSI can be used as an endpoint in clinical trials. For example, when DSI is used as an endpoint, a significant treatment response in a given patient may be defined as a decrease of 2 points or more in DSI values ​​over time, e.g., during and after treatment. The proportion of responders can be compared between the treatment group and the placebo group. The proportion of responders using DSI as an endpoint may also be compared with the proportion of responders using other tests as endpoints. Other tests, such as standard clinical tests, clinical models (e.g., MED score and CTP score), liver biopsy, hepatic venous pressure gradient (HVPG), magnetic resonance imaging (MRI), computed tomography perfusion imaging, and other imaging tests, may be insensitive or nonspecific. They may not adequately assess liver improvement after the suppression of necrotizing inflammation by treatment. In contrast, this method for determining systemic hepatic filtration rate (HFR), portal HFR, SHUNT, and DSI specifically targets cholate uptake and uses a single 90-minute non-invasive test to quantify systemic circulation, portal circulation, and portosystemic shunt, and derive DSI values ​​in a complete human subject. This method allows for real-time measurement of improvements in liver function that occur after successful therapy.

[0069] In another embodiment, an increase in at least one subsequent DSI value over time compared to a baseline DSI value may be used as an indicator of worsening liver function, worsening portal circulation, increased portosystemic shunts, increased hepatic fibrosis, increased Ishak fibrosis score, increased disease severity, and / or increased risk of clinical outcomes in the patient, and optionally, an increase in at least one subsequent DSI value over time compared to baseline is at least about 1 point.

[0070] A method is provided for determining a patient's DSI value, which includes obtaining one or more identifiable compound test results from the patient, including SHUNT values, STAT values, portal vein hepatic filtration rate (portal HFR) values, and / or systemic hepatic filtration rate (systemic HFR) values, and deriving a Disease Severity Index (DSI) value from these SHUNT values, STAT values, portal HFR, systemic HFR values, and / or systemic SHUNT values. Obtaining identifiable compound test results may include quantifying one or more identifiable compounds by LC-MS / MS.

[0071] In some embodiments, a kit of components is provided for determining one or more of the following in subjects having, suspected of having, or currently having liver impairment: STAT, portal vein HFR, systemic HFR, SHUNT, cholate removal rate, RCA20, DSI value, algebraic HR value, and / or indexed HR, the kit comprising a first component comprising one or more vials, each vial comprising a first composition comprising a single oral dose of a first identifiable compound.

[0072] The kit may further include a microsampling device, optionally comprising components selected from the group consisting of dry blood spot filter paper, capillary tubing, and volumetric microsampling devices.

[0073] The kit may further comprise a second component comprising one or more vials, each vial comprising a second composition containing a second identifiable compound in a single intravenous dose.

[0074] The kit may further comprise a third component comprising one or more vials, each vial containing a certain amount of human albumin for mixing with a single intravenous dose of a second identifiable compound before intravenous administration. The human albumin may be human serum albumin. The second composition may optionally further comprise human albumin pre-mixed with the second identifiable compound.

[0075] The kit may further comprise a fourth component comprising one or more sample collection tubes and / or transport vials, and a fifth component comprising suitable container means. The kit may comprise a sample collection tube comprising one or more sets of sterile blood-serum sample collection tubes, each set comprising enough tubes to collect multiple samples from a subject over a period of 180, 90, 60, or 45 minutes after administration of the first and second identifiable compounds.

[0076] The kit may include a first and a second identifiable compound, which is independently selected from the group consisting of identifiable bile acids, bile acid conjugates, and bile acid analogs. The first and second identifiable compounds may be stable isotope-labeled identifiable bile acids. The first and second stable isotope-labeled identifiable bile acids are 2,2,4,4- 2 H-cholic acid and 24- 13 It can be selected from C-cholic acid.

[0077] The first composition and / or the second composition may further independently comprise one or more components selected from the group consisting of pharmaceutically acceptable additives, diluents, colorants, fragrances, buffer compounds, pH adjusters, and excipients. In some embodiments, the diluent may be selected from water, sodium bicarbonate solution, non-citrus juice, or physiological saline (NS).

[0078] The first and / or second composition may contain sodium bicarbonate. The first and second compositions may independently be in a form selected from powder or solution. Both the first and second compositions may be in solution form. In some embodiments, the first composition may optionally contain a first identifiable bile acid and sodium bicarbonate, wherein the first identifiable bile acid is 2,2,4,4- 2 It is H-cholic acid. In some embodiments, the second composition optionally comprises a second identifiable bile acid and sodium bicarbonate, wherein the second identifiable bile acid is 24- 13 It is C-cholic acid.

[0079] The kit may include containers selected from one or more of the group consisting of plastic containers, reagent containers, vials, tubes, flasks, and bottles.

[0080] The kit may include a shipping box, labels, instructions for use, accompanying documents, lancets, capillary tubing, syringes, indwelling catheters, three-way stopcocks, timers, and transfer pipettes.

[0081] For example, the kit may include a shipping box containing a single box for both shipping vials to healthcare professionals and shipping samples from healthcare professionals to a reference laboratory for analysis. [Brief explanation of the drawing]

[0082] [Figure 1] A schematic diagram of portal vein HFR and SHUNT tests is shown. Healthy control subjects (upper panel) generally show low SHUNT, high portal vein HFR, and high systemic HFR, while subjects with liver disease (lower panel) show higher SHUNT, lower portal vein HFR, and lower systemic HFR. [Figure 2A]The chemical structures and ring numbering systems for C24 bile acids and C27 bile acids, as well as cholic acid, a representative C24 bile acid also known as 3α,7α,12α-trihydroxy-5β-colan-24-acid, are shown. Salts of cholic acid are called cholates. [Figure 2B] The graph shows typical cholic acid calibration curves for 0.1 μM to 10 μM for responses obtained using LC-MS / MS, with y = 2.2467x + 0.68 and R² = 0.9995. [Figure 3] Representative ion chromatograms of cholic acid (left panel) at the limit of quantification (LLOQC of 0.10 μmol / L in serum lot pool #4) monitored at a retention time of 4.04 minutes with m / z = 407.3 → 343.1, and internal standard D5-CA (right panel) at a retention time of 4.03 minutes with m / z = 412.3 → 290.2, showing the time versus peak intensity cps. The ion chromatogram on the left shows the MS / MS signal of cholic acid, and the ion chromatogram on the right shows the MS / MS signal of the internal standard cholic acid-D5. Note that the X-axis scales are different because the Analyst software always adjusts the X-axis scale based on the highest peak. [Figure 4] Representative ion chromatograms are shown for cholic acid (left panel) at the upper limit of quantitative QC (ULOQC of 10.0 μmol / L in serum lot pool #4) monitored at a retention time of 4.04 minutes with m / z = 407.3 → 343.1, and for the internal standard D5-CA (right panel) at a retention time of 4.03 minutes with m / z = 412.3 → 290.2. [Figure 5] The typical 13C-cholic acid calibration curves for 0.1 μM to 10 μM 13C-cholic acid in response to LC-MS / MS are shown, with y = 2.4233x - 0.038 and R² = 0.9981. [Figure 6]Representative ion chromatograms are shown for 13C-cholate (m / z = 408.25 [MH] → 343.1) (left panel) at the limit of quantification QC (LLOQ of 0.10 μmol / L in serum lot pool #4), and for the internal standard D5-CA (right panel) at a retention time of 4.03 minutes with m / z = 412.3 → 290.2. [Figure 7] Representative ion chromatograms are shown for 13C-cholate (m / z = 408.25 [MH] → 343.1) (left panel) at the upper limit of quantitative QC (ULOQC of 10.0 μmol / L in serum lot pool #4), and for the internal standard D5-CA (right panel) at a retention time of 4.03 minutes with m / z = 412.3 → 290.2. [Figure 8] The typical cholic acid-D4 calibration curves for 0.1 μM to 10 μM cholic acid-D4 responses obtained using LC-MS / MS are shown, with y = 1.7102x - 0.0357 and R² = 0.999. [Figure 9] Representative ion chromatograms are shown for cholate-D4 (411.25 [MH] → 347.100 Da) (left panel) at the limit of quantification QC (LLOQ of 0.10 μmol / L in serum lot pool #4), and for the internal standard D5-CA (right panel) at a retention time of 4.03 minutes with m / z = 412.3 → 290.2. [Figure 10] Representative ion chromatograms are shown for cholate-D4 (411.25 [MH] → 347.100 Da) (left panel) at the upper limit of quantitative QC (ULOQC of 10.0 μmol / L in serum lot pool #4), and for the internal standard D5-CA (right panel) at a retention time of 4.03 minutes with m / z = 412.3 → 290.2. [Figure 11]This figure shows the connection and position of the chromatography column switching valve between the preparation extraction column and the analysis column in the LC-MS / MS system. Figure 11A shows valve position 1, where HPLC pump I injects the sample into the extraction column as it flows through the injector. Figure 11B shows valve position 2, where HPLC pump II backflushes the extraction column onto the analysis column, which is eluted into the API4000MS / MS system where MRM monitoring is used. [Figure 12A] The accuracy and correlation (R²=0.8965) of STAT testing at 60 minutes to FLOW testing in early-stage CHC patients are presented, along with equations for interconverting log STAT and log FLOW values ​​to obtain estimated flow rates. [Figure 12B] A graph showing one exemplary relationship between DSI values ​​and STAT values ​​in n=1363 subjects and n=1736 tests is presented. The equation for the DSI vs. STAT relationship is derived, where y = 9.4514 ln(x) + 21.12, and x = STAT value (μM adjusted for a 75kg body weight) and y = DSI value. R² = 0.8499. [Figure 12C] A graph showing another exemplary relationship between DSI and STAT values ​​in n=1783 tests is shown. The equation for the DSI vs. STAT relationship is derived, where y = 1.3816(Ln x)² + 9.2339(Ln x) + 20.196, where x = STAT value (μM adjusted for a 75kg body weight) and y = DSI value. R² = 0.8684. [Figure 13A]The correlations between scoring systems are shown for FLOW and Ishak scoring, SHUNT and Ishak scoring, FLOW and Metavir scoring, and SHUNT and Metavir scoring, respectively. Figure 13A shows the results of previously disclosed FLOW testing in healthy controls and all stages of CHC. Data from HALT-C (late-stage CHC, stable and compensated, Ishak F2-6) were combined with data from early CHC studies (healthy controls (C) and early-stage CHC, Ishak F1-2) and studies of healthy donors in living donor liver transplantation (healthy controls (C)). F2 patient data were combined because they did not differ between studies. Portal venous blood flow (mean + / - SEM) in healthy controls and patients of all stages of CHC was graphed as a continuous feature demonstrating the ability to assess the full spectrum of the disease. The n for each group is shown above its symbol. HepQuant FLOW testing may enhance the early detection of liver disease, where it is most treatable. [Figure 13B] Figure 13B shows previously disclosed SHUNT test data for healthy controls and all stages of CHC. Data from HALT-C were combined with data from early CHC studies (healthy controls (C) and early-stage CHC, Ishak F1-F2) and healthy donor studies in living donor liver transplantation (healthy controls (C)). F2 patient data were combined as they did not differ between studies. Portosystemic shunt fractions (mean + / - SEM) for healthy controls and patients of all stages of CHC were graphed as a continuous feature demonstrating the ability to assess the full spectrum of the disease. The n for each group is shown above its symbol. The increased variability in F1 is due to the small number of patients diagnosed at this early stage. HepQuant SHUNT testing may enhance the early detection of liver disease, when most treatable. [Figure 13C]Figure 13C shows previously disclosed FLOW test data for healthy controls and all stages of CHC. Data from HALT-C (late-stage CHC, stable and compensated, METAVIR F1-4) were combined with data from early-stage CHC studies (healthy controls (C) and early-stage CHC, METAVIR F1) and healthy donor studies in living donor liver transplantation (healthy controls (C)). F1 patient data were combined because they did not differ between studies. Portal venous blood flow (mean + / - SEM) for healthy controls and patients of all stages of CHC was graphed as a continuous feature demonstrating the ability to assess the entire spectrum of the disease. The value of n for each group is indicated above its symbol. [Figure 13D] Figure 13D shows previously disclosed SHUNT test data for healthy controls and all stages of CHC. Data from HALT-C (late-stage CHC, stable and compensated, METAVIR F1-4) were combined with data from early-stage CHC studies (healthy controls (C) and early-stage CHC, METAVIR F1) and healthy donor studies in living donor liver transplantation (healthy controls (C)). F1 patient data were combined because they did not differ between studies. Portosystemic shunt fractions (mean + / - SEM) for healthy controls and patients of all stages of CHC were graphed as a continuous feature demonstrating the ability to assess the entire spectrum of the disease. The n for each group is indicated above its symbol. [Figure 14A] This shows the HFR (portal vein HFR, FLOW) of PSC patients at various stages of the disease, compared to healthy controls. [Figure 14B] This shows SHUNT for PSC patients at various stages of the disease compared to healthy controls. [Figure 14C] This shows the STATs for PSC patients at various stages of the disease, compared to healthy controls. [Figure 15]FLOW and SHUNT test results for individual healthy controls and PSC patients are presented along with FLOW cutoff values ​​(5, 10, and 20 mL / min / kg for markedly severe, moderate, and mild disease, respectively) and SHUNT cutoff values ​​(26%, 43%, and 60%, respectively for mild, moderate, and markedly severe disease, respectively). [Figure 16] FLOW and SHUNT test results for individual healthy controls and HCV patients are presented along with FLOW cutoff values ​​(5, 10, and 20 mL / min / kg for markedly severe, moderate, and mild disease, respectively) and SHUNT cutoff values ​​(26%, 43%, and 60%, respectively for mild, moderate, and markedly severe disease, respectively). [Figure 17] This graph shows the relationship between a patient's DSI value and their maximum liver volume as a percentage. A higher DSI value indicates a lower percentage of maximum liver volume. [Figure 18] As provided in Example 9 of U.S. Patent No. 9,091,701, DSI correlates linearly with the Ishak fibrosis score (liver biopsy, left panel) but is not affected by steatosis (fat biopsy score, right panel). The graph shows n for each data point. [Figure 19] This study demonstrates the performance of DSI in identifying patients with future clinical outcomes compared to the Ishak fibrosis score (liver biopsy), platelet count (CBC), and MELD (a model of end-stage liver disease scoring). At the optimal cutoff, DSI remarkably outperformed other standard diagnostic methods, including liver biopsy and MELD, for predicting future clinical outcomes. Specifically, DSI exhibited the best sensitivity, specificity, PPV, and NPV compared to liver biopsy, platelet count, and MELD. [Figure 20]This plot shows cholate test results for non-cirrodegenerative chronic hepatitis C patients (Ishak F2,3,4, n=19,63,45) with DSI cutoff values ​​of 15, 25, and 35 for mild, moderate, and severe disease, respectively. The results for cholate-based tests SHUNT (%), whole-body HFR (mL / min / kg), portal vein HFR (mL / min / kg), and DSI are shown. Portal vein HFR is plotted on the X-axis, whole-body HFR on the Y-axis, the ratio of whole-body HFR to portal vein HFR (SHUNT) is represented by a diagonal line, and DSI is shown in a shaded area. Surprisingly, non-cirrode patients with high DSI are at higher risk of outcomes, as discussed in Example 10 of U.S. Patent No. 9,091,701, where portal vein HFR, whole-body HFR, and SHUNT were measured by methods including HPLC-MS by SIM. [Figure 21] This plot shows cholate test results for non-cirrodegenerative chronic hepatitis C patients (Ishak F5,6, n=48,49) with DSI cutoff values ​​of 15, 25, and 35 for mild, moderate, and severe disease, respectively. The results for cholate-based tests SHUNT (%), whole-body HFR (mL / min / kg), portal vein HFR (mL / min / kg), and DSI are shown. Portal vein HFR is plotted on the X-axis, whole-body HFR on the Y-axis, the ratio of whole-body HFR to portal vein HFR (SHUNT) is represented by a diagonal line, and DSI is shown in a shaded area. Surprisingly, as discussed in Example 10 of U.S. Patent No. 9,091,701, patients with cirrhosis and low DSI have a lower risk of outcome, as portal vein HFR, whole-body HFR, and SHUNT were measured by methods including HPLC-MS by SIM. [Figure 22]The plot shows cholate test results for patients with primary sclerosing cholangitis (PSC) and healthy controls. Portal vein HFR is plotted on the X-axis, systemic HFR on the Y-axis, the ratio of systemic HFR to portal vein HFR (SHUNT) is represented by a diagonal line, and DSI is shown in the shaded area. Predictive DSI cutoffs for mild, moderate, and severe PSC diseases, including varicose veins and decompensation, 14, 18, and 36, as disclosed in U.S. Patent No. 9,091,701, where portal vein HFR, systemic HFR, and SHUNT were measured by methods including HPLC-MS by SIM, are shown at the interface between zones. [Figure 23] This plot shows DSI versus MELD scores in PSC patients on the waiting list for liver transplantation. DSI was superior to MELD in assessing the risk of complications and prioritizing liver transplantation for PSC patients. Despite lower MELD scores, PSC patients with DSI > 20 developed portal hypertension-related complications, and PSC patients with DSI > 40 required liver transplantation, as disclosed in U.S. Patent No. 9,091,701, where portal vein HFR, systemic HFR, and SHUNT were measured by methods including HPLC-MS by SIM. [Figure 24] The graph shows patients who achieved SVR compared to their quartiles for liver function. The probability of SVR is most correlated with DSI, as discussed in Example 13 of U.S. Patent No. 9,091,701, where portal vein HFR, systemic HFR, and SHUNT were measured by methods including HPLC-MS by SIM. 230 chronic HCV patients (Ishak F2-6) enrolled in the HALT-C trial, characterized by progressive fibrosis and failure of previous treatment with interferon-based therapy, were examined at baseline and then re-treated with PEG / RBV. Patients who achieved a sustained virological response (SVR) (n=32, including 5 cirrhosis) and non-responders (NR) were re-examined at 2 years. Examination could predict a sustained virological response (SVR) to pegylated interferon / ribavirin (PEG / RBV) and measure improvement in liver function in those who achieved SVR. [Figure 25]This graph shows baseline and continuous DSI values ​​for 13 patients ultimately diagnosed with HCC from the HALT-C supplementary study. The dashed line near the bottom of the graph represents the DSI 18.3 cutoff value. 12 out of 13 HCC cases had a baseline DSI > 18.3. The relative risk of HCC for a DSI > 18.3 is 11.4. [Figure 26] This graph shows the estimated baseline and continuous estimated DSI values ​​for 13 patients ultimately diagnosed with HCC from the HALT-C supplementary study. Estimated DSI values ​​were obtained from STAT values ​​using an equation. 12 out of 13 HCC cases had a baseline estimated DSI > 18.3. The relative risk of HCC for an estimated DSI > 18.3 is 11.4. [Figure 27] This graph shows the survival rates of 220 HALT-C patients, divided by baseline DSI tertile versus study year. Patients in tertile (A) had a baseline DSI value <15.395, patients in (B) had a DSI value between 15.395 and 19.898, and patients in (C) had a DSI value >19.898. The number of subjects for each DSI tertile for each study year is shown below the graph. The shaded areas in the graph indicate the 95% confidence interval for each tertile. [Figure 28] The predicted event rates for each of the four Poisson regression models for all 188 subjects are shown as a function of the baseline DSI. Note that models B and D have the same predicted values. This is expected, as shown in the explanatory equation for model D. [Figure 29] A coincident plot is shown regarding the relationship between baseline and 24-month DSI values ​​in 188 HALT-C patients. The plot shows the difference (dsi24-dsi0) versus (dsi0+dsi24) / 2. The coincident plot shows regression to the mean but has a positive slope. Individuals with lower baseline DSIs tended to have lower DSIs at 24 months, while those with higher baseline DSIs tended to have higher DSIs at 24 months. [Figure 30] The LUXON® MS / MS parameters are shown, including ionization mode: positive, flow rate: 6 L / min, and gas: air. [Figure 31] This shows an exemplary desorption peak of the C0.1d4-CA (d4-cholic acid) standard 413.4 / 359.4 (large peak), which has an internal standard d5-CA-245 (414.4 / 245.1) (inserted peak). [Figure 32] The image shows two exemplary mass spectra of the generated ion (MS2)12CA at 355.40 m / z, Da (left panel), and the generated ion (MS2) of d5-DA at 360.40 m / z, Da (right panel), along with their intensity, positive modes, and cps vs. m / z (mass-to-charge ratio). [Figure 33A] The graphs show the concentration ratio to area ratio from MS / MS without LC for standard samples of 12C-CA at concentrations of 0.1 μM, 0.2 μM, 0.6 μM, 1 μM, 2 μM, 6 μM, and 10 μM (n=3 for each). For 12C-CA, the linearity exceeded 0.99. [Figure 33B] The graphs show the concentration ratio to area ratio from MS / MS without LC for standard samples of 13C-CA at concentrations of 0.1 μM, 0.2 μM, 0.6 μM, 1 μM, 2 μM, 6 μM, and 10 μM (n=3 for each). For 13C-CA, the linearity exceeded 0.99. [Figure 33C] The graphs show the concentration ratio to area ratio from LC-free MS / MS for standard samples of d4-CA at concentrations of 0.1 μM, 0.2 μM, 0.6 μM, 1 μM, 2 μM, 6 μM, and 10 μM (n=3 for each). For d4-CA, the linearity exceeded 0.99. [Figure 34] Figure 34A shows the dose-normalized plasma unconjugated OCA AUC (0-24 hours) (h*ng / mL / mg) at baseline (day 1) in the clinical study. The circles represent observational data, and the red line represents linear regression fitting. R² = 0.394. OCA = obeticholic acid. Figure 34B shows the dose-normalized plasma unconjugated OCA AUC (0-24 hours) (h*ng / mL / mg) at the end of treatment (day 85) in the clinical study. The circles represent observational data, and the red line represents linear regression fitting. R² = 0.424. OCA = obeticholic acid. Oveticholic acid plasma exposure was associated with DSI measurement. [Figure 35]Figure 35A shows graphs of mean antipyrinx clearance for patients divided into three groups based on Child-Pugh's CP A5, CP A6, and CP B classes. Patients in CP B class showed lower mean antipyrinx clearance compared to the CP A5 and CP A6 patient groups. Figure 35B shows graphs of mean antipyrinx clearance for CP A5 subjects (N=85) from Figure 35A, subdivided into four groups based on DSI scores of 5-15, 15-25, 25-35, and 35-45. Patients with higher DSI scores showed lower mean antipyrinx clearance. Figure 35C shows graphs of mean antipyrinx clearance for CP A6 subjects (N=53) from Figure 35A, subdivided into four groups based on DSI scores of 5-15, 15-25, 25-35, and 35-45. Patients with higher DSI scores exhibited lower mean antipyrinx clearances. Figure 35D shows graphs of mean antipyrinx clearances for CP B subjects (N=12) from Figure 35A, subdivided into four groups based on DSI scores of 5–15, 15–25, 25–35, and 35–45. Patients with higher DSI scores generally exhibited lower mean antipyrinx clearances. [Figure 36]Figure 36A shows graphs of mean methionine breath test results for patient groups divided by Child-Pugh scores A5, A6, and B. Patients with CP class B showed lower mean methionine breath test values ​​compared to CP A5 and CP A6 patient groups. Figure 36B shows graphs of mean methionine breath test results for Child-Pugh A5 patients (N=105), subdivided into four groups based on DSI scores in the ranges of 5-15, 15-25, 25-35, and 35-45. Generally, patients in the higher DSI score groups showed lower mean 13CO2 breath scores, regardless of CP class. Figure 36C shows graphs of mean methionine breath test results for Child-Pugh A6 patients (N=63), subdivided into four groups based on DSI scores in the ranges of 5-15, 15-25, 25-35, and 35-45. Generally, patients with higher DSI scores, regardless of CP class, showed a mean 13CO2 exhaled score decrease. Figure 36D shows a graph of the mean methionine exhaled test results of Child-Pugh class B patients (N=15), subdivided into four groups based on DSI scores in the ranges of 5–15, 15–25, 25–35, and 35–45. Generally, patients with higher DSI scores, regardless of CP class, showed a mean 13CO2 exhaled score decrease. [Figure 37]Figure 37A shows a graph of the mean caffeine removal rates for patient groups divided by Child-Pugh scores A5, A6, and B. Patients with CP class B showed a lower mean caffeine removal rate compared to CP A5 and CP A6 patient groups. Figure 37B shows a graph of the mean caffeine removal rates for Child-Pugh A5 patients (N=97), further subdivided into four groups based on DSI scores in the ranges of 5-15, 15-25, 25-35, and 35-45. Generally, patients in higher DSI score groups showed a lower caffeine removal rate regardless of CP class. Figure 37C shows a graph of the mean caffeine removal rates for Child-Pugh A6 patients (N=58), further subdivided into four groups based on DSI scores in the ranges of 5-15, 15-25, 25-35, and 35-45. Generally, patients with higher DSI scores, regardless of CP class, exhibited reduced caffeine removal rates. Figure 37D shows a graph of the mean caffeine removal rates of Child-Pugh class B patients (N=13), further subdivided into four groups based on DSI scores in the ranges of 5–15, 15–25, 25–35, and 35–45. Generally, patients with higher DSI scores, regardless of CP class, exhibited reduced caffeine removal rates. [Figure 38]Figure 38A shows graphs of the mean MEGX 15-minute concentrations after lidocaine administration for three Child-Pugh score groups: CP A5, CP A6, and CP B. Patients in class CP B showed a decrease in mean MEGX 15-minute concentrations compared to the CP A5 and CP A6 patient groups. Figure 38B shows graphs of the mean MEGX 15-minute concentrations for Child-Pugh A5 patients (N=98), subdivided into four groups based on DSI scores in the ranges of 5-15, 15-25, 25-35, and 35-45. Generally, patients in different DSI score groups showed different mean MEGX 15-minute concentrations. Figure 38C shows graphs of the mean MEGX 15-minute concentrations for Child-Pugh A6 patients (N=60), subdivided into four groups based on DSI scores in the ranges of 5-15, 15-25, 25-35, and 35-45. Generally, patients in different DSI score groups exhibited different mean MEGX 15-minute concentrations. Figure 38D shows a graph of the mean MEGX 15-minute concentrations of Child-Pugh class B patients (N=13), subdivided into four groups based on DSI scores in the ranges of 5–15, 15–25, 25–35, and 35–45. Generally, patients in different DSI score groups exhibited different mean MEGX 15-minute concentrations. [Figure 39]Figure 39A shows a graph of mean galactose removal capacity versus Child-Pugh score for three groups: CP A5, CP A6, and CP B. Patients in class CP B showed a decrease in mean galactose removal capacity compared to the CP A5 and CP A6 patient groups. Figure 39B shows a graph of the mean galactose removal capacity of Child-Pugh A5 patients (N=104) subdivided into four DSI score groups based on DSI scores in the ranges of 5-15, 15-25, 25-35, and 35-45. Generally, patients in higher DSI score groups showed a decrease in mean galactose removal capacity. Figure 39C shows a graph of the mean galactose removal capacity of Child-Pugh A6 patients (N=64) subdivided into four groups based on DSI scores in the ranges of 5-15, 15-25, 25-35, and 35-45. Generally, patients with higher DSI scores exhibited a reduced mean galactose removal capacity. Figure 39D shows a graph of the mean galactose removal capacity of Child-Pugh class B patients (N=15), subdivided into four groups based on DSI scores in the ranges of 5–15, 15–25, 25–35, and 35–45. Generally, patients with higher DSI scores exhibited a reduced galactose removal capacity, regardless of CP class and score. [Figure 40] A summary of PK changes for five different drugs is presented, and patients were divided into DSI 5-15, DSI 15-25, DSI 25-35, and DSI 35-45 groups based on their DSI score for each of the following: antipyrinx clearance, methionine breath test, caffeine removal rate, lidocaine MEGX 15-minute concentration, and galactose removal. Generally, patients with the highest DSI scores (35-45) also exhibited the lowest mean PK values ​​for each of the following: antipyrinx clearance (Figure 40A), methionine breath test (Figure 40B), caffeine removal (Figure 40C), lidocaine MEGX 15-minute concentration (Figure 40D), and galactose removal ability (Figure 40E). [Figure 41]This is a functional map of indexed hepatic reserve in Fontan patients versus non-obese controls. Filled circles represent HR values ​​in individual Fontan patients (n=18), and open circles represent values ​​in individual non-obese controls. Fontan patients 17 and 20 exhibit poor liver function, with indexed HRs of only about 50% and 51%, respectively, compared to healthy non-obese controls. Fontan patient 16 exhibits an indexed HR of approximately 100% compared to non-obese controls. [Modes for carrying out the invention]

[0083] This specification provides an improved method for evaluating liver function in a patient, comprising rapidly and efficiently processing, detecting, and quantifying identifiable compounds from a patient's blood or serum sample.

[0084] A method is provided for estimating the risk of experiencing a clinical event within a one-year period for individual patients with chronic liver disease.

[0085] U.S. Patent No. 8,613,904, Everson et al., discloses a method for evaluating liver function in a patient, comprising obtaining a patient serum sample following administration of two identifiable stable isotope-labeled cholate compounds, and cumbersome sample preparation and analysis of the patient serum sample using GC-MS.

[0086] U.S. Patent No. 8,778,299, Everson, discloses a method for evaluating liver function, comprising obtaining a patient serum sample following administration of two identifiable stable isotope-labeled cholate compounds, and processing and analyzing the patient serum sample using HPLC-MS.

[0087] U.S. Patent No. 9,091,701, Everson discloses a method for determining liver function and obtaining a disease severity index (DSI) value in a patient, comprising obtaining a serum sample from the patient after administration of two identifiable stable isotope-labeled cholate compounds, and processing and analyzing the patient serum sample using HPLC-MS.

[0088] This specification provides improved methods for the rapid and efficient processing of patient samples, and for the extraction and quantification of identifiable compounds in patient blood or serum samples. For example, compared to prior art procedures, a method is provided in which unlabeled endogenous cholic acid is quantified not only in baseline samples but now in each individual sample. The original multi-step extraction procedure, including a combination of solid-phase extraction, liquid-liquid extraction, evaporation, and reconstitution, is replaced herein by an automated online extraction procedure. Detection and quantification are performed using MS / MS rather than MS using selective ion monitoring, based on analyte ion transitions in multiple reaction modes. The improved analysis makes it possible to utilize much smaller amounts of blood or serum sample at each time point. Thus, additional types of blood sample collection methods can be successfully utilized.

[0089] In this disclosure, mass spectrometry (MS or MS / MS) may be used alone or optionally in combination with chromatographic techniques to quantify identifiable compounds in a sample.

[0090] The advantages of the methods of this disclosure for analysis allow for much smaller blood or serum sample collection volumes than previously required for use in liver function tests such as the dual cholate SHUNT test, FLOW test, portal vein HFR, systemic HFR, STAT test, DSI test, RCA20, cholate removal rate, algebraic hepatic reserve, or indicator-based hepatic reserve test. A minimum of 5 microliters of blood per sample may be used. Each of these liver function tests requires administration of one or more identifiable compounds to a subject and collection of blood or serum samples at one, two or more, three or more, four or five or more time points, following oral and / or intravenous administration of one or more identifiable compounds.

[0091] Identifiable compounds. In some embodiments, one or more identifiable compounds may be administered to a subject by oral and / or intravenous administration. One or more blood or serum samples are obtained from the subject less than 3 hours after administration. The blood or serum samples are processed as provided herein, and one or more identifiable compounds are quantified in each sample as provided herein. Any identifiable compounds that are safely administered orally may have the following characteristics: approximately 100% absorption following oral administration, high hepatic extraction (>50%, >60%, >70%, >80%, or >90% in the first pass through the liver of a healthy subject), and are removed from blood or plasma solely by the liver. Identifiable compounds for the measurement of portal flow may be endogenous compounds or xenobiotics.

[0092] In some embodiments, the identifiable compound may be an identifiable bile acid. Human bile acids are generally C24 molecules consisting of a C19 cyclopentanophenanthrene (steroid) core and a carboxylate side chain. C27 molecules also exist. The basic structures and ring numbering systems of C24 and C27 bile acids are shown in Figure 2A. A representative C24 bile acid, cholic acid, is also shown. The structural diversity of human bile acids stems from several factors, namely (1) A / B ring fusion stereochemistry, cis / 5β-H or trans / 5α-H, (2) hydroxylation sites that can occur at C3, C6, C7 and C12, (3) glycine or taurine conjugates at the C24-carboxyl group, and (4) dehydrogenation and epimerization of the hydroxyl group. The first three factors are mainly due to host metabolism, and the last factor may be due to in vivo changes in the gut microbiota. Considering only host synthesis and metabolism of bile acids, 48 ​​possible BA species with oxidation sites at C3, C6, C7 and / or C12 can be produced. This number increases to 384 when considering the entire host-gut microbiota cometabolism. Lan et al., 2016, Anal Chem 88(14):7041-7048.

[0093] Identifiable bile acids may be endogenous bile acids, bile acid conjugates, labeled bile acids, isotope-labeled bile acids, or bile acid analogs. Identifiable bile acids include, for example, dehydrolithocholic acid (dehydroLCA), lithocholic acid (LCA), isodeoxycholic acid (isoDCA), isolithocholic acid (isoLCA), allolithocholic acid (alloLCA), glycolitocholic acid (GLCA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), taurolithocholic acid (TLCA), apocholic acid (apoCA), 23-nordeoxycholic acid (nor-DCA), 12-ketritocholic acid (12-ketoLCA), 7-Ketrithocholic acid (7-Ketol-LCA), 6,7-Diketritocholic acid (6,7-Dikeol-LCA), Glycodeoxycholic acid (GDCA), 6-Ketolitocholic acid (6-Ketol-LCA), Glycochenodeoxycholic acid (GCDCA), Hyodeoxycholic acid (HDCA), Ursodeoxycholic acid (UDCA), Cholic acid (CA), Taurodeoxycholic acid (TDCA), Alocholic acid (ACA), β-Hyodeoxycholic acid (β-HDCA), Murocholic acid (Muro-CA), Hyo Cholic acid (HCA), 12-dehydrocholic acid (12-DHCA), β-mulicolic acid (β-MCA), norcholic acid (NorCA), 7-ketodeoxycholic acid (7-ketoDCA), glycocholic acid (GCA), α-mulicolic acid (α-MCA), glycohyodeoxycholic acid (GHDCA), 3β-cholic acid (βCA), glycosodeoxycholic acid (GHCA), ω-mulicolic acid (ωMCA), taurocholic acid (TCA), glycohyodeoxycholic acid (GHCA), taurohyodeoxycholic acid It may be xycholic acid (THDCA), 7,12-diketritocholic acid (7,12-diketoLCA), dehydrocholic acid (DHCA), ursocholic acid (UCA), taurohyocholic acid (THCA), tauro-β-mulicolic acid (TβMCA), tauro-α-mulicolic acid (TαMCA), glycodehydrocholic acid (GDHCA), tauro-ω-mulicolic acid (TωMCA), taurohydrocholic acid (TDHCA), or isotope-labeled derivatives, analogs thereof, or epimers thereof.

[0094] For example, the identifiable bile acid may be an endogenous bile acid or a bile acid conjugate. The identifiable compound may be an identifiable cholate compound. The cholate compound may be selected from the following labeled compounds, namely cholic acid, any glycine conjugate of cholic acid, any taurine conjugate of cholic acid, chenodeoxycholic acid, any glycine conjugate of chenodeoxycholic acid, any taurine conjugate of chenodeoxycholic acid, deoxycholic acid, any glycine conjugate of deoxycholic acid, any taurine conjugate of deoxycholic acid, or litcholic acid, or any glycine or taurine conjugate thereof.

[0095] Cholates occur naturally and are not known to be harmful or adverse when administered intravenously or orally at the doses used in HQ testing. Serum cholate concentrations achieved by either intravenous or oral administration are similar to serum bile acid concentrations that occur after the intake of a fatty meal. Since cholates occur naturally in humans in pool sizes of 1–5 g, the 20 and 40 mg doses of labeled cholate used herein are unlikely to cause harm.

[0096] Identifiable bile acids may be labeled bile acids. Examples of labeled bile acids include radiolabeled bile acids, non-radiolabeled stable isotope-labeled bile acids, or fluorescently labeled bile acids. Examples of labeled bile acids include fluorescein lysicol trisodium salt (NRL-972 trisodium salt), fluorescein lysicol (NRL-972), (18)F-chenodeoxycholic acid, cholyl-lys-fluorescein (CLF), fluorescein isothiocyanate glycolate (FITC-GC), litcholyl-lysyl-fluorescein (LLF), and dansyl-labeled cholic acid.

[0097] Identifiable bile acids include, for example, taurochenodeoxycholic acid, sodium salt (taurine-2,2,3,4,4,6,6,7,8-D9-CDCA), taurochenodeoxycholic acid, sodium salt (taurine-2,2,4,4-D4-CDCA), taurocholic acid, sodium salt (taurine-13C2-CA), taurocholic acid, sodium salt (taurine-2,2,4,4-D4-CA), taurodeoxycholic acid, sodium The bile acid taurine conjugate may be a stable isotope-labeled conjugate selected from taurine-2,2,4,4,11,11-D6-DCA, taurodeoxycholic acid, sodium salt (taurine-2,2,4,4-D4-DCA), tauroursodeoxycholic acid, sodium salt (taurine-2,2,4,4-D4-UDCA), or tauroursodeoxycholic acid, sodium salt (taurine-12C2-UDCA).

[0098] Identifiable bile acids include, for example, glycochenodeoxycholic acid (glycine-2,2,3,4,4,6,6,7,8-D9-CDCA), glycochenodeoxycholic acid (glycine-2,2,4,4-D4-CDCA), glycocococholic acid (glycine-2,2,4,4-D4-CA), glycocococholic acid (glycine-1-13C-CA), and glycodeoxycholic acid (glycine-2,2,4,4,11,11 The bile acid glycine conjugate may be a stable isotope-labeled conjugate selected from -D6-DCA), glycodeoxycholic acid (glycine-2,2,4,4-D4-DCA), glycolicotocholic acid (glycine-2,2,4,4-D4-LCA), glycoursodeoxycholic acid (glycine-2,2,4,4-D4-UDCA), and glycoursodeoxycholic acid (glycine-13C2-UDCA).

[0099] Identifiable bile acids include, for example, α-mulicoleic acid (e.g., 2,2,3,4,4-D5-αMCA), β-mulicoleic acid (e.g., 2,2,3,4,4-D5-βMCA), chenodeoxycholic acid (e.g., 2,2,3,4,4,6,6,7,8-D9-CDCA), chenodeoxycholic acid (e.g., 2,2,3,4,4-D5-CDCA), chenodeoxycholic acid (e.g., 2,2,4,4-D4-CDCA), and chenodeoxycholic acid. The primary bile acids may be stable isotope-labeled primary bile acids selected from lic acid (e.g., 24-13C-CDCA), gamma-mulicoleic acid (e.g., 2,2,3,4,4-D5-γMCA), ω-mulicoleic acid (e.g., 2,2,3,4,4-D5-ωMCA), and cholic acid (e.g., 2,2,3,4,4-D5-CA, 2,2,4,4-D4-CA, 3,6,6,7,8,11,11,12-D8-CA, and 24-13C-CA).

[0100] The identifiable bile acids may be stable isotope-labeled secondary bile acids selected from, for example, deoxycholic acid (2,2,4,4,11,11-D6-DCA), deoxycholic acid (2,2,4,4-D4-DCA), deoxycholic acid (24-13C-DCA), glycoursodeoxycholic acid (glycine-13C2-UDCA), litcholic acid (2,2,4,4-D4-LCA), tauroursodeoxycholic acid, sodium salt (taurine-13C2-UDCA), ursodeoxycholic acid (2,2,4,4-D4-UDCA), and ursodeoxycholic acid (24-13C-UDCA).

[0101] The identifiable compounds may be bile acid analogs or epimers. The term “analog” refers to a structural analogue, also known as a chemical analogue, that has a similar structure to another but differs with respect to one or more components. For example, the bile acid analogue may be a synthetic or semi-synthetic bile acid analogue. The bile acid analogue may be obeticholic acid, also known as 6α-ethylchenodeoxycholic acid, or 3α,5β,6α,7α)-6-ethyl-3,7-dihydroxycolan-24-acid. Obeticholic acid (OCA) is an analogue of chenodeoxycholic acid, differing in that it has an ethyl moiety at the 6α position instead of a hydrogen residue. Chenodeoxycholic acid is an active physiological ligand of the farnesoid X receptor (FXR), which is involved in many physiological and pathophysiological processes. OCA is known to be an FXR agonist.

[0102] Identifiable compounds may be FXR agonists, such as obeticholic acid (OCA), chenodeoxycholic acid, or ethyl-3,7,23-trihydroxy-24-nor-5-colan-23-sodium sulfate.

[0103] The bile acid analog may be ursodeoxycholic acid (UDCA), also known as ursodiol. Ursodeoxycholic acid is the epimer of chenodeoxycholic acid. Ursodeoxycholic acid is thought to be a secondary bile acid that is a metabolite of intestinal bacteria. UDCA is known to be useful in the treatment of primary biliary cholangitis, in reducing gallstone formation, improving bile flow, and after bariatric surgery to prevent cholelithiasis caused by rapid weight loss due to cholesterol supersaturation in the bile.

[0104] The bile acid analog may be hyodeoxycholic acid (HDCA), also known as 3α,6α-dihydroxy-5β-colan-24-acid. Hydrodeoxycholic acid differs from deoxycholic acid in the position of its hydroxyl group; the 6α-hydroxyl group is located at position 12 in the former. HDCA is known as a secondary bile acid, a metabolite produced by intestinal bacteria.

[0105] In various embodiments, any bile acid or bile acid conjugate may be in the form of a physiologically acceptable salt, such as a sodium salt of cholic acid. In one embodiment, the term cholic acid refers to a sodium salt of cholic acid. In some preferred embodiments, cholic acid (cholate) is an identifiable cholate compound. As used herein, the terms cholate compound, cholate, and cholic acid are used interchangeably.

[0106] Examples of xenobiotics that can be administered orally and have high first-pass hepatic clearance include, but are not limited to, propanolol, nitroglycerin or derivatives of nitroglycerin, or galactose and related compounds.

[0107] In some embodiments, the identifiable compound is propranolol. Propranolol is a non-selective beta-blocker that has been shown to be effective in preventing variceal bleeding and rebleeding and is widely used as a pharmacotherapy for portal hypertension in patients with liver cirrhosis. (Suk et al. 2007, Effect of propranolol on portal pressure and systemic hemodynamics in patients with liver cirrhosis and portal hypertension: a prospective study. Gut and Liver 1(2):159-164). Propranolol is almost completely cleared by the liver. Total (+)-propranolol plasma clearance has been demonstrated to constitute a good estimate of hepatic blood flow in patients with normal liver function. (Weiss et al. 1978 (+)-Propranolol clearance, an estimation of hepatic blood flow in man, Br.J.Clin.Pharmacol. 5:457-460).

[0108] In other embodiments, the identifiable compound is isosorbide 5-nitrate. This compound is administered orally and can be detected in plasma, for example, by HPLC-EIMS. (Sun et al., High performance liquid chromatography-electrospray ionization mass spectrometric determination of isosorbide 5-mononitrate in human plasma, J.Chromatogr.B Analyt.Technol.Biomed.Sci.2007 Feb 1;846(1-2):323-8).

[0109] In some embodiments, the identifiable compound is galactose. Galactose elimination capacity (GEC) has been used as an indicator of residual liver function. In GEC testing, galactose is typically administered intravenously at a dose of 0.5 mg / kg, and intravenous samples are taken every 5 minutes over 20–60 minutes. In individuals with chronic liver disease and cirrhosis, galactose clearance is reduced. However, due to the fact that this carbohydrate has a high extraction rate, galactose metabolism depends on hepatic blood flow and hepatic function mass. (Tygstrup N, Determination of the hepatic elimination capacity (Lm) of galactose by a single injection, Scand J Lab Clin invest, 18 Suppl 92, 1966, 118-126). Since the carbohydrate galactose is metabolized almost exclusively in the liver, and near-saturated enzymatic conversion is achieved when the removal rate at blood concentrations is sufficiently high, GEC is used as a quantitative measure of hepatic metabolic capacity. One study showed that galactose elimination capacity (GEC) was a strong predictor of mortality among patients with newly diagnosed liver cirrhosis and decreased GEC. (Jepsen et al, 2009, The galactose elimination capacity and mortality in 781 Danish patients with newly-diagnosed liver cirrhosis: a cohort study. BMC Gastroenterol. 2009, 9:50).

[0110] In certain embodiments, one or more differentiateable isotopes are incorporated into a selected identifiable compound for use in assessing liver function. The differentiateable isotopes may be either radioactive isotopes or stable isotopes incorporated into the identifiable compound. (Stable isotopes) 13 C, 2 H, 15 N, 18 O) or radioactive isotope ( 14 C, 3Stable isotopes (H, Tc-99m) can be used. The advantages of stable isotopes are the absence of radioactive exposure, their natural abundance, and the specificity of the analysis used for identifying the test compound (mass determination by mass spectrometry). Stable isotope-labeled compounds are commercially available. Identifiable compounds may be stable isotope-labeled bile acids.

[0111] Stable isotope-labeled bile acids include, for example, lithocholic acid-2,2,4,4-D4 (LCA-D4), ursodeoxycholic acid-2,2,4,4-D4 (UDCA-D4), deoxycholic acid-2,2,4,4-D4 (DCA-D4), cholic acid-2,2,4,4-D4 (D4-CA;CA-D4), and 24- 13 C-Cholic acid ( 13 You can choose from C-CA), 2,2,3,4,4-d5 cholic acid (D5-CA), glycochenodeoxycholic acid-2,2,4,4-D4 (GCDCA-D4), glycodeoxycholic acid-2,2,4,4-D4 (GDCA-D4), glycocholic acid-2,2,4,4-D4 (GCA-D4), and deoxycholic acid-24-13C (DCA-24-13C), which are commercially available from companies such as Sigma-Aldrich, IsoSciences (King of Prussia, PA), CDN Isotopes, and Cambridge Isotope Laboratories, Inc.

[0112] In some embodiments, the identifiable compound for oral administration may be any identifiable cholate compound analytically identifiable from endogenous cholic acid. In one embodiment, the identifiable cholate compound is selected from any isotope-labeled cholic acid compound known in the art. The identifiable cholate compound used in any one of these assays is a stable isotope ( 13 C, 2 H, 18 O) or radioactive isotope ( 14 C, 3It can be labeled with either H). Identifiable cholate compounds can be purchased (e.g., CDN Isotopes Inc., Quebec, CA). In a preferred embodiment, the identifiable cholate is selected from any known safe, non-radioactive stable isotope of cholic acid. In a particular embodiment, the identifiable cholate compound is 2,2,4,4-, also known as cholic acid-2,2,4,4-d4 (D4-CA). 2 H is cholic acid. In another specific embodiment, the identifiable cholate compound is cholic acid-24- 13 C( 13 Also known as C-CA) 24- 13 C is cholic acid. In another specific embodiment, the identifiable compound is 2,2,3,4,4- cholic acid, also known as cholic acid-2,2,3,4,4-d5 (D5-CA). 2 It is H-cholic acid.

[0113] In other embodiments, the identifiable compound may be an unlabeled endogenous compound, such as unlabeled cholic acid. In embodiments using an unlabeled endogenous compound, the oral test dose is sufficiently large, for example, 2.5 to 7.5 mg / kg of cholate, so that the resulting serum concentration is identifiable above the baseline serum concentration of the endogenous compound.

[0114] Platforms for detecting and measuring identifiable compounds in blood samples from subjects may depend on the type of identifiable compound administered. For stable isotopes, the concentration of the identifiable compound in a blood sample can be measured by any known method, e.g., previously used gas chromatography / mass spectrometry (GC-MS) or liquid chromatography / mass spectrometry (LC-MS). As provided herein, identifiable compounds can be detected and quantified from blood or serum samples using MS, MS-MS, or LC-MS / MS with multiple reaction monitoring (MRM). For radiolabeled test compounds, for example, scintillation spectroscopy can be used. For unlabeled compounds, for example, automated analyzers, luminescence, or ELISA can be used. It is further intended that strip tests using chromogenic agents directly or indirectly sensitive to the presence and amount of the test compound may be used for home testing or point-of-care testing.

[0115] Portal vein blood flow Portal venous flow has been found to be a crucial parameter for liver assessment. The liver receives approximately 75% of its blood through the portal vein, which carries nutrients for processing and harmful compounds for detoxification. Because this low-pressure system is sensitive to the earliest destruction of the microvascular system, the early stages of CLD can be detected by decreased portal flow and increased shunting, before any other physiological effects appear. The decrease in systemic hepatic blood flow is less pronounced in high-pressure systems and decreases only in the later stages of the disease process. Unlike biopsies, which sample only 1 / 50,000th of the liver, portal flow is a measure of the entire organ. As the disease progresses, destruction of the microvascular architecture increases, leading to increased impaired portal flow, which causes major signs of advanced chronic liver disease (CLD). Impaired blood flow causes ascites, portal hypertension, and esophageal varices. Impaired blood flow causes increased shunting of toxins, which leads to hepatic encephalopathy.

[0116] Cholates are specific probes of portal blood flow and hepatic systemic blood flow. While many liver tests have attempted to use oral or IV compound clearance, only cholates have successfully assessed early and late-stage CLD. Other oral compounds are absorbed at various sites along the GI duct and do not target portal circulation. Other compounds are taken up by nonspecific transporters. Oral cholates are high-affinity ileal sodium. + It is specifically absorbed by terminal ileal epithelial cells via the diuretic salt transporter (ISBT) and directly discharged into the portal bloodstream by the MRP3 transporter (Trauner and Boyer, 2003, Bile salt transporters: Molecular characterization, function, and regulation. Physiol Rev. 83: 633-671). + A set of different high-affinity transporters, including the taurocholate cotransporter (NTCP) and organic anion transport protein (OATP), then take up cholates into hepatocytes with highly efficient first-pass extraction (Trauner and Boyer, 2003, see below), so that any cholates that escape extraction become a direct measure of portal flow. Once inside the cell, cholates are rapidly conjugated to glycine and taurine, so that the unconjugated form, which would disrupt pharmacokinetics, does not subsequently reappear in intrahepatic circulation. Other unconjugated bile salts such as deoxycholates and chenodeoxycholates would behave similarly, but they would not be safe to administer as they are far more potent solubilizers. Patient safety can be ensured by using stable isotope-labeled endogenous compounds to avoid the risk of xenobiotic or radiation exposure. Because all the proteins and systems involved are highly conserved and important, the pharmacokinetics of cholate are consistent across individuals and are not affected by sex, age, genetic makeup, diet, or concomitant medications.

[0117] Portal vein blood flow can be non-invasively and accurately quantified by utilizing the intrinsic physiology of endogenous bile acids, cholates, which can be labeled with safe, non-radioactive stable isotopes, for example. Highly conserved intestinal transporters (ISBT, MRP3) specifically target the portal circulation for oral cholates. Highly conserved hepatic transporters (NTCP, OATP) clear cholates from the portal and systemic circulation. Therefore, non-invasive quantitative assessment of portal circulation can be performed by administering a identifiable cholate compound to a patient and determining the oral clearance curve by assessing the levels of the identifiable cholate compound in blood samples taken at various multiple time points. The FLOW (portal HFR) test accurately measures portal vein blood flow from at least five blood samples taken over 90 minutes after oral administration of deuterium cholate.

[0118] In a major study involving nearly 300 CHC patients, portal flow measured by cholate testing was a better predictor of clinical outcomes than the current optimal criterion for fibrosis measured by biopsy (Everson et al., 2011). Early CHC testing revealed that impaired portal flow and increased shunt, as measured by cholate testing, were the earliest pathophysiological indicators to be detected. These results led to a new understanding of CLD, suggesting that the harmful effects lie in the destruction of the hepatic microvascular system, not fibrosis itself. This microvascular destruction impairs portal blood flow, which can be non-invasively and accurately quantified by leveraging the unique physiology of endogenous bile acid cholates.

[0119] Portosystemic shunt As shown in Figure 1, oral cholates are taken directly into the portal vein by specific intestinal transporters and removed by hepatic transporters during the first pass through the liver. IV cholates are distributed throughout the body and extracted by both the hepatic artery and portal vein.

[0120] In a healthy state, orally administered deuterated cholate is delivered to the liver via the portal circulation. Its clearance is a measure of portal circulation and is therefore called portal HFR.13 C-cholate is delivered to the liver via both hepatic artery and portal vein circulation, and is therefore called systemic HFR. SHUNT is the ratio of systemic HFR to portal vein HFR. Normal ranges for these tests are shown in the upper panel.

[0121] In the presence of disease, SHUNT increases, and portal vein HFR and systemic HFR decrease, as shown in the lower panel.

[0122] For example, a normal, healthy control typically exhibits a SHUNT (IV cholate clearance / oral cholate clearance) of approximately 20%, a portal vein HFR (oral cholate clearance per kg of body weight) of approximately 30 mL / min / kg, and a systemic HFR (intravenous cholate clearance per kg of body weight) of approximately 6 mL / min / kg. Patients with liver disease typically exhibit higher SHUNT values ​​of approximately 30% to 90%. Patients with liver disease typically exhibit lower portal vein HFR of approximately 20 mL / min / kg to approximately 2 mL / min / kg. Patients with liver disease typically exhibit lower systemic HFR of approximately 4 mL / min / kg to approximately 1 mL / min / kg.

[0123] In diseased livers, as more blood escapes extraction into the systemic circulation through intrahepatic and extrahepatic shunts, SHUNT increases, HFR or portal blood flow decreases, and STAT increases. In normal controls, effective portal blood flow (portal HFR, FLOW) is high in healthy livers due to lower vascular resistance. Portosystemic shunt (SHUNT) is minimal. Oral cholate (STAT) at 60 minutes is low. For example, in healthy controls, FLOW = 37 mL / min. -1 kg - 1. SHUNT = 18%, and STAT = 0.2 μM. However, in subjects with liver disease, inflammation, fibrosis, and increased vascular resistance, effective portal blood flow (FLOW) decreases. Portosystemic shunt (SHUNT) increases. Oral cholate (STAT) is high at 60 minutes. For example, in CHC F2 patients, FLOW = 9 mL / min -1 kg -1 SHUNT = 35%, and STAT = 1.6 μM.

[0124] Portal vein HFR (FLOW) and SHUNT tests can be used, for example, to determine portal blood flow and liver function in healthy controls and patients with chronic liver disease such as chronic hepatitis C. SHUNT and FLOW tests, which use the measurement of identifiable compounds administered orally and / or intravenously and the measurement of identifiable compounds in multiple blood or serum samples by GC-MS or HPLC-MS, are disclosed in U.S. Patents 8,613,904 and 8,778,299, respectively, which are incorporated herein by reference.

[0125] The STAT test was developed as a screening test to estimate portal blood flow and is used to screen large populations for the detection of patients with chronic liver diseases, including chronic hepatitis C, PSC, and NAFLD. The relationship between STAT and prior art methods for determining cholate clearance from portal circulation, specifically the FLOW and SHUNT tests, has been validated using a large cohort of patients with chronic hepatitis C. A method for pre-forming a STAT test, comprising measuring identifiable compounds in a single blood or serum sample using HLPC-MS, is disclosed in U.S. Patent No. 8,961,925, which is incorporated herein by reference.

[0126] In some embodiments, the STAT test value in the subject is (a) the dose in the subject (dose oral (b) receiving a single blood or serum sample collected from a subject after oral administration of an identifiable compound, wherein the sample is collected from the subject at a specific time within approximately 20 to 180 minutes after administration, and (b) measuring the concentration of the identifiable cholate compound in the sample by MS, MS / MS, or LC-MS / MS using MRM.

[0127] In some embodiments, a single blood or serum sample in a STAT test is collected at one single time point selected from about 20: 25, 30, 35, 40, 45, 50, 55, 50, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 minutes after oral administration of an identifiable cholate compound, or any time point in between.

[0128] In some embodiments, a single blood or serum sample in a STAT test is collected at one point in time selected from approximately 45 minutes, 60 minutes, or 90 minutes after oral administration of an identifiable cholate compound.

[0129] In some embodiments, a single blood or serum sample is collected approximately 60 minutes after oral administration of an identifiable cholate compound.

[0130] In some embodiments, a single blood or serum sample is collected approximately 45 minutes after oral administration of an identifiable cholate compound.

[0131] In some embodiments, a single blood or serum sample is collected approximately 90 minutes after oral administration of an identifiable cholate compound.

[0132] In some embodiments, a method for determining STAT test values ​​in subjects who have, are suspected of having, or have chronic liver disease, wherein (a) the dose in the subject (dose oral A method is provided comprising: (b) receiving a single blood or serum sample collected from a subject after orally administering an identifiable compound, wherein the sample is collected from the subject at a specific time within approximately 20 to 180 minutes after administration; and (b) measuring the concentration of the identifiable cholate compound in the sample by MS, MS-MS with MRM, or LC-MS / MS with MRM.

[0133] In some embodiments, a method is provided for determining the portal vein HFR value in a patient, the method comprising (a) a certain dose (dose oral (b) receiving multiple blood or serum samples collected from patients with or at risk of chronic liver disease after oral administration of an identifiable cholate, wherein the samples are collected from the patients at intervals over a period of time after administration; (c) measuring the concentration of the identifiable cholate in each sample by a method including MS, MS-MS using MRM, or LC-MS / MS using MRM; (d) generating an individualized oral clearance curve from the identifiable cholate concentration in each sample, including using a computer algorithm curve that fits a model identifiable cholate clearance curve; (e) calculating the area under the individualized oral clearance curve (AUC) (mg / mL / min), dividing the dose (mg) by the AUC of the orally administered stable isotope-identified cholate to obtain the oral cholate clearance in the patient; and (e) dividing the oral cholate clearance by the patient's body weight in kg to obtain the portal vein HFR value (mL / min / kg) in the patient.

[0134] Laboratory output values. Cholate concentrations (endogenous unlabeled CA, 13C-CA, and d4-CA) may be measured from timed serum samples (collected 0, 5, 20, 45, 60, and 90 minutes after oral and intravenous administration), and the concentration of each labeled cholate may be modeled as a spline curve as a function of time to calculate the area under the curve (AUC). Cholate SHUNT laboratory parameters may include DSI, indicative hepatic reserve, algebraic hepatic reserve, RISK-ACE, SHUNT%, RCA20, systemic HFR, portal vein HFR, cholate removal rate, and volume of distribution.

[0135] In short, DSI is a unitless score representing a quantitative measure of liver function. DSI (Disease Severity Index) is a score that is a function of the sum of cholate clearances from systemic and portal circulation, adjusted for disease severity from healthy individuals to end-stage liver disease. Hepatic reserve represents the percentage of maximum functional liver volume measured by DSI normalized to the DSI range for individuals with lean body mass. The Individual Risk Score (RISK-ACE) for Annual Clinical Events is based on baseline DSI (Model A) and baseline DSI + ΔDSI (Model D) that occurred over two years in the HCV population, with approximately 25% experiencing clinical events over a maximum follow-up of 8.7 years. SHUNT% represents a quantitative measure of portosystemic shunt. SHUNT% is a measure of the percentage of orally administered d4-cholate outflow. First-pass hepatic clearance of cholate in percentage of orally administered cholate is defined as (100%-SHUNT). Systemic HFR, mL min -1 kg -1 This represents the model-independent clearance of intravenously administered 13C-cholate, adjusted for body weight and calculated from dose / AUC. Portal vein HFR, mL / min -1 kg - 1 represents the model-independent apparent clearance of orally administered d4-cholate, adjusted for body weight and calculated from dose / AUC. Cholate removal rate, k elim minutes -1 This can be represented as the first stage of removal of intravenously administered 13C-cholate and is calculated from Ln / linear regression of [13C-cholate] against time (using only 5-minute and 20-minute time points). Intravenously administered 13C-cholate is rapidly delivered to the liver via the hepatic artery. In contrast, the same 13C-cholate moves slowly to the liver via the portal vein, depending on the volume of the visceral vascular bed. Therefore, the first stage of cholate removal depends more on clearance from the hepatic artery than from the portal vein. Volume of Distribution, V d , L kg -1This is the volume of the body to which cholate is distributed. This is calculated from the intercept on the Y-axis of the Ln / linear regression of [13C-cholate] against time (using only time points of 5 minutes and 20 minutes).

[0136] In some embodiments, a method is provided for determining the whole-body HFR value in a patient, wherein the whole-body HFR value is (a) a certain dose (dose oral The method may include: (b) receiving multiple blood or serum samples collected from patients with or at risk of chronic liver disease after intravenous administration of an identifiable cholate (Isotope I) to a patient, wherein the samples are collected from the patient at intervals over a period of time after administration; (c) measuring the concentration of the identifiable cholate in each sample by a method including MS, MS-MS with MRM, or LC-MS / MS with MRM; (d) generating an individualized intravenous clearance curve from the concentration of the identifiable cholate in each sample, including using a computer algorithm curve that fits a model identifiable cholate clearance curve; (e) calculating the area under the individualized intravenous clearance curve (AUC) (mg / mL / min), dividing the dose (mg) by the AUC of the intravenously administered stable isotope-identified cholate to obtain the intravenous cholate clearance in the patient; and (e) dividing the intravenous cholate clearance by the patient's body weight in kg to obtain the whole-body HFR value (mL / min / kg) in the patient.

[0137] In some embodiments, a method is provided for determining liver SHUNT values ​​in subjects who have or are suspected of having or are at risk of having liver impairment or chronic liver disease, the method comprising: (a) obtaining a plurality of blood or serum samples, the plurality of blood or serum samples being collected from the subjects at intervals of less than 3 hours after the subjects have been orally administered a first identifiable compound and simultaneously administered a second identifiable compound intravenously; (b) quantifying the first and second identifiable compounds in the samples by a method including MS, MS / MS using MRM, or LC-MS / MS using MRM; and (c) an equation, AUC oral / AUC iv ×Dose iv / dose oral ×100%, This involves using to calculate the hepatic shunt in the subject, In the formula, AUC oral AUC is the area under the curve for the serum concentration of the first identifiable compound. iv (d) the area under the curve of a second identifiable compound, which includes (d) calculating the hepatic shunt in the subject over time to one or more shunt cutoff values ​​established from a known patient population or within a patient. The hepatic shunt in the subject compared to one or more shunt cutoff values, or the hepatic shunt in the subject over time, is an indicator of the subject's relative liver function.

[0138] In some embodiments, the estimated hepatic blood flow (HBF) in a patient is expressed by the equation, HBF can be calculated as follows: HBF = (Cholelate clearance after intravenous administration) / [1 - (SHUNT / 100)) x (1 - (Hematocrit % / 100))].

[0139] Previous human studies demonstrated the clinical utility of FLOW and SHUNT tests in chronic hepatitis C (CHC). Over the years, several new liver tests have been proposed, but few studies have directly compared their effectiveness and actual clinical utility. A very large, multicenter HALT-C trial was conducted, primarily to determine the effectiveness of long-term hepatitis C virus suppression, and included supplementary studies to evaluate a range of new quantitative liver function tests. (Everson et al., 2009. Quantitative tests of liver function measure hepatic improvement after sustained virological response: Results from the HALT-C trial. Aliment Pharmacol Ther. 29:589-601). Approximately 300 patients with progressive (Ishak F2-6) but compensated CLD were tested. Another early-stage CHC test compared these tests to those of 25 healthy controls and 23 early-stage (Ishak F1-2) CHC patients to examine the entire spectrum of this CLD. Hepatic metabolic capacity was assessed using tests for caffeine, antipyrine, lidocaine, and galactose. All of these activities were reduced in patients with cirrhosis, but not significantly different from healthy controls in early-stage CHC patients. (Everson et al., 2008. The spectrum of hepatic functional impairment in compensated chronic hepatitis c: Results from the hepatitis c antiviral long-term treatment against cirrhosis trial. Aliment Pharmacol Ther. 27:798-809). These results suggest that metabolic capacity is maintained until significant loss of functional parenchyma in late-stage CLD. In HALT-C, patients were examined consecutively every two years and followed to monitor outcomes.Using a cutoff of less than 9.5 ml / min / kg, FLOW outperformed other tests in predicting clinical outcomes with the best sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and performance in ROC analysis (Quantitative liver function tests improve the prediction of clinical outcomes in chronic hepatitis C: results from the Hepatitis C Antiviral Long-term Treatment Against Cirrhosis Trial, Everson et al., Hepatology, 2012 Apr;55(4):1019-29). FLOW had a higher ROC c statistic (0.84) compared to SHUNT (0.79). Post-SVR improvement was significantly greater with FLOW (p=0.0002) than with SHUNT (p=0.0003) (Everson et al., 2009, see below). In early-stage CHC studies, FLOW decreased from 34±14 ml / min / kg (mean ± SD) in controls to 23±10 ml / min / kg in early-stage CHC (p<0.002), but the increase in SHUNT (20±6% in controls vs. 31±14% in early-stage CHC patients, p<0.0002) was statistically more significant. Other tests could not distinguish early-stage CHC patients from healthy controls. These results suggest that SHUNT and FLOW are superior to other functional tests in detecting early-stage liver disease, tracking patients, and predicting clinical outcomes.

[0140] In a typical embodiment, the concentrations of both oral and IV cholate are measured at five different time points within 90 minutes of administration, and clearance is calculated. IV clearance relative to oral clearance is the portal-systemic shunt fraction. Oral clearance per kilogram of body weight represents the portal vein hepatic filtration rate (portal HFR, FLOW), or the amount of blood delivered through the portal vein. STAT is the concentration of oral cholate over 60 minutes and can be used to accurately estimate portal HFR.

[0141] The SHUNT test non-invasively and accurately measures portal blood flow after oral administration of a identifiable cholate compound, and also measures systemic hepatic blood flow after intravenous co-administration of a second identifiable cholate compound. Therefore, the SHUNT test can be used to determine the amount of portosystemic shunt. In some embodiments, 13 The IV dose of C-cholate is administered simultaneously with the oral dose of deuterated cholate, and at least five blood samples are taken over a 90-minute period following administration.

[0142] The deuterated cholate clearance SHUNT method yielded three test results: the portal-systemic circulation SHUNT fraction (SHUNT (%)), portal hepatic filtration rate (portal HFR (mL / min / kg)), defined as FLOW in the above discussion and examples, based on identifiable cholate compounds in orally administered blood, and systemic hepatic filtration rate (systemic HFR (mL / min / kg)), based on identifiable cholate compounds in intravenously administered blood. Cholate-2,2,4,4-d4 (40 mg) was administered orally and taken up into the portal vein by specific intestinal transporters. Cholate-24- 13 C (20 mg) is administered intravenously and is taken up from the systemic circulation, mainly via the hepatic artery. Specific hepatic transporters clear cholates from the portal vein and systemic circulation. For example, highly conserved hepatic transporters (NTCP, OATP) clear cholates from the portal vein and systemic circulation.

[0143] The Shunt equation can be expressed as follows:

number

[0144] The absorption of cholate is assumed to be approximately 100%. The absorption of unbound cholate is rapid after oral administration, and in peripheral venous blood samples, d4-cholate is detected within 5 to 10 minutes after its administration, and the peak absorption is 20 to 60 minutes. Cholate is a ligand of the OATP transporter, but most of its absorption is passive by flip-flop across the intestinal epithelial cell membrane due to its detergent-like properties. The literature shows that the absorption rate is 100%.

[0145] Each of the liver function tests of the present invention is based on peripheral venous blood sampling from the whole body compartment. In the cholate SHUNT test, 13C-cholate is administered intravenously to measure cholate clearance because this dose is 100% available in the whole body compartment. The clearance of 13C-cholate is Cl iv13C-cholate -dose iv / AUC iv which can be defined by In contrast, d4-cholate is administered orally, absorbed from the intestine, and delivered to the liver via the portal vein. Considering its rapid and efficient intestinal absorption, d4-cholate that is not extracted in the first pass through the liver flows out into the whole body compartment and is then eliminated in the same manner as 13C-cholate. The clearance of d4-cholate is Cl po d4-cholate =Fx(dose po / AUC po ) which can be defined by where F is the fraction of the oral dose that escapes first pass extraction. By setting Cl iv 13C-cholate equal to Cl po d4-cholate , F can be calculated from the concentration in peripheral venous blood. As the disease progresses, if HFR p decreases relatively more than HFR s , F usually increases in relation to the development of collateral circulation. F is measured only in the cholate SHUNT test. F is indirectly assayed in both the FLOW test and the STAT test, and in the latter test, the peripheral venous concentration of d4-cholate, which increases as F increases, is measured either by AUC(FLOW) or at a single point in time (STAT).

[0146] The volume of distribution and hepatic extraction of [13C-CA] are assumed to be the same as those of [d4-CA], that is, the same subject can be given IV or PO under the same conditions and the same clearance can be obtained. Evidence to support this assumption is the finding that 13C-labeled bile acids and deuterium-labeled bile acids are handled similarly in the human body. (Everson GT. Steady-state kinetics of serum bile acids in healthy human subjects: single and dual isotope techniques using stable isotopes and mass spectrometry. J Lipid Res 1987;28:238-252.)

[0147] The three main outputs of the cholate SHUNT test, DSI, SHUNT, and HR, are unitless. DSI and HR can be indexed relative to the HFR of healthy individuals, where HFR is defined as clearance divided by body weight and is expressed per kilogram of body weight. Clearance values are normalized to the body weight of the subject, but other indexing methods can be used relative to, for example, the lean body mass of the subject, ideal body weight, body surface area, blood volume, estimated blood volume.

[0148] The unit of kilograms of body weight (kg) is in both the numerator and denominator and cancels out. Therefore, there is no need to normalize HFR to a given body weight or size for the calculation of DSI or HR.

[0149] For example, if HFR is normalized to a 75 kg body weight, HFR = Cl / body weight kg = dose / (AUC × body weight kg ) HFR adjustd75kg = dose / [(AUC×body weight kg ) / 75kg] = HFR × 75 And so, The multiplier 75 cancels out because it appears in both the numerator and denominator of DSI, HR, and SHUNT.

[0150] SHUNT, which is the ratio of clearances within an individual, is not indexed relative to the control group, but it is also unitless because the expression of clearance by body weight (or size) is present in both the numerator and denominator and cancels out. Similarly, SHUNT does not require normalization to body weight or size.

[0151] Estimation of portal vein hepatic filtration rate The STAT test is a simplified, non-invasive, and convenient test intended for screening purposes, which can reasonably estimate portal blood flow at a single point in time, for example, from a single blood sample taken 60 minutes after oral administration of an identifiable cholate compound, such as deuterated cholate.

[0152] Comparison of portal vein HFR (FLOW), SHUNT, STAT, and DSI liver function tests.

[0153] Table 1 shows a comparison of typical embodiments of the SHUNT, FLOW, STAT, and DSI tests. [Table 1]

[0154] Normal liver function values ​​have been established in previous studies in healthy controls, with a mean SHUNT of 20%, a mean HFR (FLOW) of 30, and a mean STAT of 0.4.

[0155] In diseased livers, as more blood escapes extraction into the systemic circulation through intrahepatic and extrahepatic shunts, SHUNT increases (approximately 30–90%), HFR (FLOW) or portal blood flow decreases (approximately 20–2 mL / min / kg), and STAT increases (0.6–5 μM).

[0156] Improved methods for the preparation of blood or serum samples, detection of analytes, and quantification are provided herein, enabling improved recovery of analytes from samples, increased selectivity of analytes, improved sample throughput, a significant reduction in processing time, a ten-fold reduction in the patient sample volume required, and improvement of the lower limit of quantification (LOQ). These methods and advantages can be applied to one or more of the SHUNT, FLOW, STAT, and DSI liver function tests.

[0157] Definitions As used herein, "a" or "an" can mean one or more of an item.

[0158] When referring to any numerical parameter, the term "about" means + / - 10% of the numerical value. For example, the phrase "about 60 minutes" refers to 60 minutes + / - 6 minutes.

[0159] As used herein, the term "accuracy" (measurement) refers to the closeness of agreement between the value of a measured quantity and the true value of the quantity being measured.

[0160] The term "acceptability" as used herein is based on individual criteria that set the minimum operating characteristics for a measurement procedure.

[0161] The term "precision" as used herein refers to the closeness of agreement between independent test / measurement results obtained under specified conditions.

[0162] The term "trueness" as used herein refers to the closeness of agreement between the expected value and the true value of a test result or measurement result.

[0163] The term "quantity measured" is used when referring to the quantity intended to be measured in place of an analyte (a component represented by a measurable amount).

[0164] As used herein, the term “verification” focuses on whether the specifications of a measurement procedure can be achieved, while “verification” refers to verifying that the procedure is suitable for its intended purpose.

[0165] The term "measurement procedure" refers to a detailed description of a measurement, based on a measurement model, following one or more measurement principles, including any calculations to obtain the measurement result, and following a given measurement method.

[0166] As used herein, “clearance” may mean removing a substance from one place to another.

[0167] As used herein, the term “simultaneously” refers to two or more events occurring within 20 minutes, 15 minutes, 10 minutes, 5 minutes, or 3 minutes of each other.

[0168] As used herein, the terms “patient,” “subject,” or “subjects” include, but are not limited to, humans, other mammals, or domesticated or exotic animals, such as dogs, cats, ferrets, rabbits, pigs, horses, cattle, birds, or reptiles.

[0169] The acronym "HALT-C" refers to the Long-Term Antiviral Treatment Trial for Hepatitis C Cirrhosis. The HALT-C trial was a large, prospective, randomized, controlled trial of long-term low-dose PEG interferon therapy in patients with advanced hepatitis C who had not shown sustained virological response to previous interferon-based therapy. The NIH-funded Long-Term Antiviral Treatment for Hepatitis C Cirrhosis (HALT-C) trial investigated whether long-term use of antiviral therapy (maintenance therapy) slows the progression of liver disease. In non-cirrhotic patients with significant fibrosis, effective maintenance therapy was expected to slow or halt histological progression to cirrhosis, as assessed by serial liver biopsies. However, tracking disease progression by biopsy carries risks of complications, and in some cases, death. In addition, sampling errors and variability in pathological interpretation of liver biopsies limit the accuracy of histological assessments and endpoints. Histological endpoints are unreliable because progressive fibrosis is already present and fibrotic changes related to treatment or disease progression cannot be detected. Therefore, standard endpoints for an effective response to maintenance therapy in patients with cirrhosis are prevention of clinical decompensation (ascites, variceal bleeding, and encephalopathy) and stabilization of liver function as clinically measured by the Childs-Turcotte-Pugh (CTP) score. However, clinical endpoints and the CTP score were known to be insensitive parameters of disease progression. A dual isotope technique using identifiable cholate was used in the development of the SHUNT test and in conjunction with the HALT-C trial. The term "SHUNT test" refers to the previously disclosed QLFT (Quantitative Liver Function Test) used as a comprehensive assessment of hepatic blood flow and liver function. The SHUNT test is used to determine the plasma clearance of identifiable cholate administered orally and intravenously in subjects with or without chronic liver disease. The SHUNT fraction or percentage quantifies the outflow of d4-cholate into the systemic circulation from the ratio of the clearance of intravenously administered 13C-cholate to the clearance of orally administered d4-cholate.The SHUNT test, which analyzes at least five blood samples taken from a patient at intervals of at least approximately 90 minutes after oral and intravenous administration of differentiateable cholic acid, is disclosed in Everson et al., U.S. Patent No. 8,613,904, which is incorporated herein by reference in its entirety. These studies demonstrated reduced cholic acid clearance in patients with either hepatocellular injury or portosystemic shunt. "SHUNT test value" refers to a numerical value (%). The term "SHUNT%" represents a quantitative measure of portosystemic shunt. SHUNT% is a measure of the percentage of orally administered d4-cholate efflux. First-pass hepatic clearance of cholate in percentage of orally administered cholate is defined as (100%-SHUNT). The SHUNT test method is disclosed in U.S. Patents 8,613,904, 9,639,665, 8,778,299, 9,417,230, and 10,215,746, each of which is incorporated herein by reference in whole. Analysis of samples for stable isotope-labeled cholates is performed, for example, by GC-MS after sample derivatization, or by LC-MS without sample derivatization, or by LC-MS / MS or MS / MS as disclosed herein. The ratio of AUC of orally administered cholate to intravenously administered cholate is dose-corrected to define the cholate shunt. The cholate shunt is given by formula, AUC. oral / AUC iv ×Dose iv / dose oral It can be calculated using ×100%, in the formula, AUC oral AUC is the area under the curve of serum concentration of orally administered cholic acid. iv This represents the area under the curve for cholic acid administered intravenously.

[0170] The SHUNT test allows for the measurement of hepatic first-pass removal of bile acids from the portal circulation. Hepatic flow-dependent first-pass removal of bile acids ranges from approximately 60% for unconjugated dihydroxy bile acids to approximately 95% for glycine-conjugated cholates. As used herein, free cholates have a reported first-pass removal of approximately 80%, which is in close agreement with the previously observed first-pass removal of approximately 83% in healthy controls. After hepatic uptake, cholic acid efficiently conjugates to either glycine or taurine and is secreted into the bile. Physicochemically, cholic acid can be readily separated from other bile acids and bile acid or cholic acid conjugates using chromatography.

[0171] "Cholate removal rate", k elim minutes -1 The term represents the first stage of removal of intravenously administered 13C-cholate and is calculated from Ln / linear regression of [13C-cholate] against time (using only 5-minute and 20-minute time points). Intravenously administered 13C-cholate is rapidly delivered to the liver via the hepatic artery. In contrast, the same 13C-cholate moves slowly to the liver via the portal vein, depending on the volume of the visceral vascular bed. Therefore, the first stage of cholate removal depends more on clearance from the hepatic artery than from the portal vein.

[0172] "Distribution volume", V d , L kg -1 The term represents the volume of the body in which cholate is distributed. This is calculated from the intercept on the Y-axis of the Ln / linear regression of [13C-cholate] against time (using only 5-minute and 20-minute time points).

[0173] The acronym "IV" or "iv" refers to the intravenous administration route.

[0174] The acronym "PO" refers to the route of administration by oral ingestion.

[0175] The acronym "PHM" refers to a perfused liver mass.

[0176] The acronym "SF" refers to the shunt fraction, such as liver SF or cholate SF.

[0177] The acronym "ROC" refers to Recipient Operating Characteristics. An ROC curve is a graph plot that shows the performance of a binary classifier system as the discrimination threshold changes. It is created by plotting the fraction of true positives from positive (TPR) versus the fraction of false positives from negative (FPR) for various threshold settings. Sensitivity is the probability that a person with the disease will have a positive test result or a value above the threshold. Sensitivity is defined as the true positive rate (TPR). TPR = TP / P = TP / (TP+FN). The false positive rate (FPR) is FPR = FP / N = FP / (FP+FN). Accuracy (ACC) is defined as ACC = (TP+TN) / (P+N). Specificity is the probability that a person without the disease will have a negative test result or a value below the threshold. Specificity (SPC), or true negative rate (TN), is defined as SPC = TN / N = TN / (FP + TN) = 1 - FPR. Positive predictive value (PPV) is defined as PPV = TP / (TP + FP). Negative predictive value (NPV) is defined as NPV = TN / (TN + FN).

[0178] The C statistic is the area under the ROC curve, or "AUROC" (Area Under the Recipient Action Characteristics Curve), and ranges from 0.5 (no discrimination) to a theoretical maximum of 1 (perfect discrimination).

[0179] The term "sustained virological response" (SVR) is used to describe a desired response in a patient, for example, when the hepatitis C virus is not detected in the blood six months after the end of treatment. Conventional treatments using interferon and ribavirin do not necessarily eliminate or eradicate the hepatitis C virus. Sustained virological response is associated with a very low incidence of relapse. SVR is used to evaluate new drugs and compare them to proven therapies.

[0180] "Oral Cholate Clearance" (Cl oralThe term "oral cholate clearance" refers to the clearance of orally administered cholate compounds from a subject's body, measured by blood or serum samples from the subject. Oral cholate clearance is used as a measure of portal blood flow. Orally administered cholic acid is absorbed through the epithelial-lining cells of the small intestine, binds to albumin in the portal blood, and is transported to the liver via the portal vein. Approximately 80% of cholic acid is extracted from the portal blood in the first pass through the liver. Cholic acid that escapes hepatic extraction exits the liver via the hepatic veins, is excreted into the venous lumen, returns to the heart, and is delivered into the systemic circulation. The area under the curve (AUC) of peripheral venous concentration over time after oral administration of cholic acid quantifies the proportion of cholic acid that escapes hepatic extraction and defines "oral cholate clearance."

[0181] The terms "portal vein hepatic filtration rate," "portal HFR," and "FLOW test" refer to oral cholate clearance (portal vein hepatic filtration rate, portal HFR), which is used as a measure of portal blood flow or portal circulation. This is obtained, for example, from the analysis of the concentrations of identifiable cholate compounds in at least five blood samples taken from a subject over approximately 90 minutes after oral administration of an identifiable cholate compound. The unit of the portal HFR value is typically expressed in mL / min / kg, where kg refers to the subject's body weight in kg. -1 kg - 1 may be used for the model-independent apparent clearance of orally administered d4-cholate, adjusted for body weight, and calculated from dose / AUC. FLOW testing methods are disclosed in U.S. Patents 8,778,299, 9,417,230, and 10,215,746, each of which is incorporated herein by reference in whole. "Whole-body HFR", mL min -1 kg - 1 may be used as a model for the independent clearance of intravenously administered 13C-cholate, adjusted for body weight, and calculated from dose / AUC.

[0182] The term “STAT test” refers to an estimate of portal blood flow by analysis of a single patient blood sample taken during a defined period following oral administration of a differentiateable cholate. In one embodiment, the STAT test refers to the analysis of a single blood sample taken at a specific point in time after oral administration of a differentiateable cholate. In a particular embodiment, the STAT test is a simplified and convenient test intended for screening purposes that can reasonably estimate portal blood flow (estimated flow rate) from a single blood sample taken 60 minutes after oral administration of deuterated cholate. In some embodiments, STAT is the d4-cholate concentration in the blood sample over 60 minutes. STAT correlates well with DSI and can be used to estimate DSI. STAT test values ​​are typically expressed as a concentration, e.g., micromolar (uM) concentration. STAT testing methods are disclosed in U.S. Patents 8,961,925 and 10,222,366, each of which is incorporated herein by reference in whole. Using STAT laboratory values, portal vein HFR can be estimated as provided in U.S. Patent Nos. 8,961,925 and 10,222,366. Using STAT laboratory values ​​in a patient, DSI values ​​in a patient can be estimated as provided herein.

[0183] The term "DSI test" refers to a disease severity index test derived from the results of one or more liver function tests based on hepatic blood flow. The DSI score is a function of the sum of cholate clearances from systemic and portal circulation, adjusted for disease severity ranging from healthy subjects to end-stage liver disease. The DSI is a unitless score that represents a quantitative measure of liver function. The Disease Severity Index (DSI) value in a patient may be obtained by a method comprising: (a) obtaining one or more liver function test values ​​in a patient who has or is at risk of having chronic liver disease, wherein the one or more liver function test values ​​are obtained from one or more liver function test values ​​selected from the group consisting of SHUNT, portal hepatic filtration rate (portal HFR), and systemic hepatic filtration rate (systemic HFR); and (b) obtaining the patient's DSI value using the Disease Severity Index equation (DSI equation), wherein the DSI equation comprises one or more terms and a constant for obtaining the DSI value, wherein at least one term of the DSI equation independently represents a liver function test value in the patient or a mathematically transformed liver function test value in the patient from step, and at least one term of the DSI equation multiplies by a coefficient specific to the liver function test. The DSI is an index or score that encompasses cholate clearance from both systemic and portal circulation. The DSI ranges from 0 (healthy) to 50 (severe end-stage disease) and is calculated from both HFRs. Based on the reproducibility of DSI, the minimum detectable difference indicating a change in liver function in a subject may be approximately 1.5 points, approximately 2 points, or approximately 3 points. DSI testing methods and equations are disclosed in U.S. Patents 9,091,701, 9,759,731, and 10,520,517, each of which is incorporated herein by reference in whole. Additional DSI equations have been developed and are provided herein. Methods for estimating DSI values ​​in patients from STAT test values ​​are also provided herein.

[0184] The term "hepatic reserve" refers to the percentage of maximum functional liver volume measured by DSI, and indexed hepatic reserve can be normalized to the DSI range in lean body mass subjects.

[0185] HR (algebraic) is simply an algebraic transformation of the DSI value in the object. HR = [100 - (2 × DSI)].

[0186] The hazard ratio (HR) with indicators is normalized to the outcome within a cohort of normal, non-obese controls.

[0187] One equation for liver reserve function (with indicators) begins with the DSI equation.

number

number

[0188] 30 non-obese controls (portal vein HFR 29.10 ± 9.04 mL) -1 kg -1, whole body HFR6.52±1.49mL min -1 kg -1 Based on the HFR range in ), the minimum values ​​for normal portal vein HFR and systemic HFR were set as follows. [Table 2]

[0189] By using the range from non-obese controls for all controls, it becomes possible to detect changes in HR in overweight and obese subjects regarding the potential for fatty liver disease. p and HFR s As y and z approach respectively, the heart rate (HR) approaches 100, indicating "normal hepatic reserve." As the hemoglobin rate (HFR) approaches 1, the HR approaches 0, indicating "no hepatic reserve."

[0190] "Intravenous cholate clearance" (Cl iv The term cholate shunt refers to the clearance of cholate compounds administered intravenously. Intravenously administered cholate, bound to albumin, is distributed throughout the body and delivered to the liver via both portal vein and hepatic artery blood flow. The AUC of peripheral venous concentration versus time after intravenous administration of cholate corresponds to 100% systemic delivery of cholate. The ratio of AUC of orally administered cholate to intravenously administered cholate, corrected for dose, defines the cholate shunt.

[0191] The term "RCA20" represents the amount of an identifiable compound administered intravenously, such as a identifiable cholate compound like 13C-CA, that remains in circulation 20 minutes after intravenous injection. The formula for RCA20 can be expressed as follows: Equation 11: RCA20=(1-([ 13 [C CA] t=0 -[ 13 [C CA] t=20 ) / [ 13 [C CA] t=0 ) × 100%, In the formula, RCA20 represents the amount of intravenously administered 13C-CA remaining in circulation 20 minutes after intravenous injection. 13[C CA] t=0 It is determined from the Ln / linear regression of [13C-CA] versus time. RCA20 can be compared to R15 for ICG data. Indocyanine green (ICG) clearance tests (K) and 15-minute retention rates (R15) are used, for example, as indicators of liver function in patients with cirrhosis.

[0192] The term "quantitative liver function tests" (QLFT) refers to assays that measure the liver's ability to metabolize or extract test compounds, allowing for the identification of patients with liver dysfunction in the early stages of disease, and in some cases defining the risk of cirrhosis, splenomegaly, and varicose veins. One of these assays is the cholate shunt assay, in which cholate clearance is assessed by analyzing a body fluid sample after exogenous cholate has been taken up by the body.

[0193] The term "Ishak fibrosis score" is used in reference to a scoring system that measures the degree of fibrosis (scarring) of the liver caused by chronic necrotizing inflammation. A score of 0 represents no fibrosis, and a score of 6 indicates established fibrosis. Scores of 1 and 2 indicate mild portal fibrosis, and stages 3 and 4 indicate moderate (bridging) fibrosis. A score of 5 indicates nodular formation and incomplete cirrhosis, and a score of 6 indicates clear cirrhosis.

[0194] The term “Childs-Turcotte-Pugh (CTP) score” or “Child-Pugh score” refers to a classification system used to assess the prognosis of chronic liver disease, provided in Pugh et al. Transection of the oesophagus for bleeding oesophageal varices. Br J Surg 1973;60:646-649, which is incorporated herein by reference. The CTP score includes five clinical measures of liver disease, each with a score from 1 to 3, where 3 is the most severe abnormality. Five additional scores are used to determine the CTP score. The five clinical measures include total bilirubin, serum albumin, prothrombin time international normalized ratio (PT INR), ascites, and hepatic encephalopathy. The CTP score is one of the scoring systems used to stratify the severity of end-stage liver disease. Chronic liver disease is classified into Child-Pugh classes A to C using the additional scores. Child-Pugh class A refers to a CTP score of 5-6. Child-Pugh class B refers to a CTP score of 7-9. Child-Pugh class C refers to a CTP score of 10-15. The website calculates the postoperative mortality risk for patients with cirrhosis. http: / / mayoclinic.org / meld / mayomodel9.html

[0195] The term “Model of End-Stage Hepatic Disease (MELD)” refers to a scoring system used to assess the severity of chronic liver disease. MELD was developed to predict mortality within three months post-operatively in patients who have undergone transjugular intrahepatic portosystemic shunt (TIPS) procedures for liver transplantation. MELD is also used to determine prognosis and prioritize patients for liver transplantation. MELD uses patient values ​​for serum bilirubin, serum creatinine, and the international normalized ratio (INR) of prothrombin time to predict survival. The scoring system is used by the United Network for Organ Sharing (UNOS) and Eurotransplant to prioritize liver transplant allocations, instead of the older Child-Pugh score. See UNOS (2009-01-28) “MELD / PELD calculator documentation,” incorporated herein by reference. For example, interpreting MELD scores in hospitalized patients, the three-month mortality rate is 71.3% for MELD scores of 40 or higher.

[0196] The term "standard sample" refers to a sample containing a known concentration of an analyte used for comparison purposes when analyzing a sample containing an analyte at an unknown concentration.

[0197] The term "chronic hepatitis C" (CHC) refers to a chronic liver disease caused by a viral infection that leads to inflammation, liver damage, and cirrhosis. Hepatitis C is an infectious disease caused by a bloodborne virus that attacks the liver and leads to inflammation. Many people infected with the hepatitis C virus (HCV) show no symptoms until liver damage appears during routine medical checkups, sometimes even several years later.

[0198] The term "alcoholic steatohepatitis" (ASH) refers to a chronic condition of liver inflammation caused by excessive alcohol consumption. Progressive inflammatory liver damage is associated with long-term heavy ethanol intake and can progress to cirrhosis.

[0199] The term "non-alcoholic steatohepatitis" (NASH) refers to a severe chronic condition of liver inflammation that progresses from a less severe, simple fatty liver condition called steatosis. Simple steatosis (alcoholic fatty liver) is an early, reversible consequence of excessive alcohol consumption. In people who do not drink much alcohol, the cause of fatty liver disease is less clear, but it can be associated with factors such as obesity, hyperglycemia, insulin resistance, or high levels of blood triglycerides. In certain cases, fat accumulation may be associated with inflammation and scar formation in the liver. This more severe form of the disease is called non-alcoholic steatohepatitis (NASH). NASH is associated with a much higher risk of liver fibrosis and cirrhosis than NAFLD. Patients with NASH have an increased risk of hepatocellular carcinoma. NAFLD can progress to NASH with cirrhosis and hepatocellular carcinoma.

[0200] The term "non-alcoholic fatty liver disease" (NAFLD) refers to a common chronic liver disease partially characterized by fatty liver disease accompanied by associated risk factors of obesity, metabolic syndrome, and insulin resistance. Both NAFLD and NASH are often associated with obesity, diabetes, and asymptomatic elevations of serum ALT and γ-GT. Ultrasound monitoring can suggest the presence of fatty infiltration in the liver, and differentiation between NAFLD and NASH typically requires liver biopsy.

[0201] The term "primary sclerosing cholangitis" (PSC) refers to a chronic liver disease caused by progressive inflammation and scarring of the bile ducts of the liver. Scarring of the bile ducts can obstruct the flow of bile, potentially leading to cholestasis. Inflammation can lead to cirrhosis, liver failure, and liver cancer. Chronic bile duct obstruction leads to portal vascular fibrosis, and ultimately to biliary cirrhosis and liver failure. The curative treatment is liver transplantation. Indications for transplantation include recurrent bacterial cholangitis, jaundice resistant to medical and endoscopic treatment, decompensated cirrhosis, and complications of portal hypertension (PHTN). PSC progresses to varices-ascites and encephalopathy due to chronic inflammation, fibrosis / cirrhosis, altered portal circulation, portal hypertension, and portosystemic shunts. Altered portal flow is an indicator of clinical complications.

[0202] A "quantitative ion" is a single fragment ion selected from each analyte used for the quantification of the analyte. The quantitative ion may be the strongest fragment ion, and additional ions may be confirmatory ions.

[0203] A "confirmation ion" is an ion selected from the mass spectrum of the target analyte compound. The presence of the correct amount of confirmation ion relative to the quantitative ion is evidence that the target compound has been correctly identified.

[0204] The term "ion ratio monitoring" refers to the ratio of a quantitative ion to a selected confirmatory ion. For example, if the confirmatory ion signal exceeds 50% of the quantitative ion, the ion ratio in the patient sample should not deviate by ±20-30% from the standard mean ratio.

[0205] A "calibration sample" is a sample containing a known concentration of the compound to be quantified (the target analyte compound). This can also be called a calibration standard. If the method uses an internal standard, that compound may also be present in the calibration sample.

[0206] An "internal standard" is an identifiable version or analogue of the target analyte compound. For example, the internal standard may be an isotope-labeled analyte compound or a mass-identifiable analogue of the target analyte compound. The internal standard may be added at the start of sample preparation, for example, before solid-phase extraction. The amount of the internal standard may be within the working standard curve, preferably the lower half, lower third, or lower quarter of the working standard curve.

[0207] The term "selective ion monitoring" (SIM) refers to a mass spectrometer being set to scan over a very small mass range, typically a single mass unit. The narrower the range, the more specific the SIM data becomes. Only compounds with the selected mass are detected and plotted. However, since the complete mass of a compound may not be a unique identifier, a SIM plot may show a number of peaks without unique identifiers. For example, in a complex sample, many compounds may have the same mass or the same m / z ratio.

[0208] The term "multiple reaction monitoring" (MRM) refers to a method used for analyte quantification in tandem mass spectrometry in which a specific mass of ions is selected in a first step of tandem mass spectrometry, and the ion product of the fragmentation reaction of the precursor ions is selected for detection in a second mass spectrometry step. For example, in the first step of MRM, the sample ionization method may be EI, ESI, or MALDI, utilizing m / z separation to prepare the precursor ions. The precursor ions are subjected to fragmentation, and further m / z separation is performed to prepare the fragment ions.

[0209] The term "Limit of Quantitative

[0210] Terms not otherwise defined herein can be found in "CLSI. Liquid-chromatography-mass spectrometry methods. Approved guideline, C62-A, Wayne, PA, Clinical and Laboratory Standards Institute," 2014, and "CLSI. Mass spectrometry in the clinical laboratory: general principles and guidance. Approved guideline, C50-A, Wayne, PA, Clinical and Laboratory Standards Institute," 2007.

[0211] The mass spectrometer (MS) may be, for example, any suitable mass spectrometer known in the art. The MS may be a quadrupole mass spectrometer (Q), a time-of-flight (TOF) mass spectrometer, or an ion trap mass spectrometer. The MS / MS may be a triple quadrupole LC-MS / MS mass spectrometer, where Q1 decomposes molecular ions, Q2 fragments molecules, and Q3 decomposes fragments, e.g., API 4000 (AB Sciex Instruments). A given molecule that produces a given fragment is a reaction. Multiple reaction monitoring (MRM) may be used to measure several molecules, each giving a characteristic fragment or several fragments. The MS, MS / MS, or LC-MS / MS may include any suitable ionization technique known in the art. The ionization technique may be selected from electron ionization (EI), electrospray ionization (ESI), or atmospheric pressure chemical ionization (APCI). In some embodiments, a method is used to introduce the sample into the MS or MS / MS instrument without chromatography. For example, matrix-assisted laser desorption / ionization (MALDI) can be used to introduce and simultaneously ionize a sample without chromatography. Methods for introducing the sample into MS or MS / MS may include laser diode thermal desorption (LDTD), such as with a Phytronix Luxon source. MS or MS / MS may also be involved in acoustic ejection mass spectrometry (AEMS), such as combining an open-port interface (OPI) with acoustic droplet ejection (ADE), to enable direct sample analysis from a plate without LC. An example of ADE is AB Sciex's Echo MS.

[0212] Chromatographic techniques may be optionally used in conjunction with MS or MS / MS methods for quantifying identifiable compounds in patient samples. Liquid chromatography (LC) may be used, for example, in accordance with MS or MS / MS techniques. LC may be used for chromatographic separation of sample components using any suitable solid-phase matrix. Any suitable chromatographic method may be used, including normal-phase chromatography, reversed-phase chromatography, ion-exchange chromatography, hydrophilic interaction chromatography, size exclusion chromatography, affinity chromatography, etc. For example, the LC solid phase may be C18 or C8, or other reversed-phase solid-phase matrices. Gas chromatography may be used in accordance with MS or MS / MS techniques. Chromatographic separation of sample components in the gas phase may be used. For example, GC may be used using a matrix containing silica or other solid phases.

[0213] Computer / Processor The detection, prognosis, and / or diagnostic methods used in SHUNT, FLOW, STAT, and / or DSI tests may utilize the use of a processor / computer system. For example, a general-purpose computer system comprising a processor coupled with program memory for storing computer program code for implementing the method, working memory, and software interfaces including conventional computer screens, keyboards, mice, and printers, as well as other interfaces such as network interfaces and database interfaces, is used in one embodiment described herein.

[0214] The computer system accepts user input from data input devices such as a keyboard, input data files, or a network interface, or from another system such as a system for interpreting data such as MS, MS / MS, LC-MS / MS, or GC / MS data, and provides output to output devices such as a printer, display, network interface, or data storage device. An input device, such as a network interface, receives input including the detection and quantification of identifiable cholate compounds measured from processed blood or serum samples as described herein. An output device provides output such as a display, including one or more digits and / or graphs depicting the detection and / or quantification of compounds.

[0215] The computer system is connected to a data store that stores data generated by the methods described herein. This data is stored for each measurement and / or each object, and optionally, multiple sets of each of these data types are stored corresponding to each object. One or more computers / processors can be used, for example, as separate machines, for example, to be connected to the computer system via a network, or they can include separate or integrated programs running on the computer system. Whichever method is used, these systems receive data and, in return, provide data relating to detection / diagnosis.

[0216] Embodiments provide a method for selecting a treatment for subjects having abnormal levels of one or more identifiable bile acid compounds in blood or serum samples taken at multiple or single time points after oral and / or intravenous administration, the method comprising: for subjects with liver disease or impairment, calculating an output score by inputting identifiable cholate compound levels into a function that provides a predictive relationship between cholate levels and outcomes using a computing device; and displaying the output score using a computing device.

[0217] In embodiments, the method further includes using a computing device to determine whether an output score is greater than, equal to, or less than a cutoff value, and, if the output score is greater than, equal to, or less than a cutoff value, indicating whether the subject is likely to experience a clinical outcome.

[0218] In the embodiment, the computing device comprises a processing unit and a system memory connected to the processing unit, the system memory including instructions that, when executed by the processing unit, cause the processing unit to calculate the level of identifiable cholate compounds from a single blood sample from a subject into a function that provides a predictive relationship between identifiable cholate levels in subjects with liver disease or dysfunction, and to display the output score. In the embodiment, the system memory also includes instructions that, when executed by the processing unit, cause the processing unit to determine whether the output score is greater than, equal to, or less than a cutoff value, and, if the output score is greater than or equal to the cutoff value, to display whether the subject is likely to experience a clinical outcome.

[0219] In this specification, the following acronyms apply: DPBS refers to Dulbecco phosphate-buffered saline. EDTA refers to ethylenediaminetetraacetic acid. GLP refers to Good Laboratory Practice. HPLC refers to high-performance liquid chromatography. LC refers to liquid chromatography. LC-MS / MS refers to liquid chromatography-tandem mass spectrometry. LLOQ refers to the limit of quantification. m / z refers to the mass-to-charge ratio, where m is the ionic mass in atomic mass units (amu) and z is the formal charge of the ion, which is typically +1 unless otherwise specified. MRM refers to multiple reaction monitoring. MS refers to mass spectrometry. MS / MS refers to tandem mass spectrometry. Q1 refers to quadrupole 1. Q3 refers to quadrupole 3. QAU refers to quality assurance unit. QC refers to quality control. r refers to the correlation coefficient. RSD% refers to the relative standard deviation in percentage units. StdDev refers to the standard deviation. v / v refers to volume / volume.

[0220] Sample collection and processing Improved methods for sample collection and processing are provided herein for the quantification of identifiable bile acids in patient blood or serum samples.

[0221] Conventional techniques using HPLC-MS or GC-MS required the collection of at least 0.5 mL of blood or serum sample at each time point. This was because each sample was subjected to extensive processing before analysis.

[0222] To ensure accurate liver function testing, labeled cholate test compounds must be isolated and identified from patient serum samples. Cholate compounds are amphiphilic molecules possessing both hydrophobic and hydrophilic regions. Cholates are also carboxylic acids that can exist in either an uncharged free acid form (cholic acid) or a charged carboxylic acid form (cholate), depending on pH. These properties can be utilized to isolate cholate compounds from serum. In contrast to GC-MS, HPLC-MS allows for the analysis of cholates without sample derivatization. Alternatively, GC-MS can be used for sample analysis by derivatization, such as the method of Everson and Martucci, US2008 / 0279766, incorporated herein by reference.

[0223] A method for processing and quantifying identifiable cholate compounds in blood or serum samples using HPLC-MS is provided, for example, in U.S. Patent No. 9,091,701, which is incorporated herein by reference. For example, a typical sample preparation in the prior art method may involve adding an unlabeled cholic acid internal standard to 0.5 mL of patient serum. Diluted sodium hydroxide is then added, the sample is centrifuged, and then added to a solid-phase extraction SPE cartridge pre-equilibrium with water and 10% methanol. The cartridge is washed with aqueous water and 10% methanol, and then the labeled cholate is eluted from the SPE cartridge with aqueous 90% methanol. The eluted sample is dried to remove methanol, acidified with 0.2 N HCl, and then converted to the free acid form of the cholate compound. Diethyl ether is added to the acidified sample, and then the sample is vortexed to extract the free acid form of the cholate compound into the ether phase. The upper ether layer is collected and gently dried, and the ether is removed, for example, without heating or in a fume hood under N2 gas. Before injection into HPLC-MS, the HPLC-MS mobile phase buffer is added to the dried sample. Since HPLC-MS does not require analyte derivatization, this conventional method of sample preparation is an improvement over conventional GC-MS sample preparation methods. However, the process was still cumbersome and time-consuming due to the need for multiple manual steps.

[0224] Methods for sampling and quantifying identifiable cholate compounds in blood or serum samples using HPLC-MS analysis after analyte isolation are known, for example, as provided in Example 4 of U.S. Patent No. 9,091,701, which are incorporated herein by reference. For example, an Agilent 1100 series liquid chromatograph-mass spectrometer equipped with a G1956A multimode source using an Agilent Eclipse XDB C8, 2.1 × 100 mm 3.5 μm liquid chromatograph column can be used, prepared, for example, by running the mobile phase for 30 minutes at a column temperature of 40°C. A constant composition mobile phase buffer can be used, using 60% 10 mM ammonium acetate methanol / 40% 10 mM ammonium acetate aqueous solution. MS may be performed with multimode electrospray (MM-ES) ionization with atmospheric pressure chemical ionization (APCI). Selective ion monitoring (SIM) is performed at 407.30, 408.30, and 411.30 m / z. Three QC samples are assayed by each analytical run. The concentrations of the QC samples must be within 15% accuracy. Peaks are integrated using system software.

[0225] Individualized oral and intravenous clearance curves for the patient are generated using data from selective ion monitoring of either or both intravenous and oral samples. The curves are integrated along their respective effective time ranges, and the area under each is generated. Comparison of the intravenous cholate clearance curve with the oral cholate clearance curve allows for the determination of first-pass hepatic detoxification or portosystemic shunt. The hepatic shunt fraction is given by the formula, Shunt fraction = [AUC oral / AUC IV ]*[dose IV / dose oral ]*100% It is calculated as follows: In the formula, AUC represents the area under the curve, and dose represents the amount of the administered dose (mg).

[0226] A comparison of the advantages of the improved methods provided in this disclosure for use in liver function testing reveals that they offer several advantages over prior art LC-MS methods, as disclosed, for example, in U.S. Patent Nos. 8,778,299 and 9,091,701. These are summarized in Table 2. [Table 3]

[0227] Sample collection Improved methods for identifiable bile acid quantification are provided herein that require only about 10 microliters or less, 10 microliters or more, 20 microliters or more, 30 microliters or more, 40 microliters or more, or 50 microliters or more of a patient's blood or serum sample. This represents at least a 10-fold reduction in the amount of blood or serum sample compared to prior art methods, such as those disclosed in U.S. Patent No. 9,091,701. In contrast, the methods provided herein require blood or serum sample sizes of about 20 microliters or more, about 50 microliters or more, or about 50 to about 500 microliters, or about 50 to about 100 microliters.

[0228] Blood or serum samples for use in this method may be collected from subjects by any method known in the art. See, for example, the WHO guidelines on blood collection: best practices in phlebotomy, World Health Organization, 2010, Geneva, Switzerland, or BP-EIA: Collecting, processing, and handling venous, capillary, and blood spot samples, PATH, 2005. For example, needles and syringes, or indwelling catheters, arterial blood sampling, pediatric or neonatal blood sampling, or venous puncture with capillary sampling may be used. The choice of site and procedure may depend on the amount of blood required for the procedure and the clinical tests to be performed. For example, venous sites, finger pricks, or heel pricks, also known as capillary sampling or skin punctures, may be used.

[0229] Whole blood samples are obtained by venipuncture, collected in an anticoagulant-containing buctener tube, and refrigerated during storage and shipment. Blood samples can be further processed into different fractions. For example, after whole blood collection, the blood can be allowed to coagulate at room temperature and then centrifuged. The upper portion is called serum and does not contain fibrinogen. For example, whole blood may be allowed to stand for about 15-30 minutes. The resulting blood clot may be removed by centrifugation in a refrigerated centrifuge, for example, at 1,000-2,000 × g for about 10 minutes. The resulting supernatant is used as serum. From a practical standpoint, serum may be preferred over whole blood due to the possibility of red blood cell rupture, which makes sample handling delicate. However, in the case of blood or serum samples, special packaging using refrigerators, freezers, and / or dry ice is required for storage and shipment, which can incur significant logistics costs.

[0230] Dried blood spots (DBS) Dried blood spots (DBS) are a form of biosampling in which a blood sample is blotted and dried on filter paper. DBS typically involve attaching a small amount of capillary or venous blood to a special paper card. Compared to whole blood or plasma samples, their advantages depend on the fact that sample collection is easier and logistical aspects related to sample storage and shipping can be relatively limited, without the need for refrigeration or dry ice. Wagner et al., Mass Spectrometry Reviews, 2016, 35, 361-438.

[0231] DBS typically involves collecting a few drops of capillary blood, obtained by heel pricking or finger pricking, onto a card-shaped filter paper (also known as a "Guthrie card"). The sample is simply dried without any further processing. Chemically speaking, the analyte, along with the blood components, is adsorbed onto a solid cellulose-based matrix.

[0232] Compared to conventional venipuncture, DBS requires a much smaller volume of blood, is simple, non-invasive, and inexpensive, and has a minimal risk of bacterial contamination or hemolysis. Therefore, DBS can be stored for long periods with little degradation of the analyte, which in turn facilitates transport due to the stability of the sample.

[0233] DBS sampling involves a minimum sample volume, approximately 10–100, 20–80, or 30–70 microliters per spot.

[0234] Paper cards specifically for DBS are commercially available from several manufacturers and can be classified into two groups: untreated paper and chemically treated paper. Untreated paper consists of pure cellulose and may be manufactured from 100% pure cotton linters.

[0235] Treated paper contains cellulose treated with different proprietary chemicals. These may include Whatman (now part of GE Healthcare) FTA, FTA Elute, FTA DMPK-A, Whatman FTA DMPK-B (Majumdar & Howard, 2011), and Macherey Nagel NucleoCard (Moeller et al., 2012). FTA DMPK-A is impregnated with sodium dodecyl sulfate (SDS, less than 5%) and tris(hydroxymethyl)aminomethane (less than 5%), while FTA DMPK-B is impregnated with guanidinium thiocyanate (30-50%). Alternatively, untreated paper can be immersed in a solution, impregnated with the chemicals, and dried before use.

[0236] Due to the adsorption and solid nature of DBS, the analyte is typically less reactive than in (liquid) blood. One notable advantage of DBS is that the analyte often exhibits excellent stability under ambient conditions for at least several days (and sometimes up to several months) with minimal precautions (samples packed in bags sealed with a desiccant).

[0237] Compared to other blood-based samples, DBS significantly simplifies sample handling and short-term storage, avoiding the need for specialized equipment such as centrifuges, homogenizers, refrigerators, or freezers. Furthermore, DBS can reduce or eliminate biohazard risks. DBS collection requires only pricking, is not difficult to perform, and can be easily learned by medical staff or even the patient themselves, thus offering significant advantages in sample collection. On the other hand, venous blood collection requires a blood collection specialist. The volume of blood collected can be very small (typically tens of microliters), while a standard blood-derived sample in a tube requires a volume of 0.1 to several milliliters. Therefore, DBS is appropriate when the volume of blood collected is limited, for example, in neonates, infants, or critically ill patients.

[0238] The puncture site may be washed with 70% isopropanol. The skin may be punctured with a single-use sterile lancet, and after discarding the first drop of blood (to avoid leakage from interstitial fluid), the subsequent drops should be applied directly to the paper. Optionally, an internal standard may be spiked into the blood sample before spotting. The printed circle on the paper (e.g., 12 mm Whatman 903 or Ahlstrom 226) must be filled completely and uniformly. The sample can be dried (at room temperature, horizontally for 3-4 hours). DBS samples must be shipped to the laboratory within 24 hours and must meet several visual criteria to be considered suitable for screening. DBS exhibiting coagulation, stratification, supersaturation, insufficient volume, serum rings, visible signs of hemolysis, or contamination may be systematically rejected. Filter paper cards may be packed in a gas-impermeable zip bag with a desiccant bag for shipping or storage. Samples can be frozen (below -20°C) for longer-term storage, stored at -4°C, or stored at ambient temperature for up to 14 days.

[0239] One method of eluting dried blood spots may be performed at ambient room temperature by punching out one spot at a time from each blood-permeated circle using a single-use device. Circular punches (e.g., 9 mm, 7 mm, or 6 mm in diameter) may be used. One or more dried blood spots from a single patient may be transferred to a multi-well plate. The wells may be filled with phosphate-buffered saline using 0.05% TWEEN® 20 and 0.08% sodium azide. The cell culture plate may be placed on a laboratory shaker to allow the dried blood spots to elute for approximately 4 hours or overnight. The following day, the spots will typically be almost blood-free, and a hemolytic supernatant will have formed. The eluate may be transferred to a microfusing tube and centrifuged to separate the supernatant from any debris. The supernatant may be transferred to a sample vial or multi-well format for LC / MS-MS.

[0240] In an alternative elution method, the DBS punch sample or VAMS tip may be exposed to the extraction solution to solubilize the analyte. The punch sample on the VAMS tip may optionally be pre-soaked in water. The extraction solution may be, for example, water, acetonitrile, methanol, methanol-acetonitrile, methanol-water-formic acid, methanol-water (e.g., 90% MeOH aqueous solution, 4:1 v / v), or CHCl3 / MeOH (e.g., 2:1 v / v), and may be stirred for at least 30 minutes at a temperature of about 25°C. Optionally, the punch sample in the extraction solution may be vortexed, sonicated, incubated, and centrifuged. The supernatant may be dried in a lyophilizer. The dried sample may be dissolved in the extraction solution or extracted using the extraction solution, diluted in a mobile phase buffer (e.g., acetonitrile-water-formic acid, e.g., 5:95:0.1, v / v), and transferred to a sample vial or multiwell format for LC / MS-MS.

[0241] Blood collection cards, dried blood spot (DBS) technology, or HemaSpot® devices such as the HemaSpot®-HF device may be used. For example, the HemaSpot® HF device uses a finger stick to collect and dry blood in a protective cartridge. For example, EBF blood spot collection cards (Eastern Business Forms, Inc. Mauldin, SC) may be used, such as the Five Spot blood card or Generic mulipart card, where each circle holds up to approximately 75-80 microliters of sample. Once dried, the sample is stable at ambient temperature and can be safely and easily shipped to the laboratory for analysis.

[0242] Alternatively, capillary devices such as capillary tubes are used to obtain a fixed volume of blood sample.

[0243] Volume absorption microsampling Alternatively, blood samples can be obtained using a Volume Absorption Microsampling (VAMS®) device. A VAMS® device is a handheld device that includes a hydrophilic polymer tip connected to a plastic handle that wicks up a fixed volume (approximately 10, 20, or 30 microliters) upon contact with the blood surface. VAMS effectively provides absorption of a fixed volume of blood regardless of hematocrit.

[0244] Volume-absorbent microsampling can take advantage of small-volume sampling. A small sample volume of blood sample of 10, 20, or 30 microliters or more may be used to aspirate a fixed volume of capillary, venous blood, or serum sample.

[0245] VAMS® samples can be obtained by immersing a VAMS® tip in a suitable blood or serum sample and drying it for a certain period, e.g., about 2 hours or more, before extraction. The dried VAMS® tip can be removed from the sampler by pulling the tip against the side of the extraction tube and optionally adding 200 microliters of an extraction solution, such as methanol containing an internal standard. The tube may be sealed and mixed on a lateral shaker. Aliquots of the supernatant (e.g., 50 microliters) may be diluted with the mobile phase or, e.g., 1:4 methanol water, before injection into the LC-MS / MS system. Other extraction solutions can be used as described above.

[0246] VAMS (Variable Energy Mass Capture) small-volume acquisition devices, such as the MIitra (registered trademark) cartridge (Neoteryx, LLC), are commercially available.

[0247] Sample extraction from venous samples Previous multi-step analyte extraction procedures from blood or serum samples involved cumbersome combinations of solid-phase extraction, liquid-liquid extraction, evaporation, and reconstitution. Methods for replacing several previous manual sample extraction steps with simplified, partially automated online extraction procedures are provided herein. Furthermore, unlabeled compounds, such as unlabeled cholic acid, can be quantified not only in baseline samples but also in individual samples.

[0248] Aliquotes of calibrator, quality control samples, or research samples may be added to sample vials or deep-well 96-well plates. Sample aliquot sizes may vary, for example, in the range of approximately 20 μL to 500 μL, 30 μL to 400 μL, or 40 μL to 200 μL. Protein precipitation solution is added to each vial or well. The protein precipitation solution may contain a water-miscible organic solvent such as acetonitrile, or an alcohol solvent such as methanol. The protein precipitation solution may be, for example, acetonitrile or 0.1 M ZnSO4 / methanol 60:40, or methanol. Sample aliquots may be mixed with 3 to 5 times their volume of protein precipitation solution. In some embodiments, no acid is added to the protein precipitation solution. In some embodiments, the protein precipitation solution is not acidified.

[0249] The protein precipitation solution may contain an internal standard. For example, if the identifiable compound is an identifiable bile acid, the internal standard may be a different identifiable bile acid. For example, if the analyte is d4-CA or 13C-CA, the internal standard may be d5-CA. After adding the protein precipitation solution to the sample aliquot, the sample may be vortexed, centrifuged, and the supernatant may be directly injected into an HPLC system, for example, using an LC-MS / MS system.

[0250] The sample may be vortexed for 1-10 minutes, 2-8 minutes, or about 5 minutes, then centrifuged (16,000·g, 4°C, 15 minutes, or 4,750·g, 20 minutes, using a deep-well 96-well plate), and the supernatant may be transferred, for example, to an HPLC sample vial or to a 0.5 mL 96-well injection plate.

[0251] Manual solid-phase extraction, liquid-liquid extraction, evaporation, and reconstitution processes are no longer required compared to conventional methods.

[0252] The extraction and recovery rate of the sample analyte can be determined by comparing the LC, LC-MS, or LC-MS / MS peak area and / or peak area ratio of a sample prepared in serum with that of a sample prepared in methanol.

[0253] The absolute extraction recovery rate can be assessed in human serum samples according to the protocol described by Matuszewski et al. (2003), for example, by using the analyte / internal standard ratio in the following samples as follows:

[0254] Pre-extraction spiking: Human serum samples may be spiked with identifiable internal standards of compounds at the same levels as QC samples of 0.25, 0.75, 2.5, and 7.5 μmol / L before extraction and analysis. For example, human serum contains cholic acid. The cholic acid concentration may be quantified before spiking the sample. Spike cholic acid on top of endogenous cholic acid results in concentrations of 0.25 (+ endogenous cholic acid) μmol / L, 0.75 (+ endogenous cholic acid) μmol / L, 2.5 (+ endogenous cholic acid) μmol / L, and 7.5 (+ endogenous cholic acid) μmol / L.

[0255] Post-extraction spikes: The sample is first extracted and then spiked, resulting in the same concentrations as described above for the pre-extraction spiked samples: 0.25, 0.75, 2.5, and 7.5 μmol / L internal standards. For example, cholic acid is spiked on top of endogenous cholic acid, resulting in 0.25 (+ endogenous cholic acid) μmol / L, 0.75 (+ endogenous cholic acid) μmol / L, 2.5 (+ endogenous cholic acid) μmol / L, and 7.5 (+ endogenous cholic acid) μmol / L.

[0256] Sample preparation This disclosure provides a method for patient sample preparation that is simplified compared to prior art methods. The sample can be any suitable patient sample. In some embodiments, the patient sample is a blood sample or a serum sample. Sample preparation may include offline or in-line sample preparation. For example, offline sample preparation may be performed before MS or LC-MS or LC-MS / MS. Sample preparation may optionally include protein precipitation with an organic solvent such as methanol or acetonitrile, or other organic solvents. Liquid-liquid extraction (LLE) may be performed with an organic solvent such as ether or hexane, or other organic solvents. Solid-phase extraction (SPE) may be performed with any suitable chromatographic solid-phase medium, including normal-phase, reverse-phase, ion-exchange, hydrophobic interaction, size exclusion, affinity chromatography, etc. For example, a solid-phase matrix such as C18 or C8, or other reverse-phase matrices. Liquid chromatography may be performed with a matrix such as C18 or C8, or other reverse-phase matrices. Gel electrophoresis or capillary electrophoresis using slab 1D or 2D may be performed. Sample preparation may involve simple sample dilution. Sample preparation may include in-line or automated sample preparation, which may be continuous with MS, LC-MS, or LC-MS / MS. Line sample preparation may be accompanied by solid-phase extraction (SPE) using a matrix such as C18 or C8, or other solid-phase matrices.

[0257] For each corresponding sample pair (spikes before and after extraction), the absolute extraction recovery rate is calculated as follows: Extraction recovery rate [%] = Analyte / Internal standard ratio Pre-extraction spike / Post-extraction spike × 100.

[0258] Detection and quantification of analytes An improved method is provided for detecting and quantifying identifiable bile acids in blood or serum samples. Previous methods for quantifying analytes used LC-MS with selective ion monitoring. In this disclosure, detection and quantification are based on analyte ion transitions in multiple reaction modes (MS / MS vs. MS).

[0259] LC-MS and LC-MS / MS are combinations of liquid chromatography (LC) and mass spectrometry (MS). A sample in liquid form may be injected into an LC system, and different chemical components are separated based on their different affinities to the stationary phase inside the column and the mobile phase flowing through the solid-phase column. The output of the LC column is sent to a mass spectrometer, where it is ionized, for example, by electrospray or chemical ionization.

[0260] In single mass spectrometry (MS), only precursor ions are analyzed as being generated by a source, such as an ion trap, a single quadrupole, or a time-of-flight MS. In contrast, MS / MS combines two mass spectrometers into one instrument. The first mass spectrometer filters for the precursor ions, followed by fragmentation of the precursor ions using, for example, high energy and nitrogen gas. The second mass spectrometer is used to filter the generated ions produced by fragmentation. LC-MS / MS may utilize, for example, a tandem quadrupole (triple quadrupole) mass spectrometer (QQQ) or a quadrupole time-of-flight mass spectrometer (QTOF). Advantages of MS / MS include, for example, increased sensitivity in QQQ due to noise reduction, and the ability to obtain more structural information on the analyte based on the fragmentation pattern (QTOF). LC-MS / MS increases specificity in addition to improved sensitivity when used in MRM mode scanning for both precursor and generated ions. For example, two compounds with the same molecular weight may produce the same precursor ion, but can be identified and quantified based on the different generated ions formed after fragmentation. The increased sensitivity of MS / MS to a single MS can be utilized to reduce the required sample volume of blood or serum from a subject. This method exhibits approximately a 10-fold increase in sensitivity compared to previous LC-MS methods. This increased sensitivity makes it possible to reduce the serum sample volume by approximately 10-fold. For example, a method is provided that requires approximately 50 microliters of patient serum sample, whereas previous methods, such as those disclosed in U.S. Patent No. 9,091,701, may require 0.5 mL (500 microliters) of serum sample.

[0261] One advantage of multiple reaction monitoring (MRM) is that unique fragment ions can be monitored and quantified within a complex sample matrix. MRM plots can be simple and typically contain a single peak.

[0262] Multiple reaction monitoring (MRM) is achieved by specifying the parental mass of the analyte compound for MS / MS fragmentation and then specifically monitoring the simple fragment ions. A particular experiment is known as a "transition" and can be written as (parental mass → fragment mass). MS / MS can be performed using MRM monitoring in positive or negative ionization mode. For example, when MS / MS operates in negative multiple reaction monitoring mode, cholic acid (CA) is, for example, m / z = 407.3 → 343.1 (quantification ion) and 289.2 (confirmation ion). 13 C-CA can be monitored at m / z = 408.3 → 343.1 (quantitative ion) and 289.2 (confirmative ion), D4-CA at m / z = 411.3 → 347.1 (quantitative ion) and 290.2 (confirmative ion), and the internal standard D5-CA at m / z = 412.3 → 290.2 (quantitative ion) and 348.1 (confirmative ion). In another example, MS / MS can be performed in negative mode with the Q1 and Q3 masses for each of the following: 12C-CA (Q1=407.25Da, Q3=343.1Da), 13C-CA (Q1=408.25Da, Q3=343.1Da), d4-CA (Q1=411.25Da, Q3=347.2Da), and d5-CA (Q1=412.25Da, Q3=348.1Da), as shown in Table 19. In another example, MS / MS can be performed in positive ionization mode with Q1 mass and Q3 mass for each of the following: 12C-CA (Q1=409.3Da, Q3=355.4Da), 13C-CA (Q1=410.3Da, Q3=356.3Da), d4-CA (Q1=413.4Da, Q3=359.4Da), and d5-CA (Q1=414.4Da, Q3=360.4Da, and 245.1Da), as shown in Table 17.

[0263] The supernatant sample vial or 96-well injection plate loaded with the processed sample supernatant may be added to an autosampler or manually injected into a separation system.

[0264] The separation system may be an in-line separation system coupled with a mass detection system. The separation system may include preparation components and analytical components. The separation system may include a chromatography system. The chromatography system may include LC (liquid chromatography), HPLC (high-performance liquid chromatography), or UPLC (UHPLC, ultra-high-performance liquid chromatography). The preparation components may be used to pre-purify, isolate, and / or concentrate one or more identifiable compounds in the sample, or the sample supernatant. The preparation components may include an extraction column. The preparation components may include a solid-phase resin. The separation system may include analytical components. The analytical components can be used to help purify, concentrate, and / or separate identifiable compounds from each other and from other sample components. The analytical components may include solid-phase components. The prepared solid-phase components may include a solid phase. In some embodiments, the solid-phase resins of the preparation components and the analysis components are independently selected from the group consisting of normal-phase resins, reverse-phase resins, hydrophobic-interacting solid-phase resins, hydrophilic-interacting solid-phase resins, ion-exchange solid-phase resins, size-exclusion solid-phase resins, and affinity-based solid-phase resins.

[0265] The solid-phase resin in the preparation and / or analysis components may be selected from, for example, normal-phase resins, such as silica gel resins, reverse-phase resins such as C4, C8, C18, phenyl, propyl, or other hydrophobic interacting solid phases, ion-exchange solid-phase resins, size-exclusion solid-phase resins, and affinity-based solid-phase resins. In some embodiments, the preparation and analysis components use the same solid-phase material. In some embodiments, the preparation and analysis components include different solid-phase materials.

[0266] The separation system may include the LC-MS / MS system described herein.

[0267] For example, 20 μL of sample may be injected into the extraction column. The extraction column may be a reversed-phase extraction column. For example, a C8 4.6-12.5 mm 5 μm extraction column may be used (e.g., Eclipse XDB C-8, Agilent Technologies, Palo Alto, CA). The sample may be washed with a mobile phase buffer (e.g., a constant composition buffer containing 15% methanol with 0.1% formic acid and 85% water with 0.1% formic acid). The flow rate may be 2-3 mL / min within 0.5 minutes, and the temperature of the extraction column may be set to a temperature selected from approximately 30-45°C, or 40°C.

[0268] After 0.5 minutes, or after approximately 3 to 8, or 5 column volumes, the switching valve may be activated to elute the analyte (one or more identifiable compounds) from the extraction column onto the analysis column in backflush mode. The analysis column may be a reversed-phase analysis column. The analysis column may be, for example, a 150·4.6 mm C8, 5 μm analysis column (e.g., Zorbax XDB C8, Agilent Technologies, Palo Alto, CA). Compositional elution or gradient elution of the analyte from the analysis column may be performed. For example, gradient elution may be performed using an A:B mobile phase buffer, for example, 0.1% formic acid (solvent B) and methanol containing 0.1% formic acid in HPLC-grade water (solvent A). The following gradients may be performed: 0-0.5 min: 60% solvent B, 0.5-1.5 min: 60%-98% solvent B, 1.5-4 min: hold in 98% solvent B, 4-4.1 min: 98%-60% solvent B, and hold in 60% solvent B for the next 0.5 minutes.

[0269] The mass detection system may include a mass spectrometer. The mass spectrometer may include an ion source system and a mass decomposition / detection system. The ion source system may be any suitable ion source system known in the art. In some embodiments, the ion source system is selected from the group consisting of electrospray ionization (ES), matrix-assisted laser desorption / ionization (MALDI), fast atomic bombardment (FAB), chemical ionization (CI), atmospheric pressure chemical ionization (APCI), liquid secondary ionization (LSI), laser diode thermal desorption (LDTD), and surface-enhanced laser desorption / ionization (SELDI). In one embodiment, the electrospray ionization system is a turbo electrospray ionization system. The mass decomposition / detection system may be any suitable mass decomposition / detection system known in the art. In some embodiments, the mass decomposition / detection system may be selected from the group consisting of triple quadrupole mass spectrometers (MS / MS), single quadrupole mass spectrometers (MS), Fourier transform mass spectrometers (FT-MS), and time-of-flight mass spectrometers (TOF-MS).

[0270] In one embodiment, the mass detection system includes an ion source including an electrospray ionization system. In one embodiment, the mass detection system includes a triple quadrupole mass spectrometer (MS / MS). The MS / MS may be performed in negative multiple reaction monitoring (MRM) mode, in which case cholic acid is, for example, m / z = 407.3 → 343.1 and 289.2 (identification ions). 13 C-CA can be monitored at m / z = 408.3 → 343.1 and 289.2 (confirmation ions), D4-CA at m / z = 411.3 → 347.1 and 290.2 (confirmation ions), and the internal standard D5-CA at m / z = 412.3 → 290.2 and 348.1 (confirmation ions).

[0271] This disclosure provides a method for processing, detecting, and quantifying identifiable compound sample analytes in patient blood or serum samples. The results can be used, for example, to calculate patient liver function test values ​​in SHUNT, FLOW (portal vein HFR), systemic HFR, STAT, and / or DSI tests.

[0272] Portal vein HFR (FLOW), SHUNT, DSI, and STAT tests can be used to define the severity of disease in patients with chronic liver disease.

[0273] STAT test The STAT test is a screening method for estimating portal blood flow and liver function. The STAT test is disclosed in Everson et al., U.S. Patent No. 8,961,925, which is incorporated herein by reference in its entirety. The STAT test is intended for screening purposes and is used in conjunction with the FLOW and SHUNT tests to monitor hepatic blood flow and liver function. For example, patients whose STAT screening test results exceed a cutoff level may be candidates for more comprehensive portal vein HFR, SHUNT, or DSI tests to monitor hepatic blood flow and liver function in that patient.

[0274] STAT testing differs from SHUNT and FLOW testing in that it requires only a single blood sample from the patient, resulting in less clinical staff time, instrumentation time, and fewer clinical and laboratory supplies. For example, single blood collection does not require an indwelling catheter. Furthermore, preparing a single sample is less prone to error than preparing multiple sequential samples. This test is also more comfortable for patients, reducing the time they need to spend in the clinic.

[0275] Evidence and study design for applying liver function tests to CLD.

[0276] There are expected similarities in the disease progression of chronic liver diseases. For example, when comparing NAFLD and CHC, it is feasible to assess the entire spectrum of NAFLD because its pathophysiological progression is very similar to that of CHC. The progression is typically described by four histologically explained stages of fibrosis. In both the CHC Metavir system (Group, TFMCS1994. Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C Hepatology.20:15-20) and the NASH system (Brunt et al., 1999. Nonalcoholic steatohepatitis: A proposal for grading and staging the histological lesions. Am J Gastroenterol.94:2467-2474; Kleiner et al., 2005. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology.41:1313-1321), the absence of observable fibrosis is scored as F0. Early-stage fibrosis, F1, tends to be more periportal in CHC, and may be periportal and / or perisinusoidal in NASH. In both scoring systems, F2 represents more extensive periportal fibrosis and perisinus fibrosis, F3 represents bridging fibrosis, and F4 represents cirrhosis (Group, TFMCS.1994, Brunt et al., 1999, Kleiner et al., 2005, Goodman, ZD.2007. Grading and staging systems for inflammation and fibrosis in chronic liver diseases. J Hepatol.47:598-607).Due to this similar progression pattern, portal flow impairment in NASH patients with disease stages F1–F4 is expected to be equivalent to that in corresponding Metavir-stage F1–F4 CHC patients. Our previous CHC data, stratified according to the 6-stage Ishak system, can be readily converted to the Metavir system (see Goodman et al., 2007, below), enabling estimations of the expected magnitude of the effect, the number of subjects required, and the approximate power of the proposed study, as described below. Correlations between scoring systems for FLOW and Ishak scoring, SHUNT and Ishak scoring, FLOW and Metavir scoring, and SHUNT and Metavir scoring are shown in Figures 13A–D, respectively.

[0277] The impact of liver examinations in the early stages of chronic liver disease. While most previous test developments have focused on detecting progressive fibrosis and cirrhosis, it has been argued that the most pressing need in NAFLD is the ability to distinguish early-stage NASH from simple steatohepatitis (Wilson and Chalasani, N. 2007. Noninvasive markers of advanced histology in nonalcoholic fatty liver disease: Are we there yet? Gastroenterology. 133: 1377-1378, discussion 1378-1379, and Vuppalanchi and Chalasani 2009. Nonalcoholic fatty liver disease and nonalcoholic steatohepatitis: Selected practical issues in their evaluation and management. Hepatology. 49: 306-317). FLOW and SHUNT tests can detect liver dysfunction in NASH patents and distinguish them from those with simple steatohepatitis, which is expected to be close to normal portal vein FLOW.

[0278] In contrast to the FLOW and SHUNT tests, which require at least five blood samples taken from the patient over a period of 90 minutes or more after administration of an identifiable labeled cholate, the results of a test involving a single blood sample taken after oral administration of an identifiable labeled cholate compound were surprisingly found to correlate with the results of the FLOW, SHUNT, and DSI tests. A single-point-of-time screening test is called a STAT test.

[0279] The time of a single blood sample for STAT testing from a patient can be selected from any time after oral administration of an identifiable cholate, for example, from approximately 10 to 180 minutes after administration. In one embodiment, the time is a single time selected from approximately 20 to 120 minutes after administration. In another embodiment, the time is a single time selected from approximately 30 to 90 minutes after administration. In one embodiment, the blood sample is taken from the patient at any time selected from approximately 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 minutes after oral administration of an identifiable cholate, or any time in between. In one embodiment, the time of a single blood sample is selected from approximately 45, 60, or 90 minutes after administration. In a particular embodiment, the single blood sample is taken from the patient at approximately 45 minutes after administration. For example, see Figure 5, which compares the results of a STAT test 45 minutes after administration with those of a FLOW test. In another specific embodiment, a single blood sample is taken from the patient approximately 60 minutes after oral administration of an identifiable cholate. For example, see Figure 12A, which compares the results of a STAT test 60 minutes after administration with those of a FLOW test. The cholate concentration at 60 minutes is converted to an estimated flow rate (mL / min / kg) by an equation and compared with the actual FLOW test result.

[0280] In one embodiment, the identifiable compound for oral administration may be any identifiable bile acid that can be analytically identified from endogenous bile acids. In one embodiment, the identifiable bile acid is selected from any isotope-labeled bile acid known in the art. The identifiable bile acid used in any one of these assays is a stable isotope (e.g., 13 C, 2 H, 18 O) or radioactive isotopes (for example, 14 C, 3 It may be labeled with any of H). Identifiable cholate compounds are commercially available (e.g., Sigma-Aldrich, or CDN Isotopes Inc., Quebec, CA). In a preferred embodiment, the identifiable cholate is selected from any known safe, non-radioactive stable isotope of cholic acid. In a particular embodiment, the identifiable cholate compound is 2,2,4,4- 2 It is H-cholic acid. In another specific embodiment, the identifiable cholate compound is 24- 13 It is C-cholic acid.

[0281] STAT can be used as a screening test in patients who have, are suspected of having, or are at risk of having any chronic liver disease (CLD). A STAT test result of 0.4 ± 0.1 indicates a healthy patient. For example, the STAT test can be used as a screening test for patients who have, are suspected of having, or are at risk of having NAFLD. Hepatitis can also be caused by excessive alcohol consumption, such as alcoholic steatohepatitis (ASH), or by viral infections, i.e., chronic hepatitis C (CHC). Since all these chronic liver diseases (CLDs) are characterized by similar pathophysiology, involving inflammation, cell death, and fibrosis leading to progressive destruction of the hepatic microvascular system, the STAT test works in various aspects for all CLDs. For example, in patients diagnosed with PSC, 0.7±0.5 indicates PSC without PHTN, 1.6±1.5 indicates PSC with PHTN (splenomegaly of varices), 2.2±1.4 indicates PSC with varices, and 3.7±0.9 indicates decompensated PSC (variceal bleeding or ascites). In another aspect, the STAT result indicates that the patient should be followed up with additional tests such as FLOW, SHUNT, DSI, or other diagnostic tests. See, for example, Figures 6 and 7.

[0282] In another aspect, a single-point STAT test can be used as an in vitro screening for disease progression of any chronic liver disease. For example, individual patients diagnosed with chronic hepatitis C, chronic hepatitis B, cytomegalovirus, Epstein-Barr virus, alcoholic liver disease, amiodarone toxicity, methotrexate toxicity, nitrofurantoin toxicity, NAFLD, PSC, hemochromatosis, Wilson's disease, autoimmune chronic hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, or hepatocellular carcinoma can be monitored over time using a STAT test.

[0283] In another aspect, the STAT test result is an indicator of portal blood flow in any patient. The STAT test was initially developed, particularly to screen a large number of potential patients. Individuals with suspiciously low estimated portal flow may be referred to for FLOW or SHUNT tests to more accurately assess liver damage in early-stage NASH. Patients with NASH should be regularly monitored for progression to predict the course of their disease (Soderberg et al., 2010, Decreased survival of subjects with elevated liver function test during a 28-year follow-up. Hepatology. 51:595-602; Rafiq et al., 2009, Long-term follow-up of patients with nonalcoholic fatty liver. Clin Gastroenterol Hepatol. 7:234-238). The prognostic usefulness of biopsy in NAFLD is questionable (Angulo, P. 2010. Long-term mortality in nonalcoholic fatty liver disease: Is liver histology of any prognostic significance? Hepatology. 51:373-375). As previously disclosed, FLOW and SHUNT tests have been found to be superior to biopsy in predicting outcomes in chronic liver diseases such as CHC, and are expected to be superior in NAFLD as well.

[0284] In another embodiment, the STAT test is used to monitor the effectiveness of treatment for patients with liver disease. In one embodiment, the treatment is antiviral therapy.

[0285] In another embodiment, STAT testing may help prioritize patients awaiting liver transplantation. In one embodiment, patients awaiting liver transplantation are those with PSC, NASH, or chronic HCV.

[0286] In one embodiment, the STAT test is a non-invasive in vitro test used to screen patients for liver function or liver disease, and is used to monitor patients with liver disease receiving antiviral therapy, to monitor disease progression in patients with chronic liver disease, to determine the stage of disease in patients diagnosed with HCV or PSC, to prioritize patients with liver disease for liver transplantation, to determine the selection of patients with chronic hepatitis B who should receive antiviral therapy, to assess the risk of liver decompensation in hepatocellular carcinoma (HCC) patients undergoing evaluation for liver resection, to identify subgroups of patients on waiting lists with low MELD (model of end-stage liver disease score) who are at risk of dying while waiting for an organ donor, as an endpoint in clinical trials, to replace liver biopsy in pediatric populations, to track allograft function, to measure liver function recovery in living donors, to measure functional impairment in biliary liver disease (PSC, primary sclerosing cholangitis), or in combination with ALT to identify HCV patients in early stages F0-F2.

[0287] In one embodiment, the STAT test method is used for the early detection of undiagnosed liver disease. In certain embodiments, the STAT test method disclosed herein is used to detect early-stage liver disease and to accurately monitor the progression of liver disease. Early detection by tests such as STAT leads to early intervention when it is most effective, can reduce healthcare costs, and can significantly lower morbidity and mortality rates.

[0288] In the embodiment, STAT test values ​​are obtained after oral administration of an identifiable compound to the subject, and a single blood or serum sample is collected at a single specific time point after administration.

[0289] In some embodiments, the STAT test value, expressed as the concentration of an identifiable cholate compound in the sample, can be converted to the estimated portal flow (FLOW) value in the subject, or estimated portal HFR (FLOW) (expressed as mL / min / kg), by using an equation. In embodiments, the equation is y = 0.9702x + 0.0206, and R 2 = 0.8965, where x is LOG Hepquant FLOW and y is LOG Hepquant STAT, as shown in Figure 12A.

[0290] In some embodiments, STAT test values ​​can be used to estimate DSI values ​​in subjects. For example, Figure 12B shows the relationship between DSI values ​​and STAT values ​​for n=1363 subjects and n=1736 tests. Equation, Y=9.4514 ln(x)+21.12, where x=STAT value (μM adjusted for a body weight of 75kg) and Y=DSI value. R²=0.8499. The coefficient of determination is R 2 = 0.8499. 2 R is the square of the correlation coefficient. The correlation coefficient formula can be used to indicate the strength of the linear relationship between two variables.

[0291] In another embodiment, if a patient's STAT test results exceed a threshold, the patient undergoes FLOW, SHUNT, and / or DSI tests in conjunction with the STAT test. FLOW and SHUNT tests can be used to accurately track liver disease. Patients attempting to change their diet and lifestyle may see small positive effects even within a relatively short timeframe, encouraging patience. Physicians can track patients and manage their care more effectively. Rapid and accurate evaluation of the effectiveness of new drugs and therapies significantly accelerates their development.

[0292] In one embodiment, the STAT test may be administered to any patient, for example, a patient who has, is suspected of having, or is at risk of having, chronic liver disease. In various specific embodiments, the STAT test may be administered to a patient diagnosed with or suspected of having NAFLD, PSC, hepatitis C, hepatitis B, alcoholic liver disease, and / or biliary insufficiency.

[0293] In further embodiments, the method of this disclosure may be used in combination with FLOW tests, SHUNT tests (oral cholate clearance and cholate shunt) and / or DSI (dual cholate clearance tests), and is intended to be useful in several clinical applications, such as for selecting patients with chronic hepatitis B who should receive antiviral therapy; for assessing the risk of liver decompensation in patients with hepatocellular carcinoma (HCC) undergoing evaluation for hepatectomy; for identifying subgroups of patients on waiting lists with low MELD (model of end-stage liver disease score) who are at risk of dying while waiting for an organ donor; as an endpoint in clinical trials; to replace liver biopsies in pediatric populations; to track allograft function; to measure the recovery of liver function in living donors; and to measure functional impairment in biliary liver disease (PSC, primary sclerosing cholangitis). The clinical endpoint may be a primary or secondary clinical endpoint.

[0294] In one embodiment, the STAT screening method disclosed herein may be used in combination with FLOW and SHUNT tests (oral cholate clearance and cholate shunt) or DSI tests (dual cholate clearance tests) to monitor hepatic blood flow and liver function in individual patients. Using a known patient population, various cutoff values ​​for STAT, which is a single-point screening test at a specific selected time, are established to collect a single blood sample after oral administration of an identifiable cholate.

[0295] In another embodiment, the STAT test results of individual patients are compared to established cutoff values.

[0296] In one embodiment, the STAT test may be used in patients suspected of having liver disease. STAT test results from patients within the range of approximately 0 to approximately 0.6 μM ("A" range) may predict that FLOW test results are also within the normal range for portal circulation. Patients with STAT test results within the A range may be followed up, for example, by the use of annual STAT testing. STAT test results within the range of approximately 0.6 μM to approximately 1.50 μM ("B" range) may predict that FLOW test results are within the dangerous range for portal circulation. Patients with STAT test results within the B range should be further evaluated, for example, using FLOW, SHUNT, and / or tests to assess portal circulation, cholate clearance, and shunt, respectively. STAT test results above approximately 1.50 μM ("C" range) may predict progressive disease. Patients with STAT test results within the C range should be further evaluated, for example, by EGD (upper endoscopy, gastroscopy, duodenoscopy) and HCC (hepatocellular carcinoma) screening.

[0297] In another embodiment, STAT testing may be used to periodically monitor patients for improvement or progression of liver disease. For example, depending on the results of STAT testing, quantitative improvement in a patient can be tracked using annual STAT, FLOW, SHUNT, and / or DSI tests.

[0298] In another embodiment, the STAT test can be used to screen and assess disease severity in patients diagnosed with or suspected of having chronic liver disease, such as PSC. As shown in Figure 14C, STAT showed significant differences between healthy controls and patients with mild disease, and between patients with PHTN and decompensation (ascites or variceal bleeding). The simple and convenient STAT test can be used as a screening tool to lead patients to the more precise FLOW and SHUNT tests, shown in Figures 14A and 14B, respectively. As shown in Figure 14B, the SHUNT test demonstrated significant differentiation between each subgroup, distinguishing patients with mild PSC from healthy controls, and distinguishing cohorts with and without PHTN, as well as the group with PHTN, from the group with a history of ascites or variceal bleeding.

[0299] Disease Severity Index (DSI) While various direct cutoffs for FLOW and SHUNT tests have been previously developed for specific conditions, in some cases, using the Disease Severity Index (DSI) provides a clearer picture of patient classification in chronic liver disease.

[0300] The Disease Severity Index (DSI) uses a mathematical model designed to adapt bioassay results (liver function tests) to the assessment of disease severity in individual patients. For example, the DSI equation is developed using liver function test results from a defined patient population and healthy controls. In some embodiments, the DSI equation is developed from a specific patient population. The DSI equation has one or more terms selected from SHUNT, portal vein HFR, and / or systemic HFR, depending on the type or severity of liver disease. In some embodiments, one or more DSI cutoffs are used for DSI comparison, depending on the type and severity of the disease. In some embodiments, using DSI values ​​in patients requires only a simple table lookup.

[0301] In some embodiments, a method is provided for determining a disease severity index (DSI) value in a patient who has, is suspected of having, or is at risk of having chronic liver disease, the method comprising: (a) obtaining one or more liver function test values ​​in the patient, wherein the one or more liver function test values ​​are obtained from one or more liver function tests selected from the group consisting of SHUNT, portal vein HFR, and systemic HFR; and (b) obtaining a DSI value using a disease severity index equation (DSI equation), wherein at least one term of the DSI equation independently represents a liver function test value in the patient or a mathematically transformed liver function test value in the patient, and optionally multiplying at least one term of the DSI equation by a coefficient specific to the liver function test. In some embodiments, the mathematically transformed liver function test value in the patient is selected from the logarithm, real, natural logarithm, natural real, or reciprocal of the liver function test value in the patient. In some embodiments, each term of the DSI equation represents a liver function test value or a mathematically transformed liver function test value.

[0302] DSI is a function of shunt and portal vein HFR and systemic HFR, and therefore DSI is given by the general DSI equation 1. DSI can be determined using DSI = f(shunt, portal vein HFR, systemic HFR).

[0303] Certain DSI equations are provided in U.S. Patents No. 9,091,701, No. 9,759,731, and No. 10,520,517, each of which is incorporated herein by reference in its entirety.

[0304] Additional specific DSI equations include the following exemplary equations:

[0305] DSI Equation 2 uses SHUNT, portal vein HFR, and systemic HFR patient values.

[0306] DSI equation 2. DSI = A (shunt) + B (log portal vein HFR) + C (log whole-body HFR) + D, and the constants A, B, C, and D used in DSI equation 2 are shown in Table 3. [Table 4]

[0307] DSI Equation 3 uses portal vein HFR and systemic HFR patient values.

[0308] DSI equation 3.

number

[0309] The constants and coefficients to be used in Equation 3 are shown in Table 4. [Table 5]

[0310] In some embodiments, the SHUNT test value in a patient may be used in the DSI equation, and the SHUNT test value is a certain dose (dose oral ) Administer the first identifiable cholate orally to the patient in a certain volume (dose) ivThe method involves receiving multiple blood or serum samples collected from patients with PSC after co-administering a second identifiable cholate () intravenously to the patient, wherein the samples are collected at intervals over a period of time after administration; quantifying the concentrations of the first and second identifiable cholates in each sample; generating an individualized oral clearance curve from the concentration of the first identifiable cholate in each sample using a computer algorithm curve that fits a model oral identifiable cholate clearance curve; calculating the area under the individualized oral clearance curve (AUCoral); generating an individualized intravenous clearance curve from the concentration of the second identifiable cholate in each sample using a computer algorithm curve that fits a model intravenous identifiable second cholate clearance curve; calculating the area under the individualized intravenous clearance curve (AUCiv); and the formula, AUC oral / AUC iv ×Dose iv / dose oral ×100% The shunt value in the patient is determined by a method that includes using [a specific method].

[0311] In some embodiments, the SHUNT test uses the fact that the first identifiable cholate is the first stable isotope-labeled cholic acid and the second identifiable cholate is the second stable isotope-labeled cholic acid. In some embodiments, the first and second stable isotope-labeled cholic acids are 2,2,4,4-d4 cholate and 24- 13The sample is selected from C-cholates. In some embodiments, samples are collected from patients at intervals of 2 to 7 time points after administration. In some embodiments, samples are collected from patients at 5, 20, 45, 60, and 90 minutes after administration. In some embodiments, samples are collected at intervals from the time of administration over a period selected from approximately 45 minutes to approximately 180 minutes after administration. In some embodiments, samples are collected at intervals of approximately 90 minutes or less after administration.

[0312] Portal vein HFR, systemic HFR, and / or SHUNT values ​​in a patient may be determined by the methods provided herein.

[0313] In some embodiments, portal vein HFR, systemic HFR, and / or SHUNT values ​​in a patient are provided by a method that includes measuring the concentration of identifiable compounds in each sample by a method including LC-MS / MS using MRM.

[0314] In some embodiments, the portal vein HFR value in a patient is (i) a certain dose (dose oral The oral clearance of the identifiable compound may be determined by a method comprising: (ii) receiving multiple blood or serum samples collected from patients with or at risk of chronic liver disease after orally administering an identifiable compound to the patient, wherein the samples are collected from the patient at intervals of less than 3 hours after administration; (ii) measuring the concentration of the identifiable compound in each sample; (iii) generating an individualized oral clearance curve from the concentration of the identifiable compound in each sample, including using a computer algorithm curve that fits a model identifiable compound clearance curve; (iv) calculating the area under the individualized oral clearance curve (AUC) (mg / mL / min), dividing the dose (mg) by the AUC of the orally administered identifiable compound to obtain the oral identifiable compound in the patient; and (v) obtaining the portal vein HFR value (mL / min / kg) in the patient by dividing the oral identifiable compound clearance by the patient's body weight in kg.

[0315] In some embodiments, the whole-body HFR value in the patient, (i) a certain dose (dose iv The method may include: (ii) receiving multiple blood or serum samples collected from patients with or at risk of chronic liver disease after intravenous administration of an identifiable compound to a patient, wherein the samples are collected from the patient at intervals of less than 3 hours after administration; (ii) measuring the concentration of the identifiable compound in each sample; (iii) generating an individualized intravenous clearance curve from the concentration of the identifiable compound in each sample, including using a computer algorithm curve that fits a model identifiable compound clearance curve; (iv) calculating the area under the individualized intravenous clearance curve (AUC) (mg / mL / min), dividing the dose (mg) by the AUC of the intravenously administered identifiable compound to obtain the intravenous identifiable compound in the patient; and (v) obtaining the whole-body HFR value (mL / min / kg) in the patient by dividing the intravenous identifiable compound clearance by the patient's body weight in kg.

[0316] In some embodiments, a method is provided for calculating a Disease Severity Index (DSI) value in a patient who has, is suspected of having, or is at risk of having, chronic liver disease, the method comprising: obtaining a serum sample from a patient who has, is suspected of having, or is at risk of having, chronic liver disease, wherein the patient has previously received oral administration of a first stable identifiable compound and intravenous administration of a second identifiable compound, and the blood sample is collected from the patient within an interval of less than 3 hours after the administration of the first and second identifiable compounds; assaying the serum sample to calculate the portal hepatic filtration rate (portal HFR) in mL / min / kg, where kg is the patient's body weight; calculating the systemic hepatic filtration rate (systemic HFR) in mL / min / kg, where kg is the patient's body weight; and calculating SHUNT as a percentage; and calculating a DSI value for the patient by using the DSI equation as provided herein.

[0317] In some embodiments, a peripheral venous catheter is placed in the patient, and the patient is administered orally (D4-cholate, 40 mg) and simultaneously intravenously (13C-cholate, 20 mg). Blood samples are collected at t=5, 20, 45, 60, and 90 minutes after administration. Optionally, baseline samples are collected before administration. Samples are processed, and distinguishable bile acids are measured by LC-MS / MS using MRM according to this disclosure to obtain STAT, portal vein HFR, systemic HFR, SHUNT, cholate removal rate, RCA20, DSI value, algebraic HR value, and / or indexed HR value in the subject. The DSI value may be obtained from portal vein HFR, systemic HFR, and / or SHUNT values ​​by using the DSI equation.

[0318] In some embodiments, the DSI equation may be selected from the following:

[0319] Equation 1: DSI = f(shunt, portal vein HFR, systemic HFR)

[0320] Equation 2. DSI = A (shunt) + B (log portal vein HFR) + C (log whole-body HFR) + D, and the constants and coefficients used in DSI equation 2 are shown in Table 3.

number

[0321] Equation 4: DSI=A(SHUNT)+B(log e Portal vein HFR) + C(log e (Whole body HFR) + D, In the formula, SHUNT is the SHUNT test value (%) in the patient, portal vein HFR is the portal vein hepatic flow (HFR) test value (mL / min / kg) in the patient, kg is the patient's body weight, whole-body HFR is the whole-body HFR value (mL / min / kg) in the patient, kg is the patient's body weight, A is the SHUNT coefficient, B is the portal vein HFR coefficient, C is the whole-body HFR coefficient, and D is a constant. In some embodiments, the patient's SHUNT, portal vein HFR, and whole-body HFR test values ​​are obtained on the same day. The constant D may be a positive number between 5 and 125. The SHUNT coefficient A may be a number between 0 and positive 25. The portal vein HFR coefficient B may be a number between 0 and negative 25. The whole-body HFR coefficient C may be a number between 0 and negative 25.

[0322] For example, the DSI equation is, Equation 5: DSI=9.84(SHUNT)-12.36 Log e (Portal vein HFR) +50.5, Equation 6: DSI=5.75(SHUNT)-7.22(Log e Portal vein HFR) -8.45 (Log e Whole-body HFR) +50, or Equation 7: DSI=5.34(SHUNT)-6.65(Log e Portal vein HFR) -8.57 (Log e Selected from (whole-body HFR) +44.66.

[0323] The formula for DSI can be given as a function with three coefficients and two measurement variables. One of the general equations for calculating DSI is:

number

number

[0324] Variables b, c, and HFR in the DSI equation p , HFR s These are all in mL. -1 kg -1 This is a clearance value in units of -, but the units of the equation can be reduced by factorizing the variables as ratios. HFR p This is a clear clearance that depends on the amount of orally administered d4-cholate flowing into the systemic compartment from which peripheral venous blood is sampled.

[0325] HFR range in 50 healthy controls (30 lean, 16 overweight, 4 obese) (portal vein HFR 26.57 ± 8.37 mL / min) -1 kg -1 , whole body HFR6.09±1.54mL -1 kg -1 Based on this, the maximum values ​​for portal vein HFR and systemic HFR can be set as follows: [Table 6] HFR p and HFR s As the values ​​approach b and c respectively, the DSI approaches 0 - "no disease". As the HFR approaches 1, the DSI approaches 50 - "end-stage disease".

[0326] In some embodiments, the disease severity index equation used to assess a patient's chronic liver disease includes the following: SHUNT is the patient's SHUNT test value (%), portal vein HFR is the patient's portal vein HFR test value (mL / min / kg), kg is the patient's body weight, and the patient's SHUNT and portal vein HFR test values ​​are obtained on the same day.

[0327] In some embodiments, at least one term of the DSI equation independently represents a mathematically transformed liver function test value in a patient from a step, where the mathematically transformed liver function test value in the patient is selected from the logarithm, argument, natural logarithm, natural argument, or reciprocal of the liver function test value in the patient.

[0328] In some embodiments, each term of the DSI equation independently represents a liver function test value in the patient, or a mathematically transformed liver function test value in the patient, and at least one term of the DSI equation is multiplied by a coefficient specific to the liver function test.

[0329] The constants and coefficients of the DSI equation may vary depending on the type and / or severity of the liver disease. In some embodiments, the constants and coefficients are interrelated; for example, if all are divided by 10, the DSI is 0 to 5 instead of 0 to 50, and for healthy individuals it is 1 instead of 10. In some embodiments, the constants are positive numbers from 5 to 125. In some embodiments, the SHUNT coefficient is a number from 0 to positive 25. In some embodiments, the portal vein HFR coefficient is a number from 0 to negative 25. In some embodiments, the whole-body HFR coefficient is a number from 0 to negative 25.

[0330] In some embodiments, to obtain the DSI, at least one term of the DSI equation is multiplied by a coefficient specific to each type of test. In some embodiments, the DSI in a patient is compared to one or more DSI cutoff values ​​that indicate at least one clinical outcome.

[0331] In some embodiments, each term of the DSI equation independently represents a liver function test value in the patient, or a mathematically transformed liver function test value in the patient, and at least one term of the DSI equation is multiplied by a coefficient specific to the liver function test.

[0332] In some embodiments, a patient's DSI value is used to assess the severity, status, or resolution of chronic liver disease in a patient selected from chronic hepatitis C, chronic hepatitis B, cytomegalovirus, Epstein-Barr virus, alcoholic liver disease, amiodarone toxicity, methotrexate toxicity, nitrofurantoin toxicity, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hemoglobinosis, Wilson's disease, autoimmune chronic hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis (PSC), and hepatocellular carcinoma (HCC).

[0333] In some embodiments, the DSI value may be used to identify an increased risk of portal hypertension or decompensation in patients with chronic liver disease, where DSI ≥ 18 indicates an increased risk of portal hypertension (PHTN) and DSI ≥ 36 indicates an increased risk of decompensation. In some embodiments, portal hypertension (PHTN) is defined as splenomegaly or varices, and decompensation is defined as ascites or variceal bleeding. In some embodiments, chronic liver disease is primary sclerosing cholangitis.

[0334] In some embodiments, DSI values ​​in patients with chronic liver disease can be used to predict clinical outcomes in patients with chronic liver disease, where DSI ≥ 25 indicates an increased risk of serious clinical outcomes in the patient. In some embodiments, the chronic liver disease is chronic hepatitis C.

[0335] In some embodiments, serious clinical outcomes are selected from progression of CTP, variceal bleeding, ascites, hepatic encephalopathy, ascites + encephalopathy, or liver-related death.

[0336] In some embodiments, the DSI value of a patient on the waiting list for liver transplantation (LT) may be used to prioritize patients on the waiting list for LT, with the priority of a patient on the waiting list for LT increasing in response to an increase in the patient's DSI value over time or to a patient's DSI value exceeding 40.

[0337] In some embodiments, DSI values ​​in patients with chronic liver disease may be used to predict future clinical outcomes, with DSI > 19 indicating an increased risk of clinical outcomes in the patient.

[0338] In some embodiments, a DSI equation is provided comprising two or more terms and a constant for obtaining a DSI value, wherein at least one term of the DSI equation independently represents a liver function test value in the patient or a mathematically transformed liver function test value in the patient, and at least one term of the DSI equation is multiplied by a coefficient specific to the liver function test, and the DSI equation optionally includes one or more additional terms representing values ​​from a clinical biochemical laboratory assay selected from the group consisting of serum albumin, alanine transaminase, aspartate transaminase, alkaline phosphatase, total bilirubin, direct bilirubin, gamma glutamyl transpeptidase, 5' nucleotidase, PT-INR (prothrombin time-international normalized ratio), caffeine removal, antipyrinx clearance, galactose removal capacity, MEGX formation from lycetin, metacetin-C13, and methionine-C13, and / or one or more additional terms representing a clinical feature selected from varicose veins, ascites, or hepatic encephalopathy.

[0339] In some embodiments, the DSI value in a patient can be used to determine the percentage of the patient's maximum liver volume. For example, Figure 17 shows a graph of the relationship between a patient's DSI value and the percentage of maximum liver volume. Higher DSI values ​​indicate a greater decrease in the percentage of maximum liver volume and worsening chronic liver disease severity. Conversely, lower DSI values ​​indicate a greater percentage of maximum liver volume and decreased chronic liver disease severity. For example, a DSI value of 12 may represent approximately 75% of the patient's maximum liver volume. A DSI value of 20 may represent approximately 60% of the patient's maximum liver volume. A DSI value of 25 may represent approximately 50% of the patient's maximum liver volume. A DSI value of 30 may represent approximately 30% of the patient's maximum liver volume. A DSI value of 40 may represent approximately 20% of the patient's maximum liver volume.

[0340] In some embodiments, a method for determining a disease severity index (DSI) value in a patient further includes comparing the patient's DSI value over time with one or more DSI cutoff values, one or more normal healthy controls, or one or more DSI values ​​within the patient. In some embodiments, comparing the patient's DSI value with one or more DSI cutoff values ​​indicates at least one clinical outcome. In some embodiments, the clinical outcome is selected from a group consisting of Child-Turcotte-Pugh (CTP) increase, varicose veins, encephalopathy, ascites, and liver-related death.

[0341] In some embodiments, comparing DSI values ​​within a patient over time is used to monitor the effectiveness of treatment for chronic liver disease in the patient, and a decrease in DSI values ​​over time within the patient indicates the effectiveness of the treatment.

[0342] In some embodiments, comparing DSI values ​​in patients over time is used to monitor the need for treatment of chronic liver disease in patients, where an increase in DSI values ​​over time in a patient indicates the need for treatment in that patient.

[0343] In some embodiments, the DSI value in a patient is used to monitor the need for or effectiveness of treatment for chronic liver disease in the patient, and the treatment is selected from the group consisting of antiviral therapy, antifibrotic therapy, antibiotics, immunosuppressive therapy, anticancer therapy, ursodeoxycholic acid, insulin sensitizers, interventional therapy, liver transplantation, lifestyle modifications and dietary restrictions, hypoglycemia index diet therapy, antioxidants, vitamins, transjugular intrahepatic portosystemic shunt (TIPS), catheter-guided thrombolysis, balloon dilation and stent placement, balloon dilation and drainage, weight loss, exercise, and alcohol avoidance.

[0344] In some embodiments, DSI values ​​in patients are used to assess the severity of chronic liver disease in patients, for example, CLD being chronic hepatitis C, non-alcoholic fatty liver disease, or primary sclerosing cholangitis. DSI values ​​may be used to identify an increased risk of portal hypertension or decompensation in patients with chronic liver disease, with a cutoff of DSI ≥ 18 indicating an increased risk of portal hypertension (PHTN) and a cutoff of DSI ≥ 36 indicating an increased risk of decompensation. DSI cutoff values ​​greater than 15, 25, and 35 may indicate mild, moderate, and severe chronic liver disease, respectively. DSI values ​​may be used to predict the response to treatment, such as the percentage of CHC patients who achieve SVR after treatment with antiviral drugs such as PEG / RBV, for example, a DSI cutoff of 30 may indicate limited or no ability to achieve SVR.

[0345] In some embodiments, comparing intracellular DSI values ​​over time can be used to monitor the state of chronic liver disease or the progression of chronic liver disease in a patient, and changes in intracellular DSI values ​​over time can be used to inform the patient of the state of the disease and the risk of future clinical outcomes, with an increase in intracellular DSI values ​​over time indicating a worse prognosis and a decrease in intracellular DSI values ​​over time indicating a better prognosis.

[0346] In some embodiments, a kit is provided by the method described herein for determining one or more of the following in a subject: STAT, portal vein HFR, systemic HFR, SHUNT, cholate removal rate, RCA20, DSI value, algebraic HR value, and / or indexed HR value. One or more identifiable compounds may be provided in a kit and, depending on the form, may be used to assess liver function in a healthcare facility and / or home-use kit. Accordingly, the kit may include a suitable container means and an oral dose of an identifiable compound. The oral dose of the identifiable compound may be administered to a subject in or out of a healthcare facility such as a clinic, laboratory, or hospital. In addition, a second IV dose of the identifiable compound may be administered in a healthcare facility. Sample tubes for collecting and / or shipping samples, such as blood samples, may also be included. In one embodiment, the kit may include oral and IV doses of one or more identifiable compounds, and sample tubes for collecting samples over a period of less than 3 hours after administration of the identifiable compound. In another embodiment, the kit may include an oral dose of one or more identifiable compounds and a sample tube for collecting samples over a period of less than 3 hours after administration of the identifiable compounds. In yet another embodiment, the kit may include components necessary for a 90-minute testing period after administration of one or more identifiable compounds. In a further embodiment, the kit may include components necessary for a 30-minute testing period after administration of the identifiable compounds.

[0347] The sample may be serum, whole blood, venous blood, or capillary blood. For example, the sample may be whole blood or serum collected by venipuncture, or capillary blood collected by, for example, fingertip or plantar blood sampling.

[0348] In further embodiments, the kit may further include a lancet, a capillary tube, a filter paper card for collecting a dry blood spot sample, and / or a volume absorption microsampling device.

[0349] Identifiable compounds can be administered to the target, and samples can be collected at the same health facility. The samples can be analyzed within the same facility, or shipped to a reference laboratory, hospital laboratory, or testing center for analysis.

[0350] The kit may include a point-of-care test cassette and a lateral flow test cassette. Blood, serum, or capillary blood samples can be processed and applied to the lateral flow device. If one or more identifiable compounds are present in the sample at concentrations above a threshold, these identifiable compounds can be visualized in the test.

[0351] Further suitable reagents for use with this kit may include identifiable drug diluents, intravenous identifiable compound diluents, serum albumin for intravenous samples, protein precipitation solutions, and orally identifiable compound diluents. The kit may further include, for example, one or more suitably ali-coated compositions of identifiable compounds, labeled or unlabeled, which can be used as internal or external controls to prepare standard curves for detection assays.

[0352] The kit's container means generally include at least one vial, test tube, flask, bottle, syringe, or other container means that can hold, and preferably suitably ali-coat, an identifiable drug. The kit of the present invention also typically includes means for containing the identifiable compound and any other reagent containers that are tightly sealed for commercial sale. Such containers may include injection-molded or blow-molded plastic containers that hold the desired vial. In addition, the kit may include a product or diluent for diluting the orally identifiable compound, such as fruit juice or other liquids. The juice may be a juice other than a citrus juice.

[0353] Identifiable compounds may be provided in kits and may be used in in vitro tests to assess liver function in healthcare facilities and / or in home-use kit form. For example, a patient suspected of having the disease or illness may be tested with a STAT test after undergoing a medical history or physical examination or standard laboratory tests. Low test results ("A" range) suggest that the patient should be tested annually. Intermediate results ("B" range) indicate that the patient should be tested with one of the FLOW, SHUNT, and / or DSI tests. High results ("C" range) suggest that the patient is suspected of being in an advanced stage of the disease and should undergo further screening, such as esophagogastroduodenoscopy (EGD) or hepatocellular carcinoma (HCC) screening.

[0354] The identifiable compound may be used as an assessor of hepatic blood flow and may include a suitable container means for the identifiable cholate, which may be administered in an outpatient facility, a hospital environment, or outside a hospital environment, and an oral dose. This may include a sample tube, dry blood spot filter paper, a volume absorbent sample device, a lancet, a capillary tube, and / or a lateral flow device. In one embodiment, the kit may include an oral dose of the identifiable cholate and a sample tube for the collection of a single sample after a selected period from a specific point in time about 10 to about 200 minutes after oral administration of the identifiable cholate. In a specific embodiment, one blood sample is collected during a period of about 45 minutes after administration of the identifiable cholate. In another specific embodiment, one blood sample is collected during a period of about 60 minutes after administration of the identifiable cholate. In a further embodiment, the kit may include components necessary for a 30-minute testing period after administration of the identifiable agent. The kit may further include, labeled or unlabeled, diagnostic pharmaceutical compositions containing identifiable cholates, which may be used to prepare standard curves for detection assays, or appropriately aliquoted compositions of specific drugs such as cholates. The diagnostic pharmaceutical compositions may, as known in the art, include identifiable compounds and optionally additional pharmaceutically acceptable excipients, diluents, buffering compounds, pH adjusters, colorants, flavorings, and / or other excipients.

[0355] The kit's container means generally include other container means that can hold, and preferably suitably ali-coat, at least one vial, test tube, flask, bottle, syringe, or identifiable drug. The kit of the present invention also typically includes means for containing the identifiable drug and any other reagent containers that are tightly sealed for commercial sale. Such containers may include injection-molded or blow-molded plastic containers that hold the desired vial. In addition, the kit may contain a product for diluting the identifiable oral drug.

[0356] In embodiments, the kit may further include instructions for comparing the amount of an identifiable cholate compound to one or more cutoff values ​​in order to determine the state of portal blood flow and / or liver function in a patient.

[0357] Preparation of quality control samples for the kit. The FDA provides guidance on the acceptable accuracy and precision of analytical methods. See, for example, Bioanalytical Method Validation, May 2001, Section VI. Application of Validated Method to Routine Drug Analysis. Once an analytical method is validated for routine use, its accuracy and precision must be regularly monitored to ensure that the method continues to be performed satisfactorily. To achieve this objective, several QC samples should be prepared separately and analyzed at intervals with the processed test samples, based on the total number of samples. QC samples may be run in overlapping manner at three concentrations (one near the limit of quantification (LLOQ) (i.e., 3 × LLOQ), one at a moderate concentration, and one near the upper limit of the range) and should be incorporated into each assay run. The number of QC samples (a multiple of 3) depends on the total number of samples being run. The results of the QC samples provide a basis for approving or rejecting the run. At least four of every six QC samples should be within 15% of their respective nominal values. Two of the six QC samples may be outside the 15% range of their respective nominal values, but they are not necessarily at the same concentration.

[0358] QC samples must include the high, medium, and low ranges of both standard curves. QC samples are designed to closely simulate the actual concentrations of the labeled compound found in the patient's serum over time. For example, if an identifiable compound administered intravenously is [24- 13 If the compound is [C]-CA, its sample concentration is very high at an early stage and decreases exponentially to medium and low concentrations. When an identifiable compound is administered orally, it is [2,2,4,4- 2 In the case of [H]-CA, the sample concentration is very low at the early stage, rises to its peak at the intermediate stage, and then decreases to the intermediate concentration.

[0359] In some embodiments, a kit of components is provided for estimating portal blood flow and / or liver function in a subject, the kit comprising: a first component comprising one or more vials, each vial containing a single oral dose of an identifiable compound; and a second component comprising one or more sets of labeled sterile blood / serum sample collection tubes. The kit may further comprise one or more sets of labeled transport vials. Optionally, each transport vial contains an internally identifiable compound standard.

[0360] The kit may further include a single box for both shipping vials to healthcare professionals and shipping samples from healthcare professionals to a reference laboratory for analysis.

[0361] The identifiable compounds may be identifiable bile acids, bile acid conjugates, bile acid analogs, or FXR agonists provided herein. The identifiable compounds may be in powder or solution form. The first and second identifiable compounds may be stable isotope-labeled identifiable bile acids. In some embodiments, the first and second stable isotope-labeled identifiable bile acids are 2,2,4,4- 2 H-cholic acid and 24- 13 It can be selected from C-cholic acid.

[0362] The first and / or second compositions may further independently contain one or more components selected from the group consisting of pharmaceutically acceptable additives, diluents, colorants, fragrances, buffer compounds, pH adjusters, and excipients. For example, the diluent may be selected from water, sodium bicarbonate solution, non-citrus juice, or physiological saline (NS). The first and / or second compositions may contain sodium bicarbonate. The first and second compositions may exist independently in a form selected from powder or solution. In some embodiments, both the first and second compositions may be in solution form.

[0363] In some embodiments, the first composition may optionally comprise a first identifiable bile acid and sodium bicarbonate, wherein the first identifiable bile acid is 2,2,4,4- 2 It is H-cholic acid. In some embodiments, the second composition optionally comprises a second identifiable bile acid and sodium bicarbonate, wherein the second identifiable bile acid is 24- 13 It is C-cholic acid.

[0364] The kit may include containers selected from one or more of the group consisting of plastic containers, reagent containers, vials, tubes, flasks, and bottles. The kit may include a shipping box, labels, instructions for use, accompanying documents, lancets, capillary tubing, syringes, indwelling catheters, three-way stopcocks, timers, and transfer pipettes. For example, the kit may include a shipping box containing a single box for both shipping vials to healthcare professionals and shipping samples from healthcare professionals to a reference laboratory for analysis. [Examples]

[0365] Example 1. Development of an LC-MS / MS assay. Previously, the inventors disclosed, for example, in U.S. Patent No. 8,778,299, a multi-step extraction procedure for two analytes from human serum, and the detection of the analytes using selective ion monitoring, including D4-CA and 13 We developed and partially validated an LC-MS assay to quantify C-CA.

[0366] This specification provides improved methods for sample extraction, analyte detection, and quantification using LC-MS / MS for use in liver function assays, in order to improve sample extraction efficiency, assay throughput, and validate assays for evaluating analytical assay performance to support pre-market device approval.

[0367] Previous multi-step extraction procedures, including combinations of solid-phase extraction, liquid-liquid extraction, evaporation, and reconstitution, have been replaced by the automated online extraction procedure described herein. Furthermore, the assay has been further streamlined to quantify unlabeled cholic acid in each individual sample, as well as in the baseline sample.

[0368] The assay development followed the procedures and principles described in CLSI. Liquid-chromatography-mass spectrometry methods. Approved guideline, C62-A, Wayne, PA, Clinical and Laboratory Standards Institute, 2014, and CLSI. Mass spectrometry in the clinical laboratory: general principles and guidance. Approved guideline, C50-A, Wayne, PA, Clinical and Laboratory Standards Institute, 2007.

[0369] However, the final method developed differed slightly from the method described in the original, fully executed research plan. For example, one difference compared to the previous assay was that the analyte was now detected in a negative multiple reaction mode (MRM, LC-MS / MS) instead of a single-ion mode (LC-MS).

[0370] Reference material: Cholic acid-24- 13 C( 13 C-CA) and cholic acid-2,2,4,4-d4 (D4-CA) were from Sigma Aldrich (St. Louis, MO). Cholic acid and the internal standard cholic acid-2,2,3,4,4-d5 (D5-CA) were also from Sigma Aldrich (St. Louis, MO). All reference materials were in free acid form and had valid analytical certificates.

[0371] Research sample: Human serum for assay validation was purchased from BioreclamationIVT (Westbury, NY) and Gemini Bio Products (West Sacramento, CA). 100 test samples were provided by the sponsor. These samples were stored at -70°C or below in a properly licensed and monitored freezer, and aliquots were transferred to Laboratory 1. After receipt, the samples were stored at -70°C or below in a properly licensed and monitored freezer.

[0372] Preparation of calibration and quality control samples Preparation of the stock solution and working solution Cholic acid (CA), 13 The stock solutions for C-CA, D4-CA, and the internal standard D5-CA were based on three independent weights of each compound. The stock solution (10 nmol / L) was prepared in 1.0 mol / L NaHCO3 buffer and stored below -70°C. 500 μL of the 10 nmol / L stock solution was diluted in 4500 μL of LC-MS grade water to obtain a 1 nmol / L working solution. Working solutions for quality control samples and standard curves were prepared by diluting the stock solution with methanol / 0.1 mol / L NaHCO3 buffer (80 / 20, v / v).

[0373] Preparation of internal standard solution: The internal standard solution was aliquoted and transferred to a 1.5 mL conical polypropylene tube with a snap-on cap, and stored at -70°C or below. To prepare a protein precipitate solution containing 0.5 μmol / L of D5-CA, 50 μL of the internal standard solution was added to 99.95 mL of protein precipitate solution (in this case, LC-MS grade methanol). The volume was measured according to the required volume. The protein precipitate solution was stored at +4°C for up to one week.

[0374] Preparation of calibrators and quality control (QC) samples: Serum quality control samples, calibration control samples, and "blank" samples were prepared in bulk using human serum. In the following, the "blank" sample is: 13Samples were defined as those from individuals not administered C-CA and D4-CA. Since human serum contains cholates, the sample for the cholic acid calibrator was not any of the other analytes, but was diluted using Dulbecco's Phosphate Buffer Solution (DPBS) containing 5% human serum albumin, at a ratio of 1:10 (v / v) for 0.1, 0.2, 0.6, and 1.0 μmol / L calibrators, and 1:5 (v / v) for 2.0, 6.0, and 10.0 μmol / L calibrators. Cholic acid was added to obtain the calibrator concentration. To obtain the following concentrations, two sets of calibrators were prepared, one containing cholic acid (CA) and the other containing... 13 A set containing both C-CA and D4-CA was prepared.

[0375] Serum calibration curves (0.1, 0.2, 0.6, 1.0, 2.0, 6.0, and 10 μmol / L) were constructed by plotting the nominal concentrations against the analyte region after appropriate correction for cross-ion interference. For example, see Figure 2B for a representative cholate calibration curve, Figure 5 for a 13C-cholate calibration curve, and Figure 8 for a representative d4-cholate calibration curve. A regression equation with 1 / x weighting was used. Concentrations were calculated as μmol / L serum.

[0376] D4-CA and 13 The C-CA(1~4) QC samples are: 1.0.25 μmol / L D4-CA and 7.5 μmol / L 13 C-CA 2.0.75 μmol / L D4-CA and 2.5 μmol / L 13 C-CA 3.2.5 μmol / L D4-CA and 0.75 μmol / L 13 C-CA 4.7.5 μmol / L D4-CA and 0.25 μmol / L 13 It was C-CA.

[0377] The QC samples for CA(1~4) are: 1. Endogenous level QC (human serum) 2. Human serum concentrated with CA at 0.75 μmol / L 3. Human serum concentrated with CA at 2.5 μmol / L The sample was human serum concentrated with CA at a concentration of 4.75 μmol / L.

[0378] Calibrators and quality control were prepared fresh each day of analysis by spiking a suitable working solution into human serum or diluted serum of the cholate calibrator sample.

[0379] Sample processing method A 50 μL aliquot of a research serum sample, calibrator, or quality control sample was transferred to a 1.5 mL deep-well 96-well plate. A 200 μL protein precipitate solution containing the internal standard D5-CA (0.5 μmol / L) was added to each well.

[0380] The sample was vortexed for 5 minutes, centrifuged (16,000·g, 4°C, 15 minutes or 4,750·g, 20 minutes using a deep-well 96-well plate), and the supernatant was transferred to an HPLC vial (or a 0.5 mL 96-well injection plate).

[0381] LC-MS / MS equipment: The AB Sciex API4000 LC-MS / MS analytical system was used with a turbo-electrospray interface and negative multiple reaction monitoring (MRM) mode. The HPLC system included two G1312A binary pumps, two G1379A vacuum degassers, and a G1316A thermostat column compartment with an integrated 6-port Rheodyne column switching valve (Rheodyne, Cotati, CA) (all Agilent 1100 series, Agilent Technologies, Palo Alto, CA). The switching valve connections are shown in Figures 11A-B. The system also included a Leap CTC PAL autosampler with a cooling stack (Leap Technologies, Carrboro, NC). The analytical column was 150·4.6 mm C8, 5 μm (Zorbax Eclipse XDB C8, Agilent Technologies). The online extraction column was 12.5-4.6 mm C8, 5 μm (Zorbax Eclipse XDB C8, Agilent Technologies).

[0382] The mobile phase buffer was HPLC-grade water + 0.1% formic acid / HPLC-grade methanol + 0.1% formic acid. The flow rate was 2-3 mL / min during online extraction and 1 mL / min on the analytical column. The autosampler temperature was 4°C. The extraction column temperature was 40°C.

[0383] The analytical column temperature was 40°C. Unless otherwise specified, an injection volume of 20 μL was used. The assay execution time was 4.5 minutes.

[0384] The connections and locations of the column switching valves within the LC-MS / MS system are shown in Figures 11A and 11B. As shown in Figure 11A, in Mode 1, HPLC pump I flows through the injector, injecting the sample into the extraction column. As shown in Figure 11B, in Mode 2, HPLC pump II backflushes the extraction column onto the analytical column eluted into the API4000 MS / MS system where MRM monitoring is used.

[0385] Method of analysis. 20 μL of sample was injected into a 4.6 x 12.5 mm x 5 μm extraction column (Eclipse XDB C-8, Agilent Technologies, Palo Alto, CA). After injection, the sample extraction column was washed with a mobile phase of 15% methanol containing 0.1% formic acid and 85% water containing 0.1% formic acid. The flow rate was 2-3 mL / min within 0.5 minutes, as shown in Figure 11A, and the extraction column temperature was set to 40°C. After 0.5 minutes, as shown in Figure 11B, the switching valve was activated, and the analyte was eluted from the extraction column into a 150 x 4.6 mm C8, 5 μm analysis column (Zorbax XDB C8, Agilent Technologies, Palo Alto, CA) in backflush mode. The mobile phase consisted of HPLC-grade water containing 0.1% formic acid (solvent A) and methanol containing 0.1% formic acid (solvent B). The following gradient was performed. 0-0.5 min: 60% solvent B, 0.5-1.5 min: 60%-98% solvent B, 1.5-4 min: held in 98% solvent B, 4-4.1 min: 98%-60% solvent B, and then remaining in 60% solvent B for the next 0.5 minutes. Elution detection was performed by MS / MS run in negative multiple reaction monitoring (MRM) mode. Cholic acid was detected at m / z = 407.3 → 343.1 and 289.2 (confirmation ions). 13 C-CA was monitored at m / z = 408.3 → 343.1 and 289.2 (confirmation ions), D4-CA at m / z = 411.3 → 347.1 and 290.2 (confirmation ions), and internal standard D5-CA at m / z = 412.3 → 290.2 and 348.1 (confirmation ions).

[0386] After the analysis was complete, the peaks were merged (linear fit with 1 / x weighting, generated in Excel), and the results were printed. Based on the analyte / internal standard ratio contained in each batch, a calibration curve was used to determine cholic acid. 13 The concentrations of C-cholic acid and cholic acid-D4 were quantified. Signal integration was performed using Applied Biosystems Analyst Software (version 1.6.2 or later), and quantification was performed using Microsoft Excel with a semi-validated spreadsheet.

[0387] Data processing: After the analysis was completed, the peaks were integrated and the results were printed (AB SCIEX Analyst Software, version 1.6.2). Before reporting the concentrations, the analyte and internal standard signals were corrected for analyte isotope interference. Importantly, in cholic acid... 13 The natural abundance of C (m / z = 407.3) is: 13 The molecular ion of C-CA (m / z=408.3) interferes, and the natural abundance of D4-CA (m / z=411.3) interferes with the molecular ion of the internal standard D5-CA (m / z=412.3). 13 The natural abundance of C-CA (m / z=408.3) interfered with the molecular ion of D4-CA (m / z=411.3). Calculations to correct for these isotopic interferences were based on the following: (A) Blank sample (for monitoring potential matrix interference), (B) Unlabeled cholic acid (from cholic acid calibrator if necessary) in the signal at m / z=408.3→343.1 (C) D5-CA signal at m / z = 412.3 → 348.1 (D5-CA compatible sample, f[13C]), (D)m / z=408.3→343.1 13 C-CA signal (if necessary) 13 (from the C-CA calibrator), and (E) D4-CA signal at m / z = 411.3 → 347.1 (from CA and D4-CA compatibility and / or CA calibrator).

[0388] Regarding cholic acid isotope interference 13 After correcting the C-CA signal and the D5-CA signal for D4-CA isotope interference in each individual sample, the resulting analyte / internal standard ratio was calculated and used to quantify the analyte based on the calibration curve included in each batch.

[0389] The CA quality control level was calculated based on the average during each run, or based on the QCendo signal response of individual samples, depending on the validation experiment, and was calculated against the average QCendo signal response. The QC signal (for QC1-4) was calculated by subtracting the signal response of endogenous QC from the signal of the enriched QC level (nominal CA QC signal response = enriched QC signal response - endogenous QC signal response).

[0390] Isotope crosstalk coefficient: 13 In C-cholic acid, (f[ 13 C) = 13 C-CA signal (peak area) / CA signal (peak area) For cholic acid-D4, (f[d4]) = CA-D4 signal (peak area) / CA-D5 signal (peak area), and For cholic acid-D5, (f[d5]) = CA-D5 signal (peak area) / CA-D4 signal (peak area).

[0391] These correction coefficients are either within each run, or, if a suitable coefficient cannot be determined, a predetermined set of coefficients, i.e. From a CA-compatible sample (50 μL of 10 μmol / L CA in methanol + 200 μL of methanol + internal standard), and from f[ pre-determined at 0.01331 (1.33% crosstalk) 13 C], From a CA-compatible sample (50 μL of 10 μmol / L CA in methanol + 200 μL of methanol + internal standard), and f[D4] pre-determined at 0.04603 (4.60% crosstalk), The d5 conformance was determined from a sample (5 μmol / L CA-d 450 μL in methanol + 200 μL of methanol, no internal standard) and based on the pre-determined f[D5] at 0.04342 (4.34% crosstalk).

[0392] The crosstalk correction for the analyzed signals was as follows: 13 C-cholic acid signal (c peak area [ 13 C) = Cholic acid signal (peak area) - Cholic acid signal (peak area) * f[ 13 Correction signal for [C]. Correction signal for cholic acid - D4 (c peak area [D4]) = cholic acid - D4 signal (peak area) - cholic acid - d5 signal (peak area) * f [D4]. Correction signal for cholic acid - D5 (c peak area [D5]) = cholic acid - D5 signal (peak area) - cholic acid - D4 signal (peak area) * f [D5]. No correction coefficient is needed for the cholic acid signal. The response signal was used to generate a 1 / x weighted linear calibration curve, correct the CA quality control signal response, and perform all quantifications. Cholic acid response signal = Cholic acid signal (peak area) / Corrected cholic acid signal (c peak area). 13 C-cholic acid response signal = correction 13 C-cholic acid signal (c peak area) / Corrected cholic acid signal (c peak area). Cholic acid-D4 response signal = Corrected cholic acid-D4 signal (c peak area) / Corrected cholic acid signal (c peak area).

[0393] Verification procedure: The redeveloped assay was validated to the extent deemed compliant with FDA device (PMA) applications. The validation followed applicable FDA and Clinical Laboratory Standards Institute guidelines as closely as possible.

[0394] The results of this verification met the prescribed acceptance criteria, and the assay was deemed suitable for analyzing clinical and research samples from regulated clinical trials. The following minimum criteria were met to authorize the execution of the analysis: (i) the assay was under control and, where applicable, all corrective actions were successfully resolved and documented; (ii) the system / instrument was deemed suitable for use; (iii) the linearity of the calibration curve must be better than r=0.99; (iv) the calibration sample signal-to-noise ratio was low, at least 8:1; (v) there was no significant interference in the blank sample; (vi) there was no significant carryover; (vii) the accuracy of 3 / 4 of the calibration samples (=75%) was better than ±20% than the nominal value, except for the LLOQ sample; (viii) 3 / 4 of the quality control samples in the analysis batch were within 15% of the nominal concentration.

[0395] The blank is 13 The serum samples were from individuals that had not received either C-CA or D4-CA. Since cholic acid is an endogenous compound, human serum samples always contain it, and CA concentrations were measured in these "blank" samples. For CA, it was assumed that there was "no significant interference in the blank samples" unless the cholic acid concentration in the "blank" sample was higher than 15% of the initially measured concentration.

[0396] Sample storage: Research samples, calibrators, stock solutions, and quality control samples were stored at -70°C or below, except when used for testing. Samples were stored at -70°C or below for possible further analysis.

[0397] The analysis sequence for the samples was as follows: methanol, CA-d4 system compatibility test sample, methanol, blank sample (=blank serum + methanol), double zero sample (=blank serum + internal standard), 13C-CA and CA-d4 calibrators in serum; methanol (carryover control); CA system compatibility test sample, methanol, blank sample (=1:10 diluted serum + methanol), double zero sample (=1:10 diluted serum + internal standard), CA calibrators in 1:10 diluted serum (1:5 2, 6 and 10 μmol / L calibrators); methanol (carryover control), quality controls for 13C-CA and CA-d4, methanol (carryover control); endogenous quality control; CA quality control; methanol (carryover control); validation study samples (maximum 100 samples), methanol, blank sample, zero sample, calibrator; methanol (carryover control); quality control; methanol.

[0398] One of the most significant changes compared to the previous assay protocol is that the analyte is now detected in a negative multiple reaction mode (MRM, LC-MS / MS) instead of a single-ion mode (LC-MS). The redeveloped assay was then validated to the extent deemed compliant with FDA pre-market device (PMA) applications. The validation followed applicable FDA and Clinical Laboratory Standards Institute guidelines as closely as possible.

[0399] The development and validation of the assay included the following elements: limit of quantification, range of reliable response, diurnal accuracy, diurnal inaccuracy, intermittent accuracy (20 days), intermittent inaccuracy (20 days), exclusion of carryover, dilution linearity, matrix interference and ion suppression / enhancement, absolute extraction and recovery, reanalysis of generated samples, robustness, and stability testing. The validation procedure is described in more detail in Examples 1a) to 1m) below.

[0400] Example 1a) Limit of Quantitative Previously, using the older LC-MS method under U.S. Patent No. 8,778,299, standard curve samples prepared with a matrix of normal human serum for 13C-CA ranged from a lower limit of quantification (LLOQ) of 0.1 μM to an upper limit of quantification (ULOQ) of 10.0 μM, while standard curves for 4D-CA ranged from LLOQ 0.1 μM to ULOQ 5.0 μM.

[0401] Based on previous validation of the LC-MS assay, the target LLOQ for each analyte in the current LC-MS / MS assay is 0.1 μmol / L, which is also the lowest calibrator. Therefore, the goal was to validate this LLOQ for the version of the assay developed and validated herein. The determination of the LLOQ followed the procedures and principles described in CLSI EP17-A2. Accordingly, 24 samples at a concentration of 0.1 μmol / L were analyzed. The approval target was ±20% accuracy (compared to reference concentration) as per the applicable FDA recommendation [FDA, 2013]. Based on Table 4 of CLSI EP17-A2, in an observation of n=20, 85% of the results (17 / 20 samples) should be within the approval target of 80-120% of the nominal concentration. Furthermore, for each analyte in all samples, the inaccuracy (CV%) in LLOQ should be less than 20% [FDA, 2013].

[0402] Results relative to the limit of quantification (LLOQ). Cholic acid, 13 For C-cholic acid and cholic acid-D4, the LLOQ was 0.1 μmol / L in serum (cholic acid-free serum was not available for cholic acid in a 1:10 [v / v] serum dilution in 5% albumin in Dulbecco's phosphate-buffered saline (DPBS)). A typical cholic acid calibration curve is shown in Figure 2.

[0403] In summary, the accuracy and inaccuracy (CV%) of LLOQ were (n=24). [Table 7]

[0404] For all three analytes in Table 5, the measured concentrations in all 24 samples (100%) were within 80-120% of the nominal concentration, and the total inaccuracy (CV%) was 20% or less. Therefore, a concentration of 0.1 μmol / L corresponds to cholic acid, one of the three analytes in human serum. 13 All C cholic acid and cholic acid D4 met the LLOQ acceptance criteria. Compared with the internal standard cholic acid-D5, cholic acid at 0.1 μmol / L LLOQ met the requirements. 13 Representative ion chromatograms of C-cholic acid and cholic acid-D4 are shown in Figures 3, 6, and 9, respectively.

[0405] Example 1b) Reliable reaction range (analytical measurement range) The analytical measurement range is determined by the LLOQ and the upper limit of quantification (ULOQ). The ULOQ is the highest amount of analyte in the undiluted sample and can be quantitatively determined by the acceptable inaccuracy and accuracy. Based on previous LC-MS validation, the target ULOQ for each analyte is 10 μmol / L, which is also the best calibrator. The ULOQ was verified according to the procedures and principles described in CLSI EP17-A. In accordance with the requirements of the applicable FDA guidelines (FDA, 2001 and 2013), the approved target accuracy was ±15% (compared to the nominal concentration). Here again, based on CLSI EP17-A2, in an observation of n=20, 85% of the results (17 / 20 samples) had to be within the approved target. Furthermore, the inaccuracy (CV%) for each analyte in all ULOQ samples had to be 15% or less (FDA, 2013).

[0406] To further confirm the analytical measurement range (CLSI EP06-A), 40 sets of calibrators were evaluated for each analyte. These calibration curves were also used to confirm the most appropriate fit (CLSI C62-A). Acceptance was achieved if the calibration curves consistently had a correlation coefficient of r > 0.99 and at least 75% of the non-zero calibrators met the acceptance criteria. To meet the acceptance criteria, the measured concentrations of the calibrators had to be within 85–115% of the nominal concentration, with the exception of the lowest calibrator (LLOQ, 0.1 μmol / L), which had to be within 80–120% of the nominal concentration.

[0407] The range of reliable reactions is cholic acid, one of the three analytes in human serum. 13 The calibration curves for all C-cholic acid and cholic acid-D4 were linear from 0.1 to 10 μmol / L. The correlation coefficient of the calibration curves was consistently r > 0.99 (40 calibration curves measured on 20 different days). Since the calibration curves consistently had a correlation coefficient of r > 0.99 and at least 75% of the non-zero calibrators met the acceptance criteria, the acceptance criteria were met. To meet the acceptance criteria, the measured concentrations of the calibrators had to be within 85 to 115% of the nominal concentration, with the exception of the lowest calibrator (LLOQ, 0.1 μmol / L), which had to be within 80 to 120%. As shown in Table 6 below, all calibrator levels for all three analytes met the applicable acceptance criteria. The concentration at LLOQ also met the stricter acceptance criteria of 85 to 115%. [Table 8]

[0408] Each of them is cholic acid, 13 Representative individual calibration curves for C-cholic acid and cholic acid-D4 are shown in Figures 2, 5, and 8, respectively. Cholic acid at 10 μmol / L ULOQ, 13Representative ion chromatograms of C-cholic acid and cholic acid-D4 are shown in Figures 3, 6, and 9, respectively.

[0409] Examples 1c and d) Accuracy and inaccuracy during the day In accordance with the applicable FDA guidance (FDA 2001, 2013), daytime accuracy and inaccuracy must be established. For this purpose, 10 sets of QC samples were extracted and analyzed in the same run with two sets of calibrators positioned before and after the QC samples. For each analyte and concentration level, accuracy (%) and inaccuracy (CV%) were calculated. Daytime accuracy was considered acceptable if 75% of the concentrations were within the acceptable limit of 85-115% and the CV% was 15% or less. Daytime accuracy and precision are shown in Table 7.

[0410] Examples 1e and f) Accuracy and inaccuracy over a period of days To establish day-to-day accuracy and inaccuracy (FDA 2001, 2013, CLSI C62-A), we followed the "20×2×2" protocol for single-site evaluation studies recommended by CLSI EP05-A3 (see also ISO 5725-5). This protocol uses a nested dispersion component design with two runs per test day and two replicate measurements per sample over 20 test days. A single reagent lot and a single calibration lot were used, and the tests were performed on a single instrument. Day-to-day inaccuracy was estimated using a balanced nested linear component of a two-factor variance (ANOVA, n=80) model, as described in more detail in CSI EP05-A3 (Sections 3.4 and 3.6). Within each "day," "drug" and "run" were nested within "run." Day-to-day inaccuracy of 15% or less was considered acceptable. Accuracy was calculated for each individual study day, and distribution statistics for the 20 test days were calculated. The acceptable limit for accuracy was 85–115% of the nominal concentration. Table 7 shows the accuracy and precision during the day and daytime. [Table 9]

[0411] Results for daytime and intermittent inaccuracy and accuracy. This assay met the acceptance criteria for daytime inaccuracy (CV% ≤ 15%) and accuracy (at least 80% of samples within 85–115% of nominal concentration) at six QC levels, with the exception of cholic acid at the 0.25 μmol / L QC level, where only 50% of the samples met the accuracy acceptance criterion for daytime accuracy. For intermittent inaccuracy, ≤ 15% was considered acceptable. Accuracy was calculated for each individual study day, and distribution statistics for 20 test days were calculated. The acceptable limit for accuracy was 85–115% of nominal concentration. All three analytes met the acceptance criteria for accuracy and inaccuracy, with the exception of cholic acid at the endogenous compound level in serum, where the intermittent inaccuracy was 38.6%.

[0412] Example 1) Remove carryover Carryover was assessed based on methanol samples injected after the highest calibrator during day-to-day accuracy and inaccuracy testing, as required by FDA and CLSI guidelines (FDA 2001, 2013, CLSI EP-10-A3). During these experiments, two sets of each calibrator were run on each of 20 different days. This was used for analysis of the calibrators in each set (1: cholic acid, 2: 13 This means that a total of 40 carryover blank methanol samples (C-CA and D4-CA) were included. Three analytes (cholic acid, 13 Significant carryover effects were excluded if either the C-CA or D4-CA signal and 67% of the internal standard in the methanol sample were less than 20% of the signal of the corresponding lowest calibrator (=LLOQ). Results: Consistently, carryovers of less than 0.25% were found, and less than 67% of the signal in the methanol sample was greater than 20% of the signal of the corresponding lowest calibrator (=LLOQ), which was considered acceptable.

[0413] Example 1h) Dilution linearity Acceptable dilutions that yield accurate results both within and outside the measurement range require validation. Since the sample matrix significantly affects LC-MS / MS separation and ionization chemistry, the selected diluent should be suitable for the matrix (CLSI C62-A). 13 C-CA and D4-CA were administered to obtain concentrations within the linear range of the assay. For dilution experiments, human serum samples spiked with 20 μmol / L (more than twice the ULOQ) and ULOQ (10 μmol / L) were used. Samples were diluted 1:1 and 1:5 (n=12 for each concentration level and dilution). Linearity of dilution was assumed if 75% of the diluted samples were at 85–115% of the nominal concentration [CLSI C62-A]. The inaccuracy of each dilution had to be less than 15%.

[0414] Example 1i) Matrix interference and matrix effects (ion suppression / ion enhancement). Matrix interference was examined according to CLSI EP07-A3. Therefore, the potential impact of interference on inaccuracy (CLSI EP05-A3) and accuracy (CLSI EP09-A3) was evaluated.

[0415] Matrix interference by endogenous compounds in human serum. To assess whether any physiologically present compounds in human serum interfered with the quantification of the analyte, samples from 12 different and diverse individuals were used. These samples were sex-balanced and included African American and Hispanic individuals, as well as samples from patients with cholestasis. Blank samples were extracted (in three separate steps) and analyzed using LC-MS / MS. For all samples, the retention time window of the control and 13 Significant interference was not expected if there was no signal in the internal standard for C-CA and D4-CA signals exceeding 20%, or in the internal standard for LLOQ, or in the endogenous cholic acid signal increase of 20% or less.

[0416] Matrix interference by endogenous compounds (cholesterol, triglycerides, bilirubin) Potential interference from cholesterol, triglycerides, and bilirubin was assessed in blank, zero, and QC samples. These samples were spiked to yield the following concentrations: bilirubin: 0, 30, and 60 μmol / L; triglycerides: 0, 150, and 300 μmol / L; and cholesterol: 0, 250, and 500 μmol / L. The number of observations for each QC level and spiked endogenous concentration was n=8. No interference was expected in the following cases: (i) In the blank samples (n=24), LLOQ 13 (ii) For C-CA and D4-CA signals, there were no signals in the corresponding retention time window exceeding 20%; (ii) for spiked samples, the inaccuracy (CV%) did not exceed 15%, and the accuracy was acceptable (75% of the measured concentrations were within 85-115% of the nominal concentrations).

[0417] Matrix interference during hemolysis. Potential interference with hemoglobin was examined in hemolytic human serum samples. Hemolytic serum collected from three different individuals, serum samples spiked with lysed blood cells, and serum spiked with hemoglobin were examined. Blank samples, zero samples, and samples spiked at the same concentration levels as QC (0.25, 0.75, 2.5, and 7.5 μmol / L) were prepared, extracted, and analyzed. No interference was expected in the following cases: (i) Blank and zero samples showed LLOQ 13 For C-CA, D4-CA, and D5-CA (blank only) signals, or for increases in endogenous cholic acid signals of less than 20%, there was no signal in the corresponding retention time window greater than 20%, and (ii) in spiked samples, the inaccuracy (CV%) did not exceed 15%, and the accuracy was acceptable (75% of the measured concentrations were within 85-115% of the nominal concentrations).

[0418] Drug Interference Interference with 136 drugs (100 ng / mL), abused drugs, and their major metabolites was investigated. Potential interference was evaluated in blank, zero, and QC samples. These samples were spiked at three different concentration levels using the diagnostic reagent and their selected major metabolites. The number of observations for each QC level and spiked endogenous concentration was n=6. No interference was expected in the following cases: (i) Blank and zero samples showed LLOQ 13 For C-CA, D4-CA, and D5-CA (blank only) signals, or for increases in endogenous cholic acid signals of less than 20%, there was no signal in the corresponding retention time window greater than 20%, and (ii) in spiked samples, the inaccuracy (CV%) did not exceed 15%, and the accuracy was acceptable (75% of the measured concentrations were within 85-115% of the nominal concentrations).

[0419] Isotope Interference Unlabeled cholic acid 13 C signal and D4CA 13 The stability of the 1C signal is essential for accurate calculation of analyte concentration. Therefore, the stability of unlabeled cholic acid 13 C signal and D4CA 13 It was also important to study the potential interference of the C signal. This was achieved by using the same approach described above for matrix interference, interference by endogenous compounds (cholesterol, triglycerides, and bilirubin), interference in hemolysis, and interference by 136 drugs and selected major metabolites. However, samples were spiked at the QC level with only cholic acid and D4-CA. The number of observations was the same as for the corresponding interference tests described above. 13 The potential effects on the C signal (m / z = 408.3 and m / z = 412.3) were analyzed. No internal standards were added to these samples. For cholic acid, the signal ratio was calculated at m / z = 408.3 / 407.3, and for D4-CA, the signal ratio was calculated at m / z = 412.3 / 411.3. Based on CLSI C62-A (Section 7.4) and CLSI C50-A, significant isotope interference cannot be expected if the ion ratio of each QC level does not change by more than ±20%.

[0420] Matrix effect (ion suppression / ion enhancement) The matrix components of biological samples assayed by mass spectrometry generally include salts, lipids, proteins, peptides, and small organic molecules. It is well known that any matrix component can interfere with or enhance the ionization of the analyte in a mass spectrometry experiment. The most important matrix components that alter the ionization efficiency of the analyte are salts and lipids, most specifically phospholipids (CLSI C50-A, CLSI C62-A, and CLSI EP14-02). Generally, the magnitude of matrix effects should be evaluated in the context of the total tolerance (TEa) limit required of the method, which is divided into inaccuracy, bias, and interference components (CLSI EP07-A3 and CLSI EP21-A).

[0421] Based on CLSI C62-A, the following was considered best practice: Matrix effects in at least five different natural matrix samples were evaluated by comparing signals obtained from samples spiked with post-extraction analytes with signals obtained by spiked analytes in a neat solution, following the procedures described in CLSI C-50A and Matuszewski et al. (2003). Samples were used from 12 different and diverse individuals. These samples were sex-balanced and included samples from African Americans and Hispanics, as well as samples from patients with cholestasis, hepatic fibrosis, and persistent and / or non-persistent cirrhosis. Five calibration curves were prepared with methanol / 0.1 mmol / L NaHCO3 (80 / 20, v / v). These calibrators were injected, and the average signal for each calibration level was obtained from a neat solution. These signals were compared to the average signals obtained from 12 calibration curves prepared by spiked post-extraction analytes in 12 different matrix lots (from 12 different individuals). This was achieved by extracting 12 different lots of the blank matrix and then spiking the post-extracted components before injecting them into the instrument. In addition, potential matrix effects were assessed using post-injection experiments, as described by Mueller et al. (2002).

[0422] Passing criteria. The %matrix effect (%ME) was converted to the %matrix bias using the following equation. % Matrix bias = 100 - % ME (absolute matrix effect).

[0423] To assess whether the internal standard effectively compensated for any potential matrix effects, relative matrix effects based on the analyte / internal standard ratio were also calculated. % matrix bias was evaluated over the entire range of the tolerance set at ±15% [CLSI EP7-A3]. The %CV of peak area was also evaluated to determine the extent to which the matrix contributed to the assay inaccuracy. Results were considered acceptable if the inaccuracy did not exceed 15%.

[0424] Matrix effect (ion suppression / ion enhancement) test results. Matrix effects were tested in serum collected from 12 different individuals using the following two different approaches: (1) the procedure described in CLSI C-50A and by Matuszewski et al. (2003), and (2) the post-injection column experiment described by Muller et al. (2002).

[0425] The method described by Matuszewski et al. (2003) is based on comparing the MS / MS signal of a sample spiked after extraction with the MS / MS signal after injection of a neat solution of the analyte at the corresponding concentration. The results are summarized in Table 8. Absolute matrix effects compare the analyte signals, while relative matrix effects affect the analyte / internal standard ratio. The data showed an average ion suppression of -19.7% (matrix bias) for cholic acid, 13 The MS / MS signals of C-cholic acid, cholic acid-D4, and internal standard cholic acid-D5 showed average reductions of -34.0%, -34.9%, and -41.3%, respectively. Therefore, when assessing the relative ion suppression effect, the internal standard overcompensated for the ion suppression of cholic acid, resulting in a relative average ion enhancement rate of +40.4% (relative matrix bias). The internal standard, 13 Ion suppression for C-cholic acid (average +12.7%) and cholic acid-D4 (average +11.1% matrix bias) was compensated for. [Table 10]

[0426] Overall, matrix effects did not affect the accuracy and inaccuracy of the assay, as shown in Table 8, with the sole potential exception of low concentrations of cholic acid, which could explain a high inaccuracy of 38.9% at endogenous concentration levels in serum.

[0427] Post-column injection according to the protocol described by Muller et al. (2002) was performed at an injection rate of 25 μmol / L, using cholic acid. 13 The assay was based on sequential injection of a mixture of C-cholic acid, cholic acid-D4, and cholic acid-D5 (1 μmol / L of each compound in methanol). To examine ion suppression / enhancement, 20 μL of blank extracted matrix samples were injected (from different individuals, without adding an internal standard during extraction). The decay of the MS / MS signal over the retention time of the analyte indicates ion suppression, and peak ion enhancement. The decay of the MS / MS signal over the retention time of the analyte is consistent with the ion suppression detected in matrix effect experiments following the protocol described by Matuszewski et al. (2003) above. In summary, the absolute matrix effect resulted in a negative matrix bias (ion suppression) of -19.7 to -34.9%. The ion suppression was compensated for by the internal standard cholic acid-D4, but overcompensated in the case of cholic acid (relative matrix bias +40.4%). Nevertheless, as shown in Table 8 above, the matrix effect did not have any adverse effects related to the accuracy and inaccuracy of the assay.

[0428] Interference test results. After adding the highest concentrations of interfering compounds (60 μmol / L bilirubin, 300 μmol / L triglycerides, 500 μg / L cholesterol, and 100 ng / mL drug), the LC-MS / MS signals for ion transitions of analytes within the retention time window and in blank samples were (n=24). (i) 13(ii) Cholic acid: Average signal at LLOQ was 0.23% ± 0.42% (range 0% to 1.31%), (ii) Cholic acid-D4: Average signal at LLOQ was 2.40% ± 1.73% (range 0% to 7.28%), (iii) For hemolyzed serum, the retention time window of the analyte in the blank sample and the LC-MS / MS signal during ion transition were (n=8): 13 The average signal for C-cholic acid:LLOQ was 2.29% ± 1.06% (range 0.85% to 3.65%), and the average signal for cholic acid-D4:LLOQ was 4.70% ± 2.78% (range 1.95% to 9.23%).

[0429] In all cases, this was below the acceptable limit of less than 20% of the mean signal in LLOQ, and therefore there was no evidence of interference from hemolysis by the endogenous compounds, drugs, and assays examined.

[0430] Interference testing of analytes and spiked samples at QC concentrations yielded results summarized in Table 9. As shown in Table 9, all interference levels tested at each QC level met the acceptance criteria. This provides further evidence that there was no interference from the tested endogenous compounds, drugs, and hemolysis by the assay. [Table 11]

[0431] Specificity / Selectivity. In pooled serum samples taken from 12 different solids, cholic acid was used. 13 The responses of C-cholic acid and cholic acid-D4 in ionic transitions and retention times were less than 20% of the detector response of the analytes (LLOQ) and their internal standards at the lowest calibrators. Based on these results, the assay was considered specific. Results were dual-measured from serum samples taken from 12 different individuals after the addition of 60 μmol / L bilirubin, 300 μmol / L triglycerides, 500 μg / L cholesterol, and 100 ng / mL of the drug. (Data not shown).

[0432] Example 1j) Absolute extraction recovery rate Absolute extraction and recovery rates were assessed in human serum samples collected from 12 different individuals, also for use in studies of matrix interference and matrix effects. Following the protocol described by Matuszewski et al. (2003), the analyte / internal standard ratios in the following samples were compared as follows:

[0433] Pre-extraction spiking: Twelve samples were each spiked to the same level as the QC samples of 0.25, 0.75, 2.5, and 7.5 μmol / L, then extracted and analyzed. Human serum contains cholic acid. Cholic acid concentration was quantified before spiking the samples. Spikes of cholic acid on top of endogenous cholic acid yielded 0.25 (+ endogenous cholic acid) μmol / L, 0.75 (+ endogenous cholic acid) μmol / L, 2.5 (+ endogenous cholic acid) μmol / L, and 7.5 (+ endogenous cholic acid) μmol / L.

[0434] Post-extraction spikes: Samples from 12 individuals were first extracted and then spiked to obtain the same concentrations as described above for the pre-extraction spiked samples of 0.25, 0.75, 2.5, and 7.5 μmol / L. Cholic acid was spiked on top of endogenous cholic acid to obtain 0.25 (+ endogenous cholic acid) μmol / L, 0.75 (+ endogenous cholic acid) μmol / L, 2.5 (+ endogenous cholic acid) μmol / L, and 7.5 (+ endogenous cholic acid) μmol / L.

[0435] For each corresponding sample pair (spikes before and after extraction), the absolute extraction recovery rate was calculated as follows. Extraction recovery rate [%] = analyte / internal standard ratio, pre-extraction spike / post-extraction spike × 100.

[0436] Distribution statistics for each concentration level were calculated.

[0437] Extraction and recovery. 12 different individuals and cholesterol ( 13 Cholic acid extracted from the serum of one individual containing only C-cholic acid and cholic acid-D4.13 The average recovery rates for C-cholic acid and cholic acid-D4 are summarized in Table 10. [Table 12]

[0438] There was no significant difference in extraction and recovery between subjects with and without cholestasis.

[0439] Results of extraction and recovery. Depending on the concentration tested, the average recovery rates were 90.6% to 95.9% for cholic acid. 13 The concentrations were 104.0% to 110.2% for C-cholic acid and 104.4% to 111.0% for cholic acid-D4.

[0440] Example 1k) Re-analysis of the generated sample 100 patient samples were analyzed on two different days. Passing criteria followed the standards outlined in the 2013 FDA draft guidelines. Two-thirds (67%) of the replicated sample results had to be within a 20% difference (Day 1 vs. Day 2). The percentage difference in results was determined using the following equation. Percentage difference = (Result on day 1 - Result on rerun) * 100 / Average

[0441] Example 1l) Robustness Method robustness was considered during validation to assess the effects of small variations such as temperature or humidity fluctuations, preparation of calibrator materials by different operators, instrument cleanliness, and incubation time [CLSI EP09-A3, CLSI C62-A]. Robustness was tested based on patient samples analyzed during sample reanalysis and cross-validation studies. These samples were performed by different analysts using independently prepared calibrators, samples in different experimental environments, and different instrumentation on different days. For acceptance, the difference between analyses of the same sample had to be 20% or less for at least 67% of the samples. Furthermore, assay robustness was assessed based on the reproducibility (accuracy and inaccuracy) of QC samples over 20 days. For acceptance, at least 67% of each QC sample had to be within 85-115% of the nominal (accuracy), and the CV% had to be 15% or less (inaccuracy). Sufficient robustness was assumed only if all of these acceptance criteria were met.

[0442] Example 1) Stability Test Short-term stability was investigated under the following conditions: benchtop sample stability (1 day), sample storage stability at 4°C, -20°C, and below -70°C for 48 hours, 3 days, and 1 week, protein precipitation solution stability (benchtop for 24 hours, +4°C for 1 week), and maximum 72-hour stability of extracted sample / autosampler. Samples were placed in an autosampler and reinjected to baseline after 12 hours, 24 hours, 48 ​​hours, and 72 hours. This also examined the reproducibility of the reinjection [FDA, 2013].

[0443] Long-term stability was tested under the following conditions: initial assessment over 1 month (±3 days) (at -20°C and below -70°C), long-term stability over 2 months (±3 days), 3 months (±7 days), 6 months (±14 days), and 12 months (±14 days) (at -20°C and below -70°C), and 3 freeze-thaw cycles (below -70°C). For stability testing (excluding protein precipitate solution stability), five sets of QC were analyzed at each test time point. Stability was assumed if the results were within 85-115% of the nominal concentration.

[0444] The result was cholic acid, 13 C-cholic acid and cholic acid-D4 demonstrated stability in extracted samples at -80°C for one week, during three freeze / thaw cycles (-80°C / room temperature), at -20°C for one week, at +4°C for one week, and for 24 and 48 hours (+4°C) in an autosampler. Long-term stability testing is still ongoing.

[0445] Manual reintegration. Manual integration minimized errors by the integration software that could occur in the case of blank and low-concentration samples, where the integration software tends to include obvious baseline noise in the integrated peaks. However, it was important to avoid bias by the principal investigator / analyst. Therefore, manual integration and its documentation strictly followed the rules, procedures, checks, and balances outlined in iC42 Clinical Research and Development standard operating procedure iC42-WP-303 “Manual Integration of Chromatograms,” and were meticulously reviewed by quality assurance personnel. Any manual integration of ion chromatograms was justified in writing (signed and dated) and enumerated along with the values ​​from the automated integration. Copies of all versions of the original and modified integrations were printed, dated, signed, and filed with the study information. The verification report included only results approved after manual integration.

[0446] Conclusion. The redeveloped assay was validated to the extent deemed compliant with FDA device (PMA) applications. Validation followed applicable FDA and Clinical Laboratory Standards Institute guidelines as closely as possible. The validation results met the specified acceptance criteria, and the assay was deemed suitable for analyzing clinical and research samples from regulated clinical trials. The following minimum criteria were met to authorize the execution of the analysis: The assay was controlled, and all corrective actions, where applicable, were successfully resolved and documented. Acceptable system / instrument conformity testing. Linearity of the calibration curve was better than r=0.99. The signal-to-noise ratio of low-calibration samples was at least 8:1. No significant interference was found in blank samples. No significant carryover was found. The accuracy of 3 / 4 of the calibration samples (=75%) was ±15% better than nominal, except for LLOQ samples which were better than ±20%. Three-quarters of the quality control samples in the analysis batch were within 15% of the nominal concentration.

[0447] Example 2. Cross-validation study After completing the assay described in Example 1 in Laboratory 1, which uses an LC-MS / MS with negative-mode MRM, the assay was transferred to a different laboratory (Laboratory 2) using different instrumentation. For this purpose, the following was performed in parallel on the LC-MS / MS system in Laboratory 1 and the LC-MS system in Laboratory 2, where the assay had b...

Claims

1. A method for determining the HRindexed value of a subject, wherein the method is: (i) Receiving multiple blood or serum samples obtained from a subject who has, is suspected of having, or has a chronic liver disease or liver impairment, and the samples were collected from the subject less than three hours after simultaneous oral administration of a first identifiable compound and intravenous administration of a second identifiable compound to the subject, (ii) Quantifying the concentrations of the first and second identifiable compounds in the blood or serum sample, Receiving blood or serum samples obtained from subjects who have, are suspected of having, or are currently suffering from chronic liver disease or liver impairment, wherein the samples are collected from the subjects less than three hours after oral and intravenous administration of one or more identifiable cholate compounds, and the one or more identifiable cholate compounds are stable isotope-labeled identifiable cholate compounds. The process of processing the blood or serum sample to form a processed sample, wherein the processing includes aliquoting the sample to form sample aliquots and directly adding a protein precipitate solution to the blood or serum sample aliquots to form a protein precipitate and a supernatant for each sample. The supernatant derived from the processed sample is injected into a mass detection system including a tandem mass spectrometer. Measuring the concentration of one or more identifiable cholate compounds in the supernatant of the processed sample, wherein the measurement includes mass detection, and the tandem mass spectrometer operates in multiple reaction monitoring mode. To quantify the concentration of one or more identifiable cholate compounds in the blood or serum sample, This includes quantification, (iii) HFR in the subject ｐ Value and HFR ｓ The values ​​are determined from the concentrations of the first and second identifiable compounds in the blood or serum sample, (iv) said HFR ｐ and HFR ｓ This involves converting the value into an indexed hepatic reserve (HR indexed, HRi) value. [Math 1] And, During the ceremony, X is a scaling multiplier of 20 to 35 to obtain a range of 100 (normal hepatic reserve) to 0 (no hepatic reserve). y is the minimum portal vein HFR, determined by subtracting one standard deviation (SD) of the mean values ​​of lean body mass in multiple healthy controls ranging from 15 to 40 from the mean portal vein HFR. z is the minimum whole-body HFR, determined by subtracting one standard deviation (SD) of the mean lean body mass in multiple healthy controls ranging from 4 to 10 from the mean whole-body HFR, including the conversion, The aforementioned portal vein HFR (HFRp), mL －１ kg －１ However, it is calculated from the first identifiable compound dose / AUC adjusted for the body weight in kg of the subject, Whole body HFR (HFRs), mL min －１ kg －１ However, it is calculated from a second identifiable compound dose / AUC adjusted for the body weight in kg of the subject, and A method wherein the simultaneous administration of the first and second identifiable compounds is carried out within 5 minutes of each other.

2. The process further includes injecting the supernatant onto a chromatography system. The method according to claim 1, wherein the chromatography system includes a liquid chromatography (LC) system.

3. The mass detection system includes a mass spectrometer comprising an ion source system and a mass decomposition / detection system, The method according to claim 1, wherein the ion source system is selected from the group consisting of electrospray ionization (ES), matrix-assisted laser desorption / ionization (MALDI), fast atomic bombardment (FAB), chemical ionization (CI), atmospheric pressure chemical ionization (APCI), liquid secondary ionization (LSI), laser diode thermal desorption (LDTD), and surface-enhanced laser desorption / ionization (SELDI).

4. The method according to claim 3, wherein the mass resolution / detection system is selected from the group consisting of a triple quadrupole mass spectrometer (MS / MS), a quadrupole ion trap mass spectrometer, and a quadrupole time-of-flight mass spectrometer (TOF-MS).

5. The aforementioned process, The supernatant is injected into the preparation component, This includes eluting the aforementioned preparation components onto the analytical components to form the eluate, The preparation component includes a solid-phase resin, and each of the analysis components includes a solid-phase resin. The method according to claim 2, wherein the solid-phase resin of the preparation and analysis component is selected from the group consisting of normal-phase resins, reverse-phase resins, hydrophobic-interacting solid-phase resins, hydrophilic-interacting solid-phase resins, ion-exchange solid-phase resins, size-exclusion solid-phase resins, and affinity-based solid-phase resins.

6. The method according to claim 1, wherein the protein precipitate solution contains a water-miscible organic solvent.

7. The method according to claim 6, wherein the water-miscible organic solvent is selected from the group consisting of methanol, ethanol, isopropanol, acetonitrile, and acetone.

8. The method according to claim 1, further comprising adding an identifiable compound of an internal standard to the blood or serum sample.

9. The method according to claim 1, wherein the volume of the blood or serum sample is 10 to 500 μL, 20 to 400 μL, 30 to 300 μL, 30 to 200 μL, or 40 to 100 μL.

10. The blood or serum sample is obtained in the form of a dried blood spot sample, a capillary blood sample, or a dried volume absorption microsampling device sample. The method according to claim 9, wherein the process includes exposing the sample to an extraction solution to form the supernatant, and diluting the supernatant before injection.

11. The method according to claim 1, wherein the one or more stable isotope-labeled, identifiable cholate compounds can exhibit a high hepatic extract of at least 50%, 60%, or 70% in the first pass through the liver of a healthy subject after oral administration.

12. The one or more stable isotope-labeled cholate compounds are 2,2,4,4-d4-cholic acid (D4-CA; CA-D4), 24- １３ C-cholic acid ( １３ C-CA), 2,2,3,4,4-d ５ cholic acid (D ５ -CA), 3,6,6,7,8,11,11,12-d8 cholic acid (D8-CA), lithocholic acid-2,2,4,4-D4 (LCA-D4), ursodeoxycholic acid-2,2,4,4-D4 (UDCA-D4), ursodeoxycholic acid (24-13C-UDCA), deoxycholic acid-2,2,4,4-D4 (DCA-D4), glycochenodeoxycholic acid-2,2,4,4-D4 (GCDCA-D4), glycochenodeoxycholic acid (glycine-2,2,3,4,4,6,6,7,8-D9-CDCA), glycodeoxycholic acid -2,2,4,4-D4 (GDCA-D4), Glycocholic acid-2,2,4,4-D4 (GCA-D4), Glycocholic acid (Glycine-1-13C-CA), Deoxycholic acid-24-13C (DCA-24-13C), Deoxycholic acid (2,2,4,4,11,11-D6-DCA), α-Mulicoleic acid (2,2,3,4,4-D5-αMCA), β-Mulicoleic acid (2,2,3,4,4-D5-βMCA), Chenodeoxycholic acid (2,2,3,4,4,6,6,7,8-D9-CDCA), Chenodeoxycholic acid (2,2,3,4 Taurochenodeoxycholic acid (2,2,4,4-D4-CDCA), chenodeoxycholic acid (24-13C-CDCA), γ-mulicolic acid (2,2,3,4,4-D5-γMCA), omega-mulicolic acid (2,2,3,4,4-D5-ωMCA), taurochenodeoxycholic acid, sodium salt (taurine-2,2,3,4,4,6,6,7,8-D9-CDCA), taurochenodeoxycholic acid, sodium salt (taurine-2,2,4,4-D4-CDCA), taurocholic acid, sodium salt (taurine-13C2 -CA), taurocholic acid, sodium salt (taurine-2,2,4,4-D4-CA), taurodeoxycholic acid, sodium salt (taurine-2,2,4,4,11,11-D6-DCA), taurodeoxycholic acid, sodium salt (taurine-2,2,4,4-D4-DCA), tauroursodeoxycholic acid, sodium salt (taurine-2,2,4,4-D4-UDCA), tauroursodeoxycholic acid, sodium salt (taurine-13C2-UDCA), glycolitocholic acid (glycine-2,2,4,4-D4-LCA), 11,The method according to claim 11, selected from the group consisting of 12-double hydrogenated chenodeoxycholic acid (D2-chenodeoxycholic acid, D2-CA), glycoursodeoxycholic acid (glycine-2,2,4,4-D4-UDCA), and glycoursodeoxycholic acid (glycine-13C2-UDCA).

13. The method according to claim 1, wherein the extraction and recovery rate of the identifiable cholate compound from the blood or serum sample is >80%, >90%, or >95%.

14. The method according to claim 1, wherein the blood or serum sample is collected at one or more time points after the oral administration of the identifiable compound, selected from the group consisting of baseline, 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, and 180 minutes, or any time point in between.

15. The first and second identifiable compounds are identifiable bile acids, bile acid conjugates, or bile acid analogs. The method according to claim 1, wherein the plurality of samples are collected at baseline, 5, 20, 45, 60, and 90 minutes after the simultaneous administration.

16. A second or subsequent HRindexed value is determined in the subject after a predetermined time interval. The increase in the second or subsequent HRindexed value indicates improvement in liver function in the subject, and the increase in HRindexed is at least 2 percent, at least 3 percent, at least 4 percent, or at least 5 percent, or more, and The method according to claim 1, wherein the second or subsequent decrease in the HRindexed value indicates deterioration of liver function in the subject.

17. A kit comprising components for quantifying one or more identifiable compounds in one or more blood or serum samples from a subject in order to determine an indexed HR value in the subject, wherein the subject has, is suspected of having, or has developed liver damage, and the kit is, A first component comprising one or more vials, each vial comprising a first composition comprising a first identifiable compound in a single oral dose, A second component comprising one or more vials, each vial comprising a second composition comprising a second identifiable compound in a single intravenous dose, and Includes, The first and second identifiable compounds are stable isotope-labeled identifiable bile acids, and the first and second stable isotope-labeled identifiable bile acids are 2,2,4,4- ２ H-cholic acid and 24- １３ A kit selected from C-cholic acid.

18. The third component further comprises one or more vials, each vial containing a certain amount of human albumin for mixing with the second identifiable compound in a single intravenous dose before intravenous administration. The kit according to claim 17, wherein the human albumin is human serum albumin.

19. A fourth component comprising one or more sample collection tubes and / or transport vials, A fifth component comprising suitable container means, The kit according to claim 18, wherein the sample collection tubes comprise one or more sets of sterile blood-serum sample collection tubes, each set comprising enough tubes to collect multiple samples from the subject over a period of 180 minutes, 90 minutes, 60 minutes, or 45 minutes after administration of the first and second identifiable compounds.

20. The kit according to claim 17, wherein the first composition and / or the second composition further independently comprises one or more components selected from the group consisting of pharmaceutically acceptable additives, diluents, colorants, fragrances, buffer compounds, pH adjusters, and excipients.

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