WO2018007422A1 - Identification of human non-alcoholic fatty liver disease (nafld) subtypes - Google Patents

Identification of human non-alcoholic fatty liver disease (nafld) subtypes Download PDF

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WO2018007422A1
WO2018007422A1 PCT/EP2017/066715 EP2017066715W WO2018007422A1 WO 2018007422 A1 WO2018007422 A1 WO 2018007422A1 EP 2017066715 W EP2017066715 W EP 2017066715W WO 2018007422 A1 WO2018007422 A1 WO 2018007422A1
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Cristina ALONSO SÁNCHEZ
Ibon MARTÍNEZ ARRANZ
José María Mato De La Paz
María Luz Martínez Chantar
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One Way Liver,S.L.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
    • G01N33/6812—Assays for specific amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2570/00—Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin

Definitions

  • the present invention relates to the field of diagnostics and, more in particular the non-invasive diagnosis of non-alcoholic steatohepatitis (NASH) and steatosis in different subsets of non-alcoholic fatty liver disease (NAFLD) patients showing different serum metabolomic profiles.
  • This invention relates to methods based on signatures of serum metabolomic biomarkers for differentiating NASH from simple steatosis in different subsets of NAFLD patients.
  • Non-alcoholic fatty liver disease encompasses a wide range of conditions characterized by the build-up of fat in the liver cells in absence of alcohol abuse. At one end of the scale is the relatively harmless simple fatty liver, or steatosis, that does not cause significant liver damage. If left unattended, this condition may progress to more advanced conditions, some of which may be life threatening.
  • NASH Nonalcoholic steatohepatitis
  • NAFLD is a significant development in NAFLD, corresponding to an aggressive condition characterized by swelling and tenderness in the liver. With intense, on-going inflammation a build up of scar tissue (fibrosis) may form, eventually leading to cirrhosis where irregular bumps, known as nodules, replace the smooth liver tissue and the liver becomes harder. The effect of this, together with continued scarring from fibrosis, means that the liver will run out of healthy cells to support normal functions. This can lead to complete liver failure.
  • NAFLD is the most common cause of chronic liver disease world wide. It is considered a direct consequence of the rising global epidemic of obesity and the associated increase in the prevalence of diabetes. Most people with a fatty liver are overweight or obese. As more and more people lead inactive lives and carry extra weight around with them, so the number of cases of fatty liver, in particular NASH is rising.
  • NAFLD may be suspected in subjects with one or more components of the metabolic syndrome, especially obesity and type 2 diabetes, and elevated serum aminotransferase levels [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] in the absence of alcohol abuse or other common causes of liver disease.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • the only widely accepted test for distinguishing NASH from other forms of disease is a liver biopsy. This process involves passing a fine hollow needle through the skin and into the liver, withdrawing a small tissue of sample that is submitted for histological examination. Apart from the obvious discomfort induced by this invasive procedure, assessment is often subjective and prone to sampling error.
  • WO 2008/021192 describes a non-invasive method for the diagnosis and monitoring of liver diseases such as NASH and steatosis based on the determination of levels of fatty acids and eicosanoids in a body fluid of the patient.
  • this method is limited to the identification of lipid species and requires complex fractionation steps of the body fluids before the metabolites can be detected.
  • WO 2012/143514 relates to a method for the diagnosis of liver damage, among this liver damage is comprised NAFLD, in a subject comprising determining in a biological sample of said subject the levels of a panel of metabolic markers.
  • the method has been performed considering the metabolites detected in rat serum extracts and comparing their levels with the degree of apoptosis present in the liver cells. Liver damage therefore can be of any nature.
  • WO2013113992 and WO2015089102 disclose non-invasive tools using plasma biomarkers for differentiating non-alcoholic steatohepatitis (NASH) from non-alcoholic fatty liver (NAFL), and non-alcoholic fatty liver disease (NAFLD) from normal controls.
  • NASH non-alcoholic steatohepatitis
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-invasive methods to diagnose NASH, in order to better understand where is the patient located within the spectrum of phenotypes that can progress to cirrhosis.
  • NASH is a histological definition that groups together defects in diverse biochemical processes causing hepatic fat accumulation, inflammation, necrosis and fibrosis.
  • the identification of the types of mechanisms leading to NASH and the discovery of non-invasive biomarkers of NASH subtypes are central for the development of effective treatments and precise diagnosis.
  • the authors of the present invention have identified a number of metabolic markers in the serum samples from subjects previously diagnosed with NAFLD. These metabolic markers are significantly differentiated between two different mice genotypes, the Matla (-/-) mice model that spontaneously develops NASH on a normal diet and that of wild type (WT) mice, and used in serum samples of NAFLD patients to classify three clusters, M-subtype, non-M-subtype and I-subtype. These metabolic markers can then be used in a rapid non-invasive subclassifying diagnostic method for differentiating different subsets of NAFLD patients. Further serum metabolic profiles wouldn been identified that allow distinguishing between NASH and steatosis for each NAFLD patients cluster.
  • the invention relates to a method to profile a subject suffering from non-alcoholic fatty liver disease (NAFLD) as M-subtype, non-M- subtype or I-subtype that comprises determining in a biological sample from said subject the levels of one or more metabolic markers as defined in Table 1, wherein
  • NAFLD non-alcoholic fatty liver disease
  • step (iii) if the subject is not profiled as M-subtype according to step (i) and is not profiled as non-M-subtype according to step (ii), then the subject is profiled as I- subtype.
  • the invention relates to a method to diagnose non-alcoholic steatohepatitis (NASH) or steatosis in a subject suffering from NAFLD and profiled as M-subtype according to the first method above that comprises determining in a biological sample from said subject the levels of one or more metabolic markers according to Table 2, wherein
  • NASH non-alcoholic steatohepatitis
  • Table 2 wherein
  • the invention relates to a method to diagnose non-alcoholic steatohepatitis (NASH) or steatosis in a subject suffering from NAFLD and profiled as non-M-subtype according to the first method above that comprises determining in a biological sample from said subject the levels of one or more metabolic markers according to Table 3, wherein
  • NASH non-alcoholic steatohepatitis
  • Table 3 wherein
  • the invention relates to a method to diagnose non-alcoholic steatohepatitis (NASH) or steatosis in a subject suffering from NAFLD and profiled as I-subtype according to the first method above that comprises determining in a biological sample from said subject the levels of one or more metabolic markers according to Table 4, wherein
  • NASH non-alcoholic steatohepatitis
  • FIG. 1 Volcano plot representation indicating the -loglO(p-value) and log2(fold-change) of individual serum metabolic ion features of NASH compared to simple steatosis.
  • AA amino acids
  • SFA MUFA, PUFA
  • oxFA oxidized FA
  • DG diglycerols
  • TG triglycerides
  • ChoE cholesteryl esters
  • BA bile acids
  • PE phosphatidylethanolamines
  • LPE lyso-PE
  • PC phosphatidylcholines
  • LPC lyso-PC
  • PI phosphatidylinositols
  • LPI lyso-PI
  • Cer ceramides
  • SM sphingomyelins
  • CMH monohexosylceramides
  • FSB free sphingoid bases
  • DAG diacylglycerols
  • TAG triacylglycerols.
  • the authors of the present invention have developed a method to determine different subsets of NAFLD subjects based on the levels of metabolic markers as shown in Table 1, and wherein a NAFLD subject is classified in three major clusters (M- subtype, non-M-subtype and I-subtype).
  • This method has been developed by analysis of the differences between genotypes Matla (-/-) mice and wild type (WT) mice, as shown in the Example (see Table 5, corresponding to the metabolites showing significant differences between Matl (-/-) mice and wildtype, and that are able to subclassifying three clusters or subtypes of NAFLD patients; see also Table 12, showing the platforms used for the analysis of said metabolites significantly differentiated).
  • the most significant serum metabolites between said genotypes were selected based on an unpaired Student's t-test (or Welch ' s t test where unequal variances were found). Then, selected metabolites were analyzed in serum samples from 377 patients with biopsy proving NAFLD. A hierarchical clustering algorithm based on metabolites was used to visualize the differences between samples, as well as the ward's minimum variance method as agglomeration method. The silhouette cluster analysis showed three well separated groups: M-subtype, Non-M-subtype and I-subtype ( Figure 1).
  • the invention relates to a method to profile a subject suffering from non-alcoholic fatty liver disease (NAFLD) as M-subtype, non-M- subtype or I-subtype (first method of the invention) that comprises determining in a biological sample from said subject the levels of one or more metabolic markers as defined in Table 1 , wherein
  • NAFLD non-alcoholic fatty liver disease
  • step (iii) if the subject is not profiled as M-subtype according to step (i) and is not profiled as non-M-subtype according to step (ii), then the subject is profiled as I- subtype.
  • the first method of the invention comprises determining in a biological sample from the subject suffering from NAFLD whose profile as M-subtype, non-M-subtype or I-subtype is to be determined, the levels of at least 20% of the markers as defined in Table 1 , wherein
  • step (iii) if the subject is not profiled as M-subtype according to step (i) and is not profiled as non-M-subtype according to step (ii), then the subject is profiled as I- subtype.
  • the first method of the invention comprises determining in a biological sample from the subject suffering from NAFLD whose profile as M-subtype, non-M-subtype or I-subtype is to be determined the levels of at least 40% of the markers as defined in Table 1 , wherein
  • step (iii) if the subject is not profiled as M-subtype according to step (i) and is not profiled as non-M-subtype according to step (ii), then the subject is profiled as I- subtype.
  • the first method of the invention comprises determining in a biological sample from the subject suffering from NAFLD whose profile as M-subtype, non-M-subtype or I-subtype is to be determined the levels of at least 60% of the markers as defined in Table 1, wherein
  • step (iii) if the subject is not profiled as M-subtype according to step (i) and is not profiled as non-M-subtype according to step (ii), then the subject is profiled as I- subtype.
  • the first method of the invention comprises determining in a biological sample from the subject suffering from NAFLD whose profile as M-subtype, non-M-subtype or I-subtype is to be determined the levels of at least 80% of the markers as defined in Table 1, wherein
  • step (iii) if the subject is not profiled as M-subtype according to step (i) and is not profiled as non-M-subtype according to step (ii), then the subject is profiled as I- subtype.
  • the first method of the invention comprises determining in a biological sample from the subject suffering from NAFLD whose profile as M-subtype, non-M-subtype or I-subtype is to be determined the levels of all the markers as defined in Table 1, wherein
  • step (iii) if the subject is not profiled as M-subtype according to step (i) and is not profiled as non-M-subtype according to step (ii), then the subject is profiled as I- subtype.
  • profile relates to characterize a subject suffering from NAFLD as belonging to an M-subtype, non-M subtype or I-subtype based on particular levels of metabolites according to the present invention and shown in Table 1.
  • M-subtype relates to the subtype of NAFLD subject showing a metabolomic profile similar to that observed by the inventors in Matla (-/-) mice.
  • the subtype M is characterized by having increased levels of at least one, at least 20%>, at least 40%>, at least 60%>, at least 80%> or all the metabolic markers according to Table 1(a) with respect to the cut-off value according to Table 1(a) and/or decreased levels of at least one, at least 20%>, at least 40%, at least 60%, at least 80% or all the metabolic markers according to Table 1(b) with respect to the cut-off value according to Table 1(b).
  • non-M subtype as used herein, relates to the subtype of NAFLD subject showing a metabolomic profile which is the opposite to M-subtype metabolomic profile observed by the inventors in Matla (-/-) mice.
  • the subtype non-M is characterized by having increased levels of at least one, at least 20%, at least 40%, at least 60%, at least 80% or all the metabolic markers according to Table 1(c) with respect to the cut-off value according to Table 1(c).
  • the term "I-subtype", or intermediate subtype relates to the subtype of NAFLD subject showing a metabolomic profile that is intermediate to the metabolic profile of those NAFLD subjects showing a M-subtype and to the metabolic profile of those NAFLD subjects showing a non-M-subtype.
  • the I-subtype is characterized by not having increased levels of at least one, at least 20%, at least 40%, at least 60%, at least 80% or all the metabolic markers according to Table 1(a) with respect to the cut-off value according to Table 1(a) and/or not having decreased levels of at least one, at least 20%>, at least 40%>, at least 60%>, at least 80% or all the metabolic markers according to Table 1(b) with respect to the cut- off value according to Table 1(b) and not having increased levels of at least one, at least 20%, at least 40%, at least 60%, at least 80% or all the metabolic markers according to Table 1(c) with respect to the cut-off value according to Table 1(c).
  • similar levels refers to levels that differ in less than 1%, less than 0,75%, less than 0,5%, less than 0,25%, less than 0,01% or less than 0,001% to the levels of the metabolic markers in the Matla (-/-) mice.
  • non-alcoholic fatty liver disease refers to a group of conditions having in common the accumulation of fat in the hepatocytes. NAFLD ranges from simple fatty liver (steatosis), to non-alcoholic steatohepatitis (NASH), to cirrhosis (irreversible, advanced scarring of the liver).
  • NAFLD non-alcoholic steatohepatitis
  • NAFLD includes any stage or degree of progression of the disease.
  • subject refers to all the animals classified as mammals and includes but is not limited to domestic and farm animals, primates and humans, for example, human beings, non- human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents.
  • the subject is a male or female human being of any age or race.
  • the subject who suffers from NAFLD is a mammal, preferably a human.
  • sample or “biological sample”, as used herein, refers to biological material isolated from a subject.
  • the biological sample may contain any biological material suitable for detecting the desired biomarker and may comprise cellular and/or non cellular material from the subject.
  • the sample can be isolated from any suitable biological tissue or fluid such as, for example, liver tissue, blood, blood plasma, serum, urine or cerebral spinal fluid (CSF).
  • the samples used for the determination of the level(s) of the metabolic markers are samples which can be obtained using minimally invasive procedures.
  • the sample is a bio fluid from a subject.
  • the sample is a biofluid selected from the group consisting of blood, plasma, serum, isolated lipoprotein fraction, saliva, urine, lymph fluid, cerebrospinal fluid and bile.
  • the sample is a biofluid selected from blood, plasma and serum.
  • the sample is a serum sample.
  • lipid marker refers to small molecule compounds, such as substrates for enzymes of metabolic pathways, intermediates of such pathways or the products obtained by a metabolic pathway, the occurrence or amount of which is characteristic for a specific situation, for example NAFLD.
  • the abbreviated names of the lipid metabolites correspond to the lipid family to which it belongs followed by a lipid number of the fatty acid side chains. The lipid family is further described by the reference number of said lipid family in the LIPID MAPS structure database (http://www.lipidmaps.org/data/databases.html) using the LIPID MAPS Classification System (Fahy E. et al, Journal of Lipid Research 2009, 50: S9-S14).
  • the lipid number is a number with the format N:n, where "N” corresponds to the number of carbons in the fatty acid chains and "n” corresponds to the number of double bonds in the fatty acid chains.
  • N corresponds to the number of carbons in the fatty acid chains
  • n corresponds to the number of double bonds in the fatty acid chains.
  • the lipid metabolic markers of tables la, lb and lc, tables 2a and 2b, tables 3a and 3b and tables 4a and 4b are intended to refer to any isomer thereof, including structural and geometric isomers.
  • structural isomer refers to any of two or more chemical compounds, having the same molecular formula but different structural formulas.
  • geometric isomer or “stereoisomer” as used herein refers to two or more compounds which contain the same number and types of atoms, and bonds (i.e., the connectivity between atoms is the same), but which have different spatial arrangements of the atoms, for example cis and trans isomers of a double bond, enantiomers, and diastereomers.
  • the abbreviated name of the amino acid corresponds to the amino acid name to which it belongs followed by an accession number as described in the Human Metabolome Database HMDB (version 3.6) (http://www.hmdb.ca).
  • Metabolic markers according to the first method of the invention are shown in Table 1. Cut-off values are indicated as percentage values, wherein 0% corresponds to the level of the metabolic marker as determined in a sample from one or more healthy subjects or in a sample from one or more subjects not suffering from NAFLD. Negative percentage cut-off values represent values of metabolites which are lower than the value as determined in a sample from one or more healthy subjects or in a sample from one or more subjects not suffering from NAFLD. Table 1.Metabolic markers for profiling M, non-M and I subtypes of NAFLD subjects according to the first method of the invention.
  • the levels of one or more metabolic markers according to Table 1 are determined in a biological sample from a subject whose M-, non-M- or I-subtype profile is to be determined.
  • one metabolic marker according to Table 1 is determined.
  • At least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty- four or at least twenty- five, at least thirty, at least thirty-five, at least forty, at least forty-five, at least fifty, at least fifty-five, or at least sixty metabolic markers according to Table 1 are determined.
  • the first method of the invention can be carried out by determining the levels of a variable number of metabolites as defined in table 1 in the biological sample of the subject under study. The determination of levels of combinations of the metabolic markers may allow greater sensitivity and specificity in profiling different subtypes of NAFLD patients.
  • step (i) of the first method of the invention if the levels of at least one metabolic marker according to Table 1(a) are increased with respect to the cut-off value according to Table 1(a) and/or the levels of at least one metabolic marker according to Table 1(b) are decreased with respect to the cut-off value according to Table 1(b), then the subject is profiled as M-subtype.
  • the subject is profiled as M-subtype.
  • the subject is profiled as M-subtype if the levels of all metabolic markers according to Table 1(a) are increased with respect to the cut-off value according to Table 1(a) and/or the levels of all metabolic markers according to Table 1(b) are decreased with respect to the cut-off value according to Table 1(b), then the subject is profiled as M-subtype.
  • step (ii) of the first method of the invention if the level of at least one metabolic marker according to Table 1(c) is increased with respect to the cut-off value according to Table 1(c), then the subject is profiled as non-M-subtype.
  • the levels of at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, of the metabolic markers according to Table 1(c) are increased with respect to the cut-off value according to Table 1(c), then the subject is profiled as non-M-subtype.
  • the levels of all metabolic markers according to Table 1(c) are increased with respect to the cut-off value according to Table 1(c)
  • the subject is profiled as non-M-subtype.
  • steps (i) and (ii) according to the first method of the invention may be performed in any order, i.e, step (ii) following step (i) or alternatively step (i) following step (ii) or at the same time.
  • step (i) is performed firstly and step (ii) is performed secondly.
  • the subject suffering from NAFLD to be profiled according to the first method of the invention is not profiled as M-subtype according to step (i) (i.e.
  • level refers to the quantity of a biomarker detectable in a sample. It will be understood that the biological sample can be analyzed as such or, alternatively, the metabolites may be first extracted from the sample prior to analysis and the metabolite extract is then analyzed. If the metabolites are extracted prior to analysis, different extraction methods are available to the skilled person. The selection of one or other extraction method will depend on the class of metabolites/small molecules that are targeted from a particular analysis. Suitable extraction methods include “Extraction of free metabolite pools", “Vapor Phase Extraction”, and “Total Metabolite Extraction”. The first type of extraction, “Extraction of free metabolite pools”, is mainly used in metabolomics research.
  • free intracellular metabolite pools are obtained from a biological sample through methanol- water extraction for polar metabolites, or chloroform, methanol, chloroform/methanol extraction for non-polar metabolites.
  • the second type of extraction “Vapor Phase Extraction”, refers to the extraction of metabolites that are volatile at room temperature. The metabolites are expelled from the biological sample in the vapor phase. These metabolites are either measured directly by connecting the flask or reactor in which the vapors are generated to the analytical instrument or by absorbing first the vapors in charcoal/solvent and then analyzing the acquired solution.
  • Total Metabolite Extraction refers to the extraction of the free metabolite pools along with the metabolites that have been incorporated in cellular macromolecules, e.g. lipids, proteins, etc.
  • the present invention provides extraction of a particular class of metabolites from macromolecules (e.g. amino acids from proteins or sugars from cell wall components).
  • the present invention also provides a combined high-throughput method which extracts all metabolites simultaneously.
  • the metabolite quantification can be carried out directly in the biological sample.
  • the sample may be prepared to enhance detectability of the markers.
  • a blood serum sample from the subject can be preferably fractionated by, e.g., Cibacron blue agarose chromatography and single stranded DNA affinity chromatography, anion exchange chromatography, affinity chromatography (e.g., with antibodies) and the like.
  • the method of fractionation depends on the type of detection method used. Any method that enriches for the metabolite of interest can be used.
  • preparation involves fractionation of the sample and collection of fractions determined to contain the biomarkers.
  • Methods of pre-fractionation include, for example, size exclusion chromatography, ion exchange chromatography, heparin chromatography, affinity chromatography, sequential extraction, gel electrophoresis and liquid chromatography.
  • the analytes also may be modified prior to detection. These methods are useful to simplify the sample for further analysis. For example, it can be useful to remove high abundance proteins, such as albumin, from blood before analysis.
  • a sample can be pre-fractionated by removing proteins that are present in a high quantity or that may interfere with the detection of markers in a sample.
  • Proteins in general may be removed by using conventional techniques such as precipitation using organic solvents such as methanol precipitation, ethanol, acetonitrile, acetone or combinations thereof, in particular, combination of methanol, acetone and acetonitrile, acid precipitation using, for example, trichloroacetic acid or perchloric acid, heat denaturation and any combination of organic solvent, acid and heat precipitation.
  • organic solvents such as methanol precipitation, ethanol, acetonitrile, acetone or combinations thereof, in particular, combination of methanol, acetone and acetonitrile
  • acid precipitation using, for example, trichloroacetic acid or perchloric acid, heat denaturation and any combination of organic solvent, acid and heat precipitation.
  • serum albumin or other proteins abundant in serum such as apolipoproteins, glycoproteins, immunoglobulins may obscure the analysis of markers since they are present in a high quantity. Thus, it may be sufficient to remove one or more of the
  • the blood serum or plasma sample can be pre-fractionated by removing serum albumin.
  • Serum albumin can be removed using a substrate that comprises adsorbents that specifically bind serum albumin.
  • a column which comprises, e.g., Cibacron blue agarose (which has a high affinity for serum albumin) or anti-serum albumin antibodies can be used.
  • a sample can be pre-fractionated by isolating proteins that have a specific characteristic, e.g. are glycosylated.
  • a blood serum or plasma sample can be fractionated by passing the sample over a lectin chromatography column (which has a high affinity for sugars).
  • affinity adsorbents which are suitable for pre- fractionating blood serum or plasma samples.
  • An example of one other type of affinity chromatography available to prefractionate a sample is a single stranded DNA spin column. These columns bind proteins which are basic or positively charged. Bound proteins are then eluted from the column using eluants containing denaturants or high pH. Thus there are many ways to reduce the complexity of a sample based on the binding properties of the proteins in the sample, or the characteristics of the proteins in the sample.
  • a sample can be fractionated using a sequential extraction protocol.
  • sequential extraction a sample is exposed to a series of adsorbents to extract different types of biomolecules from a sample.
  • the determination of the level of the one or more metabolic markers is carried out by mass spectrometry.
  • mass spectrometry refers to an analytical technique to identify unknown compounds including: (1) ionizing the compounds and potentially fractionating the compounds parent ion formed into daughter ions; and (2) detecting the charged compounds and calculating a mass-to-charge ratio (m/z).
  • the compounds may be ionized and detected by any suitable means.
  • a "mass spectrometer” includes means for ionizing compounds and for detecting charged compounds.
  • mass spectrometry is used, in particular gas chromatography coupled to mass spectrometry (GC-MS), liquid chromatography coupled to mass spectrometry (LC-MS), direct infusion mass spectrometry or Fourier transform ion-cyclotrone resonance mass spectrometry (FT-ICR-MS), capillary electrophoresis coupled to mass spectrometry (CE-MS), high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS), ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS), supercritical fluid chromatography coupled to mass spectrometry (SFC-MS), flow injection analysis with mass spectrometry (FIA-MS), including quadrupole mass spectrometry, any sequentially coupled mass spectrometry, such as MS-MS or MS-MS-MS, inductively coupled plasma mass spectrometry (ICPMS), pyrolysis mass spectrometry (Py-MS), ion mobility mass spectrometry or time-of-f
  • LCMS is used as described in detail below. Said techniques are disclosed in, e.g., Nissen, Journal of Chromatography A, 703, 1995: 37- 57, US 4,540,884 or US 5,397,894.
  • the above mentioned ionization methods generally produce an ion resulting from the addition of one or more atoms or by cleavage of the molecule. These ions can then be used as surrogate markers for the metabolites used in the method of the invention.
  • surrogate marker means a biological or clinical parameter that is measured in place of the biologically definitive or clinically most meaningful parameter.
  • the ions resulting from the addition of a proton or a hydrogen nucleus [M+H] ⁇ +> where M signifies the molecule of interest, and H signifies the hydrogenion, which is the same as a proton.
  • Some ionization methods will also produce analogous ions. Analogous ions may arise by the addition of an alkaline metal cation, rather than the proton discussed above.
  • a typical species might be [M+Na] ⁇ +>, [M+NH4] ⁇ +> or [M+K] ⁇ +>.
  • the analysis of the ionized molecules is similar irrespective of whether one is concerned with a protonated ion as discussed above or dealing with an added alkaline metal cation.
  • a proton adds one mass unit (typically called one Dalton), in case of the hydrogen ion (i.e., proton), 23 Daltons in case of sodium, 18 Daltons in the case of ammonia or 39 Daltons in case of potassium.
  • These additional weights or masses are simply added to the molecular weight of the molecule of interest and the MS peak occurs at the point for the molecular weight of the molecule of interest plus the weight of the ion that has been added.
  • These ionization methods can also produce negative ions.
  • the most common molecular signal is the deprotonated molecule [M-H] ⁇ ->, in this case the mass is one Dalton lower than the molecular weight of the molecule of interest.
  • multiply charged ions are of the general identification type of [M+nH] ⁇ n+>, where small n identifies the number of additional protons that have been added.
  • the sample (or the eluent when the sample has been fractionated prior to the mass spectrometry) may be introduced into a high resolution mass spectrometer (for example, a LCT PremierTM, Waters Corp., Milford, USA) by electrospray ionization, with capillary and cone voltages set in the positive and negative ion modes to 3200 V and 30 V, and 2800 V and 50 V, respectively.
  • a high resolution mass spectrometer for example, a LCT PremierTM, Waters Corp., Milford, USA
  • An appropriate test mixture of standard compounds may be analyzed before and after the entire set of randomized injection in order to examine the retention time stability, mass accuracy and sensitivity of the system throughout the course of the run.
  • the biological sample is fractionated by liquid chromatography prior to the determination of the level(s) of the metabolic marker(s).
  • chromatography refers to a method for mixture component separation that relies on differences in the flowing behavior of the various components of a mixture/solution carried by a mobile phase through a support/column coated with a certain stationary phase. Specifically, some components bind strongly to the stationary phase and spend longer time in the support, while other components stay predominantly in the mobile phase and pass faster through the support.
  • the criterion based on which the various compounds are separated through the column is defined by the particular problem being investigated and imposed by the structure, composition and binding capacity of the stationary phase.
  • a stationary phase could be constructed such that the linear and low molecular weight molecules elute faster than the aromatic and high-molecular weight ones.
  • the components elute from the support they can be immediately analyzed by a detector or collected for further analysis.
  • a vast number of separation methods, and in particular chromatography methods, are currently available, including Gas Chromatography (GC), Liquid Chromatography (LC), Ion Chromatography (IC), Size-Exclusion Chromatography (SEC), Supercritical-Fluid Chromatography (SFC), Thin-Layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC), Ultra High Performance Liquid Chromatography (UHPLC), and Capillary Electrophoresis (CE).
  • GC Gas Chromatography
  • LC Liquid Chromatography
  • IC Ion Chromatography
  • SEC Size-Exclusion Chromatography
  • SFC Supercritical-Fluid Chromatography
  • TLC Thin-Layer Chromatography
  • HPLC High Performance Liquid Chromato
  • GC can be used to separate volatile compounds or derivatized compounds that, otherwise, are non- volatile compounds.
  • LC is an alternative chromatographic technique useful for separating ions or molecules that are dissolved in a solvent.
  • the principle of GC and LC separation is the same, their main difference lies on the phase in which the separation occurs (gas vs. liquid phase).
  • GC is used primarily to separate molecules up to 650 atomic units heavy, while, in principle, a LC can separate any molecular weight compound.
  • Suitable types of liquid chromatography that can be applied in the method of the invention include, without limitation, reverse phase chromatography, normal phase chromatography, affinity chromatography, ion exchange chromatography, hydrophilic interaction liquid chromatography (HILIC), size exclusion chromatography and chiral chromatography. These techniques are well known in the art and can be applied by the person skilled in the art without further ado.
  • the first method of the invention involves the determination of the level(s) of the metabolite(s) in the sample.
  • the expression "determining the levels of a metabolic marker” or “determining the levels of a metabolite”, as used herein, refers to ascertaining the absolute or relative amount or concentration of the metabolite in the sample. There are many ways to collect quantitative or relational data on metabolites, and the analytical methodology does not affect the utility of metabolite concentrations in predicting phenotype or assessing metabolism.
  • Suitable methods for determining the levels of a given metabolite include, without limitation, refractive index spectroscopy (RI), Ultra-Violet spectroscopy (UV), fluorescent analysis, radiochemical analysis, Infrared spectroscopy (IR), Nuclear Magnetic Resonance spectroscopy (NMR), Light Scattering analysis (LS), Mass Spectrometry, Pyrolysis Mass Spectrometry, Nephelometry, Dispersive Raman Spectroscopy, gas chromatography combined with mass spectroscopy, liquid chromatography combined with mass spectroscopy, supercritical fluid chromatography combined with mass spectroscopy, MALDI combined with mass spectroscopy, ion spray spectroscopy combined with mass spectroscopy, capillary electrophoresis combined with mass spectrometry, NMR combined with mass spectrometry and IR combined with mass spectrometry.
  • RI refractive index spectroscopy
  • UV Ultra-Violet spectroscopy
  • IR Infrared
  • the level(s) of the metabolic marker(s) are determined by mass spectrometry.
  • the biological sample is fractionated by liquid chromatography prior to the determination of the levels of the metabolic markers.
  • the liquid chromatography is performed on a C 18 column at 40°C.
  • the column may be eluted with a 19 minute gradient using a mobile phase at a flow rate of 140 ⁇ 7 ⁇ initially consisting of 100% solvent A (0.05% formic acid), with a linear increase of solvent B (acetonitrile containing 0.05% formic acid) up to 50% over two minutes, and a linear increase to 100% B over the next 1 1 min before returning to the initial composition in readiness for the subsequent injection which preceded a 45 s system recycle time.
  • the liquid chromatography is performed on a CI 8 column at 60°C.
  • the column may be eluted with a 17 min linear gradient of solvents A (water, acetonitrile and 10 mM ammonium formate), and B (acetonitrile, isopropanol and 10 mM ammonium formate).
  • solvents A water, acetonitrile and 10 mM ammonium formate
  • B acetonitrile, isopropanol and 10 mM ammonium formate
  • the first method of the invention comprises comparing the level(s) of the metabolic marker(s) according to Table 1 with a reference value, particularly with a cutoff value.
  • reference value relates to a predetermined criteria used as a reference for evaluating the values or data obtained from the samples collected from a subject.
  • the reference value or reference level can be an absolute value, a relative value, a value that has an upper or a lower limit, a range of values, an average value, a median value, a mean value, or a value as compared to a particular control or baseline value.
  • a reference value can be based on an individual sample value or can be based on a large number of samples, such as from population of subjects of the chronological age matched group, or based on a pool of samples including or excluding the sample to be tested.
  • the levels of the metabolic markers according to Table 1 are compared to their corresponding cut-off value according to Table 1.
  • Cut-off values according to Table 1 are expressed as percentage values of increased or decreased level with respect to those values determined in a control sample, i.e. in a sample from one or more healthy subjects, or in a sample from one or more subjects not suffering from NAFLD at any stage.
  • a cut-off value of 30% for a particular metabolic marker corresponds to a 30% increase of the level of said metabolic marker when compared to its level in a control sample.
  • a cut-off value of -30% for a particular metabolic marker corresponds to a 30% decrease of the level of said metabolic marker when compared to its level in a control sample.
  • the level of a particular metabolic marker as determined in a sample from a subject whose M-, non-M- or I- subtype profile is to be determined can be compared to said reference value or cut-off value, and thus assigned a level of "increased” or "decreased” level.
  • An increase in the level of a metabolic marker above the reference value or above the cut-off value of at least 1.05-fold, 1.1-fold, 1.5-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or even more compared to the reference value or the cut-off value is considered as "increased" level.
  • a decrease in the level of a metabolic marker below the reference value or the cut-off value of at least 0.99-fold, 0.95-fold, 0.9-fold, 0.75-fold, 0.2-fold, 0.1-fold, 0.05-fold, 0.025-fold, 0.02-fold, 0.01-fold, 0.005-fold or even less compared with the reference value or the cut-off value is considered as "decreased" level.
  • the authors of the present invention have identified metabolites showing different levels between non-alcoholic steatohepatitis (NASH) and steatosis subjects.
  • a set of metabolites showing different levels between NASH and steatosis has been assessed for subtypes M, non-M and I as profiled according to the first method of the invention in NAFLD subjects.
  • the present invention also relates to diagnostic methods of NASH or steatosis in a subject suffering from NAFLD, wherein said subject has been profiled as M-subtype, non-M subtype or I-subtype according to the first method of the invention.
  • Metabolites showing differential levels between NASH and steatosis in NAFLD patients profiled as M-subtype are shown in Table 2.
  • Information concerning the platforms used for the analysis of metabolites upregulated (Table 13) and downregulated (Table 14) in the M-subtype NAFLD cluster of patients between groups of NASH vs. steatosis is shown in the Example.
  • the invention relates to a method to diagnose nonalcoholic steatohepatitis (NASH) or steatosis in a subject suffering from NAFLD and profiled as M-subtype according to the first method of the invention (herein referred to as second method of the invention) that comprises determining in a biological sample from said subject the levels of one or more metabolic markers according to Table 2, wherein
  • NAFLD "subject”, “biological sample”, “metabolic marker”, “level”, “increased” and “decreased” have been previosusly described in the context of the first method of the invention.
  • the subject who suffers from NAFLD is a mammal, preferably a human.
  • the sample is a bio fluid from a subject.
  • the sample is a serum sample.
  • the levels of the metabolic markers are determined by mass spectrometry.
  • the sample is fractionated by liquid chromatography prior to the determination of the level(s) of the metabolic marker(s).
  • the levels of metabolite markers according to Table 2 are determined by means of Platforms 1, 2 and/or 3 as described in the Example section.
  • non-alcoholic steatohepatitis or “NASH”, as used herein, relates to a significant form of chronic liver disease characterized by inflammatory and fatty infiltration of the liver that is not associated with alcohol consumption.
  • steatosis also known as fatty change, fatty degeneration or adipose degeneration, relates to the process describing the abnormal retention of lipids within a cell.
  • diagnosis refers both to the process of attempting to determine and/or identify a possible disease in a subject, i.e. the diagnostic procedure, and to the opinion reached by this process, i.e. the diagnostic opinion. As such, it can also be regarded as an attempt at classification of an individual's condition into separate and distinct categories that allow medical decisions about treatment and prognosis to be made. It is to be understood that the method, in a preferred embodiment, is a method carried out in vitro, i.e. not practiced on the human or animal body.
  • non-alcoholic steatohepatitis (NASH) or steatosis is performed in a subject suffering from NAFLD, particularly in a subject suffering from NAFLD and profiled as M-subtype, non-M-subtype or I-subtype by means of the first method of the invention for profiling NAFLD subjects.
  • NAFLD non-alcoholic steatohepatitis
  • This diagnosis does not claim to be correct in 100% of the analyzed samples. However, it requires that a statistically significant amount of the analyzed samples are classified correctly.
  • the amount that is statistically significant can be established by a person skilled in the art by means of using different statistical tools; illustrative, non-limiting examples of said statistical tools include determining confidence intervals, determining the p-value, the Student's t-test or Fisher ' s discriminating functions, etc.
  • Preferred confidence intervals are at least 90%, at least 91%, at least 98%>, at least 99%>.
  • the p- values are, preferably less than 0.1, less than 0.05, less than 0.01 , less than 0.005 or less than 0.0001.
  • the teachings of the present invention preferably allow correctly diagnosing in at least 60%, in at least 70%, in at least 80%, or in at least 90% of the subjects of a determining group or population analyzed.
  • the levels of one or more metabolic markers according to Table 2 are determined in a biological sample from a subject suffering from NAFLD and profiled as M-subtype according to the first method of the invention.
  • one metabolic marker according to Table 2 is determined.
  • the second method of the invention can be carried out by determining the levels of a variable number of metabolites as defined in table 2 in the biological sample of the subject under study. The determination of levels of combinations of the metabolic markers may allow greater sensitivity and specificity in the diagnosis.
  • the subject is diagnosed with NASH.
  • the subject is diagnosed with NASH.
  • the subject is diagnosed with NASH.
  • the subject is diagnosed with steatosis.
  • the subject is diagnosed with steatosis.
  • the subject is diagnosed with steatosis.
  • At least 20% of the metabolic markers according to Table 2 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as M-subtype according to the first method of the invention, wherein
  • At least 40% of the metabolic markers according to Table 2 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as M-subtype according to the first method of the invention, wherein
  • At least 60% of the metabolic markers according to Table 2 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as M-subtype according to the first method of the invention, wherein
  • the subject is diagnosed with steatosis.
  • at least 80% of the metabolic markers according to Table 2 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as M-subtype according to the first method of the invention, wherein
  • all metabolic markers according to Table 2 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as M-subtype according to the first method of the invention, wherein
  • the method further comprises the administration of a therapeutically effective amount of a compound suitable for the treatment of NASH to the subject diagnosed with NASH, and the administration of a therapeutically effective amount of a compound suitable for the treatment of steatosis to the subject diagnosed with steatosis.
  • therapeutically effective amount relates to the sufficient amount of a compound according to the present invention, i.e. a compound for the treatment of NASH or a compound for the treatment of steatosis, to provide the desired effect, i.e.
  • the effective amount produces the amelioration of one or more symptoms of the disease that is being treated.
  • Metabolic markers according to second method of the invention are shown in Table 2. Cut-off values are indicated as percentage values, wherein 0% corresponds to the level of the metabolic marker as determined in a sample from one or more healthy subjects or in a sample from one or more subjects not suffering from NAFLD. Negative percentage cut-off values represent values of metabolites which are lower than the value as determined in a sample from one or more healthy subjects or in a sample from one or more subjects not suffering from NAFLD.
  • X-HOl 3E relates to HODE family isomers wherein the position of the double bond has not been identified; they can be distinguished by their retention time (RT) according to Table 13.
  • x-HETE/EET relates to HETE/EET family isomers wherein the position of the double bond has not been identified; they can be distinguished by their RT according to Table 13.
  • x-DiHETrE relates to DiHETrE family isomers wherein the position of the double bond has not been identified; they can be distinguished by their RT according to Table 13.
  • x- OxoODE relates to OxoODE family isomers wherein the position of the double bond has not been identified; they can be distinguished by their RT according to Table 13. PE(22:5/0:0) can be distinguished by their retention time (RT) and mass-to-charge ratio (MZ) according to Table 14.
  • the invention relates to a method to diagnose NASH or steatosis in a subject suffering from NAFLD and profiled as non-M-subtype according to the first method of the invention (herein referred to as third method of the invention) that comprises determining in a biological sample from said subject the levels of one or more metabolic markers according to Table 3, wherein (i) if the level of at least one metabolic marker according to Table 3(a) is increased with respect to the cut-off value according to Table 3(a) and/or the level of at least one metabolic marker according to Table 3(b) is decreased with respect to the cutoff value according to Table 3(b), then the subject is diagnosed with NASH, and
  • Metabolites showing differential levels between NASH and steatosis in NAFLD patients profiled as non-M-subtype are shown in Table 3.
  • Information concerning the platforms used for the analysis of metabolites upregulated (Table 15) and downregulated (Table 16) in the non-M-subtype NAFLD cluster of patients between groups of NASH vs. steatosis is shown in the Example.
  • NAFLD "subject”, “biological sample”, “metabolic marker”, “level”, “increased” and “decreased” have been previosusly described in the context of the first method of the invention.
  • diagnosis “NASH” and “steatosis” have been previously described as well in the context of the second method of the invention.
  • the subject who suffers from NAFLD is a mammal, preferably a human.
  • the sample is a biofluid from a subject.
  • the sample is a serum sample.
  • the levels of the metabolic markers are determined by mass spectrometry.
  • the sample is fractionated by liquid chromatography prior to the determination of the level(s) of the metabolic marker(s).
  • the levels of metabolite markers according to Table 3 are determined by means of Platforms 1, 2 and/or 3 as described in the Example section.
  • the subject is diagnosed with NASH.
  • the subject is diagnosed with NASH.
  • the subject is diagnosed with NASH.
  • the subject is diagnosed with steatosis.
  • the subject is diagnosed with steatosis.
  • the subject is diagnosed with steatosis.
  • At least 20% of the metabolic markers according to Table 3 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as non-M-subtype according to the first method of the invention, wherein (i) if the levels of at least 20% of the metabolic markers according to Table 3(a) are increased with respect to the cut-off value according to Table 3(a) and/or the levels of at least 20% of the metabolic markers according to Table 3(b) are decreased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with NASH, and
  • At least 40% of the metabolic markers according to Table 3 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as non-M-subtype according to the first method of the invention, wherein
  • At least 60% of the metabolic markers according to Table 3 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as non-M-subtype according to the first method of the invention, wherein
  • At least 80% of the metabolic markers according to Table 3 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as non-M-subtype according to the first method of the invention, wherein
  • all metabolic markers according to Table 3 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as non-M-subtype according to the first method of the invention, wherein
  • the method further comprises the administration of a therapeutically effective amount of a compound suitable for the treatment of NASH to the subject diagnosed with NASH, and the administration of a therapeutically effective amount of a compound suitable for the treatment of steatosis to the subject diagnosed with steatosis.
  • a therapeutically effective amount has been described previously in the context of the second method of the invention.
  • Metabolic markers according to the third method of the invention are shown in
  • Cut-off values are indicated as percentage values, wherein 0% corresponds to the level of the metabolic marker as determined in a sample from one or more healthy subjects or in a sample from one or more subjects not suffering from NAFLD.
  • Negative percentage cut-off values represent values of metabolites which are lower than the value as determined in a sample from one or more healthy subjects or in a sample from one or more subjects not suffering from NAFLD.
  • NAFLD patients profiled as non-M-subtype RT: retention time
  • x-HETE/EET relates to HETE/EET family isomers wherein the position of the double bond has not been identified; they can be distinguished by their RT according to Table 15.
  • TG(54:6) relates to two isomers where the addition of the carbon atoms and double bonds coincides, they can be distinguished by their retention time (RT) and mass-to-charge ratio (MZ) according to Table 16.
  • the invention relates to a method to diagnose NASH or steatosis in a subject suffering from NAFLD and profiled as I-subtype according to the first method of the invention (herein referred to as fourth method of the invention) that comprises determining in a biological sample from said subject the levels of one or more metabolic markers according to Table 4, wherein
  • Metabolites showing differential levels between NASH and steatosis in NAFLD patients profiled as I-subtype are shown in Table 4.
  • Information concerning the platforms used for the analysis of metabolites upregulated (Table 17) and downregulated (Table 18) in the I-subtype NAFLD cluster of patients between groups of NASH vs. steatosis is shown in the Example.
  • NAFLD "subject”, “biological sample”, “metabolic marker”, “level”, “increased” and “decreased” have been previosusly described in the context of the first method of the invention.
  • diagnosis “NASH” and “steatosis” have been previously described as well in the context of the second method of the invention.
  • the subject who suffers from NAFLD is a mammal, preferably a human.
  • the sample is a biofluid from a subject.
  • the sample is a serum sample.
  • the levels of the metabolic markers are determined by mass spectrometry.
  • the sample is fractionated by liquid chromatography prior to the determination of the level(s) of the metabolic marker(s).
  • the levels of metabolite markers according to Table 4 are determined by means of Platforms 1, 2 and/or 3 as described in the Example section.
  • the subject is diagnosed with NASH.
  • the subject is diagnosed with NASH.
  • the subject is diagnosed with NASH.
  • the subject is diagnosed with steatosis.
  • the subject is diagnosed with steatosis.
  • the subject is diagnosed with steatosis.
  • At least 20% of the metabolic markers according to Table 4 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as I-subtype according to the first method of the invention, wherein
  • At least 40% of the metabolic markers according to Table 4 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as I-subtype according to the first method of the invention, wherein
  • At least 60% of the metabolic markers according to Table 4 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as I-subtype according to the first method of the invention, wherein
  • At least 80% of the metabolic markers according to Table 4 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as I-subtype according to the first method of the invention, wherein
  • all metabolic markers according to Table 4 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as I-subtype according to the first method of the invention, wherein
  • the method further comprises the administration of a therapeutically effective amount of a compound suitable for the treatment of NASH to the subject diagnosed with NASH, and the administration of a therapeutically effective amount of a compound suitable for the treatment of steatosis to the subject diagnosed with steatosis.
  • a therapeutically effective amount has been described previously in the context of the second method of the invention.
  • Metabolic markers according to the fourth method of the invention are shown in
  • Cut-off values are indicated as percentage values, wherein 0% corresponds to the level of the metabolic marker as determined in a sample from one or more healthy subjects or in a sample from one or more subjects not suffering from NAFLD.
  • Negative percentage cut-off values represent values of metabolites which are lower than the value as determined in a sample from one or more healthy subjects or in a sample from one or more subjects not suffering from NAFLD.
  • X-HODE relates to HODE family isomers wherein the position of the double bond has not been identified; they can be distinguished by their retention time (RT) according to Table 18.
  • RT retention time
  • Matla (-/-) mice and age matched wild type animals were used. Animals were breed and housed in the CIC bioGUNE animal unit, accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Only Matla (-/-) mice showing elevated serum liver enzymes (ALT and AST) and testing positive for hepatic lipid accumulation, determined by high-frequency ultrasound imaging, were used in this study. At the end of the study, blood samples were collected.
  • Submandibular and retroorbital blood samples were collected at the beginning and at the end of the experiment. Blood samples were deposited in serum separator gel tubes (Microtainer, Becton-Dickinson, Franklin Park, NJ) and centrifuged (6000 rpm, 15 min, 4 °C) for serum separation.
  • serum separator gel tubes Merotainer, Becton-Dickinson, Franklin Park, NJ
  • Samples from subjects previously diagnosed with NAFLD Serum was prepared by incubation of venous blood of the patient in tubes with clot activator for 30 minutes before centrifugation (2500 g, 15 min); supematants were aliquoted into microtubes and stored at -80°C until metabolomic analysis.
  • Serum samples were divided into aliquots and analyzed in three different metabolomic platforms:
  • Platform 1 - Methanol extract Proteins were precipitated from the defrosted serum samples (75 ⁇ ) by adding 300 ⁇ . of methanol in 1.5 mL microtubes on ice. The extraction solvent was spiked with the following compounds not detected in non-spiked human serum extracts: NEFA(19:0) (NEFA, non-esterified fatty acids), tryptophan- d5(indole-d5), dehydrocholic acid and PC(13 :0/0:0). After brief vortex mixing, the samples were incubated overnight at -20°C. Supematants (300 ⁇ ) were collected after centrifugation at 16000 x g for 15 min and solvent removed. The dried extracts were then reconstituted in 120 ⁇ . of methanol, centrifuged (16000xg 5 min), and transferred to vials for UPLC-MS analysis.
  • NEFA non-esterified fatty acids
  • tryptophan- d5(indole-d5) tryptophan-
  • Platform 3 - Amino acids 10 ⁇ aliquots of the extracts prepared for platform 1 were transferred to microtubes and derivatized for amino acid analysis.
  • a UPLC-single quadrupole-MS amino acid analysis system was combined with two separate UPLC-time-of-flight (TOF)-MS based platforms analyzing methanol and chloroform/methanol serum extracts. Each platform involves the use of a different UPLC-MS method.
  • Platform 1 Chromatography was performed on a 1.0 mm i.d. x 100 mm ACQUITY 1.7 ⁇ CI 8 BEH column (Waters Corp., Milford, MA) using an ACQUITY UPLC system (Waters Corp., Milford, MA). The column was maintained at 40 °C and eluted with a 18 min gradient.
  • the mobile phase at a flow rate of 140 ⁇ 7 ⁇ , initially consisted of 100% solvent A (0.05%> formic acid), with a linear increase of solvent B (acetonitrile containing 0.05% formic acid) up to 50% over two minutes, and a linear increase to 100% B over the next 11 min before returning to the initial composition in readiness for the subsequent injection which preceded a 45 s system recycle time.
  • the volume of sample injected onto the column was 2 ⁇ ..
  • the eluent was introduced into the mass spectrometer (LCT-PremierXE, Waters Corp., Milford, MA) by electrospray ionization, with capillary and cone voltages set in the negative ion mode to 2800 and 50 V, respectively.
  • the nebulization gas was set to 600 L/h at a temperature of 350 °C.
  • the cone gas was set to 30 L/h, and the source temperature was set to 120 °C.
  • Centroid data were acquired from m/z 50-1000 using an accumulation time of 0.2 s per spectrum.
  • the eluent was introduced into the mass spectrometer (Xevo G2 QTof, Waters Corp., Milford, MA) by electrospray ionization, with capillary and cone voltages set in the positive ion mode to 3200 and 30 V, respectively.
  • the nebulization gas was set to 1000 L/h at a temperature of 500 °C.
  • the cone gas was set to 30 L/h, and the source temperature was set to 120 °C. Centroid data were acquired from m/z 50-1200 using an accumulation time of 0.2 s per spectrum.
  • the nebulization gas was set to 600 L/h at a temperature of 350 °C.
  • the cone gas was set to 10 L/h, and the source temperature was set to 120 °C.
  • Selected ion recording (SIR) was used to analyze desired metabolites.
  • An appropriate test mixture of standard compounds may be analyzed along the entire set of randomized sample injections in order to examine the retention time stability and sensitivity of the system throughout the course of the run which lasted a maximum of 48 h per batch of samples injected.
  • the selected ions then traversed an argon-pressurized cell, with a collision energy voltage (typically between 5 and 50 V) applied in accordance with the extent of ion fragmentation required.
  • a collision energy voltage typically between 5 and 50 V
  • Subsequent TOF analysis of the fragment ions generated accurate mass generally ⁇ 3 ppm MS/MS or pseudo MS/MS/MS spectra corrected in real time by reference to leucine enkephalin, infused at 10 ⁇ 7 ⁇ through an independent reference electrospray, sampled every 10 s.
  • Nomenclature in volcano plots AA, amino acids; SFA, MUFA, PUFA, saturated, monounsaturated and polyunsaturated fatty acids, respectively; oxFA, oxidized FA; DG, diglycerols; TG, triglycerides; ChoE, cholesteryl esters; BA, bile acids; PE, phosphatidylethanolamines; LPE, lyso-PE; PC, phosphatidylcholines; LPC, lyso-PC; PI, phosphatidylinositols; LPI, lyso-PI; Cer, ceramides; SM, sphingomyelins; CMH, monohexosylceramides; FSB, free sphingoid bases.
  • a hierarchical clustering algorithm based on metabolites ion intensity was used to visualize the differences in metabolite signatures between samples, as well as the ward's minimum variance method as agglomeration method. Biochemically related compounds were generally found to cluster together. The maximum of the average of the individual silhouette widths was calculated for the clusters as described in Rousseeuw PJ (1987 J. Comput. Appl. Math. 20: 53-65).
  • the cluster analysis was calculated with the cluster R package as described in Maechler M, et al (2015, Cluster: Cluster Analysis Basics and Extensions. R package version 2.0.3).
  • Table 14 Platform used, retention time, mass-to charge, adduct and molecular formula for each metabolite downregulated serum metabolites in the M-subtype NAFLD cluster of patients between groups of NASH vs. steatosis.
  • Table 15 Platform used, retention time, mass-to charge, adduct and molecular formula for each metabolite upregulated serum metabolites in the non-M-subtype NAFLD cluster of patients between groups of NASH vs. steatosis.
  • Table 17 Platform used, retention time, mass-to charge, adduct and molecular formula for each metabolite upregulated serum metabolites in the I-subtype NAFLD cluster of patients between groups of NASH vs. steatosis.
  • the cutoff is the best threshold value for discriminate between Steatosis and NASH.
  • the cutoff(%) is the best threshold in percentage between Steatosis increase compared to QC and NASH increase compared to QC.
  • Last column shows the NASH value (mean) minus the Steatosis value (mean),

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Abstract

The present invention is directed to a profiling method for subtypes in non- alcoholic fatty liver disease (NAFLD) patients and to diagnostic methods within those subtypes to discriminate non-alcoholic steatohepatitis (NASH) versus steatosis patients.

Description

IDENTIFICATION OF HUMAN NON-ALCOHOLIC FATTY LIVER DISEASE
(NAFLD) SUBTYPES
FIELD OF THE INVENTION
The present invention relates to the field of diagnostics and, more in particular the non-invasive diagnosis of non-alcoholic steatohepatitis (NASH) and steatosis in different subsets of non-alcoholic fatty liver disease (NAFLD) patients showing different serum metabolomic profiles. This invention relates to methods based on signatures of serum metabolomic biomarkers for differentiating NASH from simple steatosis in different subsets of NAFLD patients.
BACKGROUND OF THE INVENTION
Non-alcoholic fatty liver disease (NAFLD) encompasses a wide range of conditions characterized by the build-up of fat in the liver cells in absence of alcohol abuse. At one end of the scale is the relatively harmless simple fatty liver, or steatosis, that does not cause significant liver damage. If left unattended, this condition may progress to more advanced conditions, some of which may be life threatening. Nonalcoholic steatohepatitis (NASH) is a significant development in NAFLD, corresponding to an aggressive condition characterized by swelling and tenderness in the liver. With intense, on-going inflammation a build up of scar tissue (fibrosis) may form, eventually leading to cirrhosis where irregular bumps, known as nodules, replace the smooth liver tissue and the liver becomes harder. The effect of this, together with continued scarring from fibrosis, means that the liver will run out of healthy cells to support normal functions. This can lead to complete liver failure.
NAFLD is the most common cause of chronic liver disease world wide. It is considered a direct consequence of the rising global epidemic of obesity and the associated increase in the prevalence of diabetes. Most people with a fatty liver are overweight or obese. As more and more people lead inactive lives and carry extra weight around with them, so the number of cases of fatty liver, in particular NASH is rising.
There is currently no specific laboratory test for NASH, making it extremely difficult to diagnose since it is a silent disease and even people who go on to develop fibrosis and cirrhosis may undergo liver damage for many years before symptoms become apparent.
NAFLD may be suspected in subjects with one or more components of the metabolic syndrome, especially obesity and type 2 diabetes, and elevated serum aminotransferase levels [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] in the absence of alcohol abuse or other common causes of liver disease. The only widely accepted test for distinguishing NASH from other forms of disease is a liver biopsy. This process involves passing a fine hollow needle through the skin and into the liver, withdrawing a small tissue of sample that is submitted for histological examination. Apart from the obvious discomfort induced by this invasive procedure, assessment is often subjective and prone to sampling error.
Many methods have been described to date in an effort to classify and/or diagnose patients within the main stages of the NAFLD, including steatosis, NASH and cirrhosis.
Several predictive panels based on the multivariate analysis of well-established clinical and laboratory variables (such as age, body mass index (BMI), ALT, AST, glucose, insulin resistance, albumin) have been proposed as non-invasive markers for the quantitative assessment of fibrosis (FibroTest, NAFLD fibrosis score), steatosis (SteatoTest) and NASH (NashTest) and more recently the ELF test for the assessment of liver fibrosis in patients with NAFLD (Guha IN, et al, 2008, Hepatology 47:455- 460).
WO 2008/021192 describes a non-invasive method for the diagnosis and monitoring of liver diseases such as NASH and steatosis based on the determination of levels of fatty acids and eicosanoids in a body fluid of the patient. However, this method is limited to the identification of lipid species and requires complex fractionation steps of the body fluids before the metabolites can be detected.
WO 2012/143514 relates to a method for the diagnosis of liver damage, among this liver damage is comprised NAFLD, in a subject comprising determining in a biological sample of said subject the levels of a panel of metabolic markers. However, the method has been performed considering the metabolites detected in rat serum extracts and comparing their levels with the degree of apoptosis present in the liver cells. Liver damage therefore can be of any nature. WO2013113992 and WO2015089102 disclose non-invasive tools using plasma biomarkers for differentiating non-alcoholic steatohepatitis (NASH) from non-alcoholic fatty liver (NAFL), and non-alcoholic fatty liver disease (NAFLD) from normal controls. However, none of these documents distinguish between NASH/steatosis within different subtypes of NAFLD patients.
Clearly there is a need for non-invasive methods to diagnose NASH, in order to better understand where is the patient located within the spectrum of phenotypes that can progress to cirrhosis. In addition, NASH is a histological definition that groups together defects in diverse biochemical processes causing hepatic fat accumulation, inflammation, necrosis and fibrosis. The identification of the types of mechanisms leading to NASH and the discovery of non-invasive biomarkers of NASH subtypes are central for the development of effective treatments and precise diagnosis.
BRIEF DESCRIPTION OF THE INVENTION
The authors of the present invention have identified a number of metabolic markers in the serum samples from subjects previously diagnosed with NAFLD. These metabolic markers are significantly differentiated between two different mice genotypes, the Matla (-/-) mice model that spontaneously develops NASH on a normal diet and that of wild type (WT) mice, and used in serum samples of NAFLD patients to classify three clusters, M-subtype, non-M-subtype and I-subtype. These metabolic markers can then be used in a rapid non-invasive subclassifying diagnostic method for differentiating different subsets of NAFLD patients. Further serum metabolic profiles habe been identified that allow distinguishing between NASH and steatosis for each NAFLD patients cluster.
Thus, in a first aspect, the invention relates to a method to profile a subject suffering from non-alcoholic fatty liver disease (NAFLD) as M-subtype, non-M- subtype or I-subtype that comprises determining in a biological sample from said subject the levels of one or more metabolic markers as defined in Table 1, wherein
(i) if the level of at least one metabolic marker according to Table 1(a) is increased with respect to the cut-off value according to Table 1(a) and/or the level of at least one metabolic marker according to Table 1(b) is decreased with respect to the cutoff value according to Table 1(b), then the subject is profiled as M-subtype, (ii) if the level of at least one metabolic marker according to Table 1(c) is increased with respect to the cut-off value according to Table 1(c), then the subject is profiled as non-M-subtype, and
(iii) if the subject is not profiled as M-subtype according to step (i) and is not profiled as non-M-subtype according to step (ii), then the subject is profiled as I- subtype.
In a further aspect, the invention relates to a method to diagnose non-alcoholic steatohepatitis (NASH) or steatosis in a subject suffering from NAFLD and profiled as M-subtype according to the first method above that comprises determining in a biological sample from said subject the levels of one or more metabolic markers according to Table 2, wherein
(i) if the level of at least one metabolic marker according to Table 2(a) is increased with respect to the cut-off value according to Table 2(a) and/or the level of at least one metabolic marker according to Table 2(b) is decreased with respect to the cut- off value according to Table 2(b), then the subject is diagnosed with NASH, and
(ii) if the level of at least one metabolic marker according to Table 2(a) is decreased with respect to the cut-off value according to Table 2(a) and/or the level of at least one metabolic marker according to Table 2(b) is increased with respect to the cutoff value according to Table 2(b), then the subject is diagnosed with steatosis.
In a further aspect, the invention relates to a method to diagnose non-alcoholic steatohepatitis (NASH) or steatosis in a subject suffering from NAFLD and profiled as non-M-subtype according to the first method above that comprises determining in a biological sample from said subject the levels of one or more metabolic markers according to Table 3, wherein
(i) if the level of at least one metabolic marker according to Table 3(a) is increased with respect to the cut-off value according to Table 3(a) and/or the level of at least one metabolic marker according to Table 3(b) is decreased with respect to the cutoff value according to Table 3(b), then the subject is diagnosed with NASH, and
(ii) if the level of at least one metabolic marker according to Table 3(a) is decreased with respect to the cut-off value according to Table 3(a) and/or the level of at least one metabolic marker according to Table 3(b) is increased with respect to the cutoff value according to Table 3(b), then the subject is diagnosed with steatosis. In a further aspect, the invention relates to a method to diagnose non-alcoholic steatohepatitis (NASH) or steatosis in a subject suffering from NAFLD and profiled as I-subtype according to the first method above that comprises determining in a biological sample from said subject the levels of one or more metabolic markers according to Table 4, wherein
(i) if the level of at least one metabolic marker according to Table 4(a) is increased with respect to the cut-off value according to Table 4(a) and/or the level of at least one metabolic marker according to Table 4(b) is decreased with respect to the cutoff value according to Table 4(b), then the subject is diagnosed with NASH, and
(ii) if the level of at least one metabolic marker according to Table 4(a) is decreased with respect to the cut-off value according to Table 4(a) and/or the level of at least one metabolic marker according to Table 4(b) is increased with respect to the cutoff value according to Table 4(b), then the subject is diagnosed with esteatosis. DESCRIPTION OF DRAWINGS
Figure. l.Heatmap representation of the serum metabolomic profile obtained from 377 patients with biopsy-confirmed NAFLD. Each data point corresponds to the relative ion abundance of a given metabolite (vertical axis) in an individual patient's serum extract. Metabolite selection is based on the top 50 serum metabolites that more significantly differentiated between Matla (-/-) mice compared to WT mice. The hierarchical clustering is based on optimum average silhouette width, obtaining the classification of the samples into three clusters: a first cluster (n=l 16) showing a serum metabolomic profile similar to that observed in the Matla (-/-) mice (M-subtype), a second cluster (n= 115) showing an opposite metabolomic profile (non-M-subtype) and a third cluster (n=146) presenting an intermediate metabolomic profile (I-subtype).
Figure 2. Volcano plot representation indicating the -loglO(p-value) and log2(fold-change) of individual serum metabolic ion features of NASH compared to simple steatosis. A. Analysis of M-subtype cohort: NASH (N=57) and simple steatosis (N=59). B. Analysis of non-M-subtype cohort: NASH (N=73) and simple steatosis (N=42). C. Analysis of I-subtype cohort: NASH (N=l 17) and simple steatosis (N=29). Each metabolite is represented by a symbol. AA, amino acids; SFA, MUFA, PUFA, saturated, monounsaturated and polyunsaturated fatty acids; oxFA, oxidized FA; DG, diglycerols; TG, triglycerides; ChoE, cholesteryl esters; BA, bile acids; PE, phosphatidylethanolamines; LPE, lyso-PE; PC, phosphatidylcholines; LPC, lyso-PC; PI, phosphatidylinositols; LPI, lyso-PI; Cer, ceramides; SM, sphingomyelins; CMH, monohexosylceramides; FSB, free sphingoid bases; DAG, diacylglycerols; TAG, triacylglycerols.
DESCRIPTION OF THE INVENTION
Profiling method of the invention
The authors of the present invention have developed a method to determine different subsets of NAFLD subjects based on the levels of metabolic markers as shown in Table 1, and wherein a NAFLD subject is classified in three major clusters (M- subtype, non-M-subtype and I-subtype). This method has been developed by analysis of the differences between genotypes Matla (-/-) mice and wild type (WT) mice, as shown in the Example (see Table 5, corresponding to the metabolites showing significant differences between Matl (-/-) mice and wildtype, and that are able to subclassifying three clusters or subtypes of NAFLD patients; see also Table 12, showing the platforms used for the analysis of said metabolites significantly differentiated). The most significant serum metabolites between said genotypes were selected based on an unpaired Student's t-test (or Welch's t test where unequal variances were found). Then, selected metabolites were analyzed in serum samples from 377 patients with biopsy proving NAFLD. A hierarchical clustering algorithm based on metabolites was used to visualize the differences between samples, as well as the ward's minimum variance method as agglomeration method. The silhouette cluster analysis showed three well separated groups: M-subtype, Non-M-subtype and I-subtype (Figure 1).
Thus, in a first aspect, the invention relates to a method to profile a subject suffering from non-alcoholic fatty liver disease (NAFLD) as M-subtype, non-M- subtype or I-subtype (first method of the invention) that comprises determining in a biological sample from said subject the levels of one or more metabolic markers as defined in Table 1 , wherein
(i) if the level of at least one metabolic marker according to Table 1(a) is increased with respect to the cut-off value according to Table 1(a) and/or the level of at least one metabolic marker according to Table 1(b) is decreased with respect to the cutoff value according to Table 1(b), then the subject is profiled as M-subtype,
(ii) if the level of at least one metabolic marker according to Table 1(c) is increased with respect to the cut-off value according to Table 1(c), then the subject is profiled as non-M-subtype, and
(iii) if the subject is not profiled as M-subtype according to step (i) and is not profiled as non-M-subtype according to step (ii), then the subject is profiled as I- subtype.
In a particular embodiment, the first method of the invention comprises determining in a biological sample from the subject suffering from NAFLD whose profile as M-subtype, non-M-subtype or I-subtype is to be determined, the levels of at least 20% of the markers as defined in Table 1 , wherein
(i) if the levels of at least 20% of metabolic markers according to Table 1(a) are increased with respect to the corresponding cut-off value according to Table 1(a) and/or the levels of at least 20% of metabolic markers according to Table 1(b) are decreased with respect to the corresponding cut-off value according to Table 1(b), then the subject is profiled as M-subtype,
(ii) if the levels of at least 20% of metabolic markers according to Table 1 (c) are increased with respect to the corresponding cut-off value according to Table 1(c), then the subject is profiled as non-M-subtype, and
(iii) if the subject is not profiled as M-subtype according to step (i) and is not profiled as non-M-subtype according to step (ii), then the subject is profiled as I- subtype.
In a particular embodiment, the first method of the invention comprises determining in a biological sample from the subject suffering from NAFLD whose profile as M-subtype, non-M-subtype or I-subtype is to be determined the levels of at least 40% of the markers as defined in Table 1 , wherein
(i) if the levels of at least 40% of metabolic markers according to Table 1(a) are increased with respect to the corresponding cut-off value according to Table 1(a) and/or the levels of at least 40% of metabolic markers according to Table 1(b) are decreased with respect to the corresponding cut-off value according to Table 1(b), then the subject is profiled as M-subtype, (ii) if the levels of at least 40% of metabolic markers according to Table 1(c) are increased with respect to the corresponding cut-off value according to Table 1(c), then the subject is profiled as non-M-subtype, and
(iii) if the subject is not profiled as M-subtype according to step (i) and is not profiled as non-M-subtype according to step (ii), then the subject is profiled as I- subtype.
In a particular embodiment, the first method of the invention comprises determining in a biological sample from the subject suffering from NAFLD whose profile as M-subtype, non-M-subtype or I-subtype is to be determined the levels of at least 60% of the markers as defined in Table 1, wherein
(i) if the levels of at least 60% of metabolic markers according to Table 1(a) are increased with respect to the corresponding cut-off value according to Table 1(a) and/or the levels of at least 60% of metabolic markers according to Table 1(b) are decreased with respect to the corresponding cut-off value according to Table 1(b), then the subject is profiled as M-subtype,
(ii) if the levels of at least 60% of metabolic markers according to Table 1 (c) are increased with respect to the corresponding cut-off value according to Table 1(c), then the subject is profiled as non-M-subtype, and
(iii) if the subject is not profiled as M-subtype according to step (i) and is not profiled as non-M-subtype according to step (ii), then the subject is profiled as I- subtype.
In a particular embodiment, the first method of the invention comprises determining in a biological sample from the subject suffering from NAFLD whose profile as M-subtype, non-M-subtype or I-subtype is to be determined the levels of at least 80% of the markers as defined in Table 1, wherein
(i) if the levels of at least 80% of metabolic markers according to Table 1(a) are increased with respect to the corresponding cut-off value according to Table 1(a) and/or the levels of at least 80% of metabolic markers according to Table 1(b) are decreased with respect to the corresponding cut-off value according to Table 1(b), then the subject is profiled as M-subtype, (ii) if the levels of at least 80% of metabolic markers according to Table 1(c) are increased with respect to the corresponding cut-off value according to Table 1(c), then the subject is profiled as non-M-subtype, and
(iii) if the subject is not profiled as M-subtype according to step (i) and is not profiled as non-M-subtype according to step (ii), then the subject is profiled as I- subtype.
In a more particular embodiment, the first method of the invention comprises determining in a biological sample from the subject suffering from NAFLD whose profile as M-subtype, non-M-subtype or I-subtype is to be determined the levels of all the markers as defined in Table 1, wherein
(i) if the levels of all metabolic markers according to Table 1(a) are increased with respect to the corresponding cut-off value according to Table 1(a) and/or the levels of all metabolic markers according to Table 1(b) are decreased with respect to the corresponding cut-off value according to Table 1(b), then the subject is profiled as M-subtype,
(ii) if the levels of all metabolic markers according to Table 1(c) are increased with respect to the corresponding cut-off value according to Table 1(c), then the subject is profiled as non-M-subtype,
(iii) if the subject is not profiled as M-subtype according to step (i) and is not profiled as non-M-subtype according to step (ii), then the subject is profiled as I- subtype.
The term "profile", or "classify", as used herein, relates to characterize a subject suffering from NAFLD as belonging to an M-subtype, non-M subtype or I-subtype based on particular levels of metabolites according to the present invention and shown in Table 1. The term "M-subtype", as used herein, relates to the subtype of NAFLD subject showing a metabolomic profile similar to that observed by the inventors in Matla (-/-) mice. In a particular embodiment, the subtype M is characterized by having increased levels of at least one, at least 20%>, at least 40%>, at least 60%>, at least 80%> or all the metabolic markers according to Table 1(a) with respect to the cut-off value according to Table 1(a) and/or decreased levels of at least one, at least 20%>, at least 40%, at least 60%, at least 80% or all the metabolic markers according to Table 1(b) with respect to the cut-off value according to Table 1(b). The term "non-M subtype", as used herein, relates to the subtype of NAFLD subject showing a metabolomic profile which is the opposite to M-subtype metabolomic profile observed by the inventors in Matla (-/-) mice. In a particular embodiment, the subtype non-M is characterized by having increased levels of at least one, at least 20%, at least 40%, at least 60%, at least 80% or all the metabolic markers according to Table 1(c) with respect to the cut-off value according to Table 1(c). The term "I-subtype", or intermediate subtype, relates to the subtype of NAFLD subject showing a metabolomic profile that is intermediate to the metabolic profile of those NAFLD subjects showing a M-subtype and to the metabolic profile of those NAFLD subjects showing a non-M-subtype. In a particular embodiment, the I-subtype is characterized by not having increased levels of at least one, at least 20%, at least 40%, at least 60%, at least 80% or all the metabolic markers according to Table 1(a) with respect to the cut-off value according to Table 1(a) and/or not having decreased levels of at least one, at least 20%>, at least 40%>, at least 60%>, at least 80% or all the metabolic markers according to Table 1(b) with respect to the cut- off value according to Table 1(b) and not having increased levels of at least one, at least 20%, at least 40%, at least 60%, at least 80% or all the metabolic markers according to Table 1(c) with respect to the cut-off value according to Table 1(c). The term "similar levels", as used herein, refers to levels that differ in less than 1%, less than 0,75%, less than 0,5%, less than 0,25%, less than 0,01% or less than 0,001% to the levels of the metabolic markers in the Matla (-/-) mice.
The term "non-alcoholic fatty liver disease" or " NAFLD", as used herein, refers to a group of conditions having in common the accumulation of fat in the hepatocytes. NAFLD ranges from simple fatty liver (steatosis), to non-alcoholic steatohepatitis (NASH), to cirrhosis (irreversible, advanced scarring of the liver). The term "NAFLD" includes any stage or degree of progression of the disease.
The terms "subject", "patient" or "individual"' are used herein interchangeably to refer to all the animals classified as mammals and includes but is not limited to domestic and farm animals, primates and humans, for example, human beings, non- human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents. Preferably, the subject is a male or female human being of any age or race. In a particular embodiment of the first method of the invention, the subject who suffers from NAFLD is a mammal, preferably a human. The term "sample" or "biological sample", as used herein, refers to biological material isolated from a subject. The biological sample may contain any biological material suitable for detecting the desired biomarker and may comprise cellular and/or non cellular material from the subject. The sample can be isolated from any suitable biological tissue or fluid such as, for example, liver tissue, blood, blood plasma, serum, urine or cerebral spinal fluid (CSF). Preferably, the samples used for the determination of the level(s) of the metabolic markers are samples which can be obtained using minimally invasive procedures. In a particular embodiment, the sample is a bio fluid from a subject. In a more particular embodiment, the sample is a biofluid selected from the group consisting of blood, plasma, serum, isolated lipoprotein fraction, saliva, urine, lymph fluid, cerebrospinal fluid and bile. In a more particular embodiment, the sample is a biofluid selected from blood, plasma and serum. In a preferred embodiment, the sample is a serum sample.
The term "metabolic marker" or "metabolite", as used herein, refers to small molecule compounds, such as substrates for enzymes of metabolic pathways, intermediates of such pathways or the products obtained by a metabolic pathway, the occurrence or amount of which is characteristic for a specific situation, for example NAFLD. The abbreviated names of the lipid metabolites correspond to the lipid family to which it belongs followed by a lipid number of the fatty acid side chains. The lipid family is further described by the reference number of said lipid family in the LIPID MAPS structure database (http://www.lipidmaps.org/data/databases.html) using the LIPID MAPS Classification System (Fahy E. et al, Journal of Lipid Research 2009, 50: S9-S14). The lipid number, as the skilled person knows, is a number with the format N:n, where "N" corresponds to the number of carbons in the fatty acid chains and "n" corresponds to the number of double bonds in the fatty acid chains. In the case of glycerophospholipids, since they contain two fatty acids per lipid, when only one number is given it means that both fatty acids are the same. The lipid metabolic markers of tables la, lb and lc, tables 2a and 2b, tables 3a and 3b and tables 4a and 4b are intended to refer to any isomer thereof, including structural and geometric isomers. The term "structural isomer", as used herein, refers to any of two or more chemical compounds, having the same molecular formula but different structural formulas. The term "geometric isomer" or "stereoisomer" as used herein refers to two or more compounds which contain the same number and types of atoms, and bonds (i.e., the connectivity between atoms is the same), but which have different spatial arrangements of the atoms, for example cis and trans isomers of a double bond, enantiomers, and diastereomers. The abbreviated name of the amino acid corresponds to the amino acid name to which it belongs followed by an accession number as described in the Human Metabolome Database HMDB (version 3.6) (http://www.hmdb.ca).
Metabolic markers according to the first method of the invention are shown in Table 1. Cut-off values are indicated as percentage values, wherein 0% corresponds to the level of the metabolic marker as determined in a sample from one or more healthy subjects or in a sample from one or more subjects not suffering from NAFLD. Negative percentage cut-off values represent values of metabolites which are lower than the value as determined in a sample from one or more healthy subjects or in a sample from one or more subjects not suffering from NAFLD. Table 1.Metabolic markers for profiling M, non-M and I subtypes of NAFLD subjects according to the first method of the invention.
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
According to the first method of the invention, the levels of one or more metabolic markers according to Table 1 are determined in a biological sample from a subject whose M-, non-M- or I-subtype profile is to be determined. In a particular embodiment, one metabolic marker according to Table 1 is determined. In a more particular embodiment, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty- four or at least twenty- five, at least thirty, at least thirty-five, at least forty, at least forty-five, at least fifty, at least fifty-five, or at least sixty metabolic markers according to Table 1 are determined. In a particular embodiment, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the metabolic markers according to Table 1 are determined. In a more particular embodiment, all metabolic markers according to Table 1 are determined. Thus, it will be understood that the first method of the invention can be carried out by determining the levels of a variable number of metabolites as defined in table 1 in the biological sample of the subject under study. The determination of levels of combinations of the metabolic markers may allow greater sensitivity and specificity in profiling different subtypes of NAFLD patients.
According to step (i) of the first method of the invention, if the levels of at least one metabolic marker according to Table 1(a) are increased with respect to the cut-off value according to Table 1(a) and/or the levels of at least one metabolic marker according to Table 1(b) are decreased with respect to the cut-off value according to Table 1(b), then the subject is profiled as M-subtype. Particularly, if the levels of at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, of the metabolic markers according to Table 1(a) are increased with respect to the cut-off value according to Table 1(a) and/or the levels of at least 20%>, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, of the metabolic markers according to Table 1(b) are decreased with respect to the cut-off value according to Table 1(b), then the subject is profiled as M-subtype. In a particular preferred embodiment, if the levels of all metabolic markers according to Table 1(a) are increased with respect to the cut-off value according to Table 1(a) and/or the levels of all metabolic markers according to Table 1(b) are decreased with respect to the cut-off value according to Table 1(b), then the subject is profiled as M-subtype.
According to step (ii) of the first method of the invention, if the level of at least one metabolic marker according to Table 1(c) is increased with respect to the cut-off value according to Table 1(c), then the subject is profiled as non-M-subtype. Particularly, if the levels of at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, of the metabolic markers according to Table 1(c) are increased with respect to the cut-off value according to Table 1(c), then the subject is profiled as non-M-subtype. In a particular preferred embodiment, if the levels of all metabolic markers according to Table 1(c) are increased with respect to the cut-off value according to Table 1(c), then the subject is profiled as non-M-subtype. The skilled person acknowledges that steps (i) and (ii) according to the first method of the invention may be performed in any order, i.e, step (ii) following step (i) or alternatively step (i) following step (ii) or at the same time. In a particular embodiment, step (i) is performed firstly and step (ii) is performed secondly. According to the invention, if the subject suffering from NAFLD to be profiled according to the first method of the invention is not profiled as M-subtype according to step (i) (i.e. when the level of at least one metabolic marker as defined in Table 1 is determined in a sample from the subject to be profiled and the level of at least one metabolic marker accoding to Table 1(a) is not increased with respect to the cut-off value according to Table 1(a) and the level of at least one metabolic marker according to Table 1(b) is not decreased with respect to the cut-off value according to Table 1(b)) and said subject is not profiled as non-M-subtype either (i.e. when the level of at least one metabolic marker as defined in Table 1 is determined in a sample from the subject to be profiled and the level of at least one metabolic marker according to Table 1(c) is not increased with the respect to the cut-off value according to Table 1(c)), then said subject is profiled as I-subtype.
The term "level", as used herein, refers to the quantity of a biomarker detectable in a sample. It will be understood that the biological sample can be analyzed as such or, alternatively, the metabolites may be first extracted from the sample prior to analysis and the metabolite extract is then analyzed. If the metabolites are extracted prior to analysis, different extraction methods are available to the skilled person. The selection of one or other extraction method will depend on the class of metabolites/small molecules that are targeted from a particular analysis. Suitable extraction methods include "Extraction of free metabolite pools", "Vapor Phase Extraction", and "Total Metabolite Extraction". The first type of extraction, "Extraction of free metabolite pools", is mainly used in metabolomics research. In this case free intracellular metabolite pools are obtained from a biological sample through methanol- water extraction for polar metabolites, or chloroform, methanol, chloroform/methanol extraction for non-polar metabolites. The second type of extraction, "Vapor Phase Extraction", refers to the extraction of metabolites that are volatile at room temperature. The metabolites are expelled from the biological sample in the vapor phase. These metabolites are either measured directly by connecting the flask or reactor in which the vapors are generated to the analytical instrument or by absorbing first the vapors in charcoal/solvent and then analyzing the acquired solution. The third type of extraction, "Total Metabolite Extraction", refers to the extraction of the free metabolite pools along with the metabolites that have been incorporated in cellular macromolecules, e.g. lipids, proteins, etc. The present invention provides extraction of a particular class of metabolites from macromolecules (e.g. amino acids from proteins or sugars from cell wall components). The present invention also provides a combined high-throughput method which extracts all metabolites simultaneously.
Alternatively, the metabolite quantification can be carried out directly in the biological sample. In this case, the sample may be prepared to enhance detectability of the markers. For example, to increase the detectability of markers, a blood serum sample from the subject can be preferably fractionated by, e.g., Cibacron blue agarose chromatography and single stranded DNA affinity chromatography, anion exchange chromatography, affinity chromatography (e.g., with antibodies) and the like. The method of fractionation depends on the type of detection method used. Any method that enriches for the metabolite of interest can be used. Typically, preparation involves fractionation of the sample and collection of fractions determined to contain the biomarkers. Methods of pre-fractionation include, for example, size exclusion chromatography, ion exchange chromatography, heparin chromatography, affinity chromatography, sequential extraction, gel electrophoresis and liquid chromatography. The analytes also may be modified prior to detection. These methods are useful to simplify the sample for further analysis. For example, it can be useful to remove high abundance proteins, such as albumin, from blood before analysis. In yet another embodiment, a sample can be pre-fractionated by removing proteins that are present in a high quantity or that may interfere with the detection of markers in a sample. Proteins in general may be removed by using conventional techniques such as precipitation using organic solvents such as methanol precipitation, ethanol, acetonitrile, acetone or combinations thereof, in particular, combination of methanol, acetone and acetonitrile, acid precipitation using, for example, trichloroacetic acid or perchloric acid, heat denaturation and any combination of organic solvent, acid and heat precipitation. In the case of a blood, plasma or serum sample, serum albumin or other proteins abundant in serum such as apolipoproteins, glycoproteins, immunoglobulins may obscure the analysis of markers since they are present in a high quantity. Thus, it may be sufficient to remove one or more of the above proteins in order to detect the metabolites or minor proteins. For this purpose, the blood serum or plasma sample can be pre-fractionated by removing serum albumin. Serum albumin can be removed using a substrate that comprises adsorbents that specifically bind serum albumin. For example, a column which comprises, e.g., Cibacron blue agarose (which has a high affinity for serum albumin) or anti-serum albumin antibodies can be used. In yet another embodiment, a sample can be pre-fractionated by isolating proteins that have a specific characteristic, e.g. are glycosylated. For example, a blood serum or plasma sample can be fractionated by passing the sample over a lectin chromatography column (which has a high affinity for sugars). Many types of affinity adsorbents exist which are suitable for pre- fractionating blood serum or plasma samples. An example of one other type of affinity chromatography available to prefractionate a sample is a single stranded DNA spin column. These columns bind proteins which are basic or positively charged. Bound proteins are then eluted from the column using eluants containing denaturants or high pH. Thus there are many ways to reduce the complexity of a sample based on the binding properties of the proteins in the sample, or the characteristics of the proteins in the sample.
In yet another embodiment, a sample can be fractionated using a sequential extraction protocol. In sequential extraction, a sample is exposed to a series of adsorbents to extract different types of biomolecules from a sample.
In a particular embodiment, the determination of the level of the one or more metabolic markers is carried out by mass spectrometry. As used herein, "mass spectrometry" (MS analysis) refers to an analytical technique to identify unknown compounds including: (1) ionizing the compounds and potentially fractionating the compounds parent ion formed into daughter ions; and (2) detecting the charged compounds and calculating a mass-to-charge ratio (m/z). The compounds may be ionized and detected by any suitable means. A "mass spectrometer" includes means for ionizing compounds and for detecting charged compounds.
Preferably, mass spectrometry is used, in particular gas chromatography coupled to mass spectrometry (GC-MS), liquid chromatography coupled to mass spectrometry (LC-MS), direct infusion mass spectrometry or Fourier transform ion-cyclotrone resonance mass spectrometry (FT-ICR-MS), capillary electrophoresis coupled to mass spectrometry (CE-MS), high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS), ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS), supercritical fluid chromatography coupled to mass spectrometry (SFC-MS), flow injection analysis with mass spectrometry (FIA-MS), including quadrupole mass spectrometry, any sequentially coupled mass spectrometry, such as MS-MS or MS-MS-MS, inductively coupled plasma mass spectrometry (ICPMS), pyrolysis mass spectrometry (Py-MS), ion mobility mass spectrometry or time-of-flight mass spectrometry (TOF), electrospray ionization mass spectrometry (ESIMS), ESI- MS/MS, ESI- (MS)<n>, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOFMS), desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS)<n>, atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS/MS, and APPI-(MS)<n>, quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), and iontrap mass spectrometry, where n is an integer greater than zero. Most preferably, LCMS is used as described in detail below. Said techniques are disclosed in, e.g., Nissen, Journal of Chromatography A, 703, 1995: 37- 57, US 4,540,884 or US 5,397,894. The above mentioned ionization methods generally produce an ion resulting from the addition of one or more atoms or by cleavage of the molecule. These ions can then be used as surrogate markers for the metabolites used in the method of the invention. The term "surrogate marker", as used herein, means a biological or clinical parameter that is measured in place of the biologically definitive or clinically most meaningful parameter.
Typically, the ions resulting from the addition of a proton or a hydrogen nucleus, [M+H]<+> where M signifies the molecule of interest, and H signifies the hydrogenion, which is the same as a proton. Some ionization methods will also produce analogous ions. Analogous ions may arise by the addition of an alkaline metal cation, rather than the proton discussed above. A typical species might be [M+Na]<+>, [M+NH4]<+> or [M+K]<+>. The analysis of the ionized molecules is similar irrespective of whether one is concerned with a protonated ion as discussed above or dealing with an added alkaline metal cation. The major difference is that the addition of a proton adds one mass unit (typically called one Dalton), in case of the hydrogen ion (i.e., proton), 23 Daltons in case of sodium, 18 Daltons in the case of ammonia or 39 Daltons in case of potassium. These additional weights or masses are simply added to the molecular weight of the molecule of interest and the MS peak occurs at the point for the molecular weight of the molecule of interest plus the weight of the ion that has been added. These ionization methods can also produce negative ions. The most common molecular signal is the deprotonated molecule [M-H]<->, in this case the mass is one Dalton lower than the molecular weight of the molecule of interest. In addition, for some compounds it will be produced multiply charged ions. These are of the general identification type of [M+nH]<n+>, where small n identifies the number of additional protons that have been added.
Preferably, the sample (or the eluent when the sample has been fractionated prior to the mass spectrometry) may be introduced into a high resolution mass spectrometer (for example, a LCT Premier™, Waters Corp., Milford, USA) by electrospray ionization, with capillary and cone voltages set in the positive and negative ion modes to 3200 V and 30 V, and 2800 V and 50 V, respectively. An appropriate test mixture of standard compounds may be analyzed before and after the entire set of randomized injection in order to examine the retention time stability, mass accuracy and sensitivity of the system throughout the course of the run.
In another particular embodiment, the biological sample is fractionated by liquid chromatography prior to the determination of the level(s) of the metabolic marker(s). The term "chromatography", as used herein, refers to a method for mixture component separation that relies on differences in the flowing behavior of the various components of a mixture/solution carried by a mobile phase through a support/column coated with a certain stationary phase. Specifically, some components bind strongly to the stationary phase and spend longer time in the support, while other components stay predominantly in the mobile phase and pass faster through the support. The criterion based on which the various compounds are separated through the column is defined by the particular problem being investigated and imposed by the structure, composition and binding capacity of the stationary phase. For example, a stationary phase could be constructed such that the linear and low molecular weight molecules elute faster than the aromatic and high-molecular weight ones. As the components elute from the support, they can be immediately analyzed by a detector or collected for further analysis. A vast number of separation methods, and in particular chromatography methods, are currently available, including Gas Chromatography (GC), Liquid Chromatography (LC), Ion Chromatography (IC), Size-Exclusion Chromatography (SEC), Supercritical-Fluid Chromatography (SFC), Thin-Layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC), Ultra High Performance Liquid Chromatography (UHPLC), and Capillary Electrophoresis (CE). GC can be used to separate volatile compounds or derivatized compounds that, otherwise, are non- volatile compounds. LC is an alternative chromatographic technique useful for separating ions or molecules that are dissolved in a solvent. The principle of GC and LC separation is the same, their main difference lies on the phase in which the separation occurs (gas vs. liquid phase). In addition, GC is used primarily to separate molecules up to 650 atomic units heavy, while, in principle, a LC can separate any molecular weight compound. Suitable types of liquid chromatography that can be applied in the method of the invention include, without limitation, reverse phase chromatography, normal phase chromatography, affinity chromatography, ion exchange chromatography, hydrophilic interaction liquid chromatography (HILIC), size exclusion chromatography and chiral chromatography. These techniques are well known in the art and can be applied by the person skilled in the art without further ado.
Once the sample has been processed, the first method of the invention involves the determination of the level(s) of the metabolite(s) in the sample. The expression "determining the levels of a metabolic marker" or "determining the levels of a metabolite", as used herein, refers to ascertaining the absolute or relative amount or concentration of the metabolite in the sample. There are many ways to collect quantitative or relational data on metabolites, and the analytical methodology does not affect the utility of metabolite concentrations in predicting phenotype or assessing metabolism. Suitable methods for determining the levels of a given metabolite include, without limitation, refractive index spectroscopy (RI), Ultra-Violet spectroscopy (UV), fluorescent analysis, radiochemical analysis, Infrared spectroscopy (IR), Nuclear Magnetic Resonance spectroscopy (NMR), Light Scattering analysis (LS), Mass Spectrometry, Pyrolysis Mass Spectrometry, Nephelometry, Dispersive Raman Spectroscopy, gas chromatography combined with mass spectroscopy, liquid chromatography combined with mass spectroscopy, supercritical fluid chromatography combined with mass spectroscopy, MALDI combined with mass spectroscopy, ion spray spectroscopy combined with mass spectroscopy, capillary electrophoresis combined with mass spectrometry, NMR combined with mass spectrometry and IR combined with mass spectrometry.
In a particular embodiment, the level(s) of the metabolic marker(s) are determined by mass spectrometry. In a still more particular embodiment, the biological sample is fractionated by liquid chromatography prior to the determination of the levels of the metabolic markers. In one still more particular embodiment, the liquid chromatography is performed on a C 18 column at 40°C. The column may be eluted with a 19 minute gradient using a mobile phase at a flow rate of 140 μΙ7ιηίη initially consisting of 100% solvent A (0.05% formic acid), with a linear increase of solvent B (acetonitrile containing 0.05% formic acid) up to 50% over two minutes, and a linear increase to 100% B over the next 1 1 min before returning to the initial composition in readiness for the subsequent injection which preceded a 45 s system recycle time. In another still more particular embodiment, the liquid chromatography is performed on a CI 8 column at 60°C. The column may be eluted with a 17 min linear gradient of solvents A (water, acetonitrile and 10 mM ammonium formate), and B (acetonitrile, isopropanol and 10 mM ammonium formate). The mobile phase, at a flow rate of 400 μΤ/ηώι, initially consisted of 40%> solvent B, increasing up to 100% at 10 minutes. After 5 minutes the mobile phase was reset to the initial composition in readiness for the subsequent injection which preceded a 45 s system recycle time.
The first method of the invention comprises comparing the level(s) of the metabolic marker(s) according to Table 1 with a reference value, particularly with a cutoff value.
The term "reference value", as used herein, relates to a predetermined criteria used as a reference for evaluating the values or data obtained from the samples collected from a subject. The reference value or reference level can be an absolute value, a relative value, a value that has an upper or a lower limit, a range of values, an average value, a median value, a mean value, or a value as compared to a particular control or baseline value. A reference value can be based on an individual sample value or can be based on a large number of samples, such as from population of subjects of the chronological age matched group, or based on a pool of samples including or excluding the sample to be tested.
According to the first method of the invention, the levels of the metabolic markers according to Table 1 are compared to their corresponding cut-off value according to Table 1. Cut-off values according to Table 1 are expressed as percentage values of increased or decreased level with respect to those values determined in a control sample, i.e. in a sample from one or more healthy subjects, or in a sample from one or more subjects not suffering from NAFLD at any stage. For example, a cut-off value of 30% for a particular metabolic marker corresponds to a 30% increase of the level of said metabolic marker when compared to its level in a control sample. Similarly, a cut-off value of -30% for a particular metabolic marker corresponds to a 30% decrease of the level of said metabolic marker when compared to its level in a control sample.
Once a reference value or a cut-off value is established, the level of a particular metabolic marker as determined in a sample from a subject whose M-, non-M- or I- subtype profile is to be determined can be compared to said reference value or cut-off value, and thus assigned a level of "increased" or "decreased" level.
An increase in the level of a metabolic marker above the reference value or above the cut-off value of at least 1.05-fold, 1.1-fold, 1.5-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or even more compared to the reference value or the cut-off value is considered as "increased" level. On the other hand, a decrease in the level of a metabolic marker below the reference value or the cut-off value of at least 0.99-fold, 0.95-fold, 0.9-fold, 0.75-fold, 0.2-fold, 0.1-fold, 0.05-fold, 0.025-fold, 0.02-fold, 0.01-fold, 0.005-fold or even less compared with the reference value or the cut-off value is considered as "decreased" level.
Diagnostic methods of the invention
The authors of the present invention have identified metabolites showing different levels between non-alcoholic steatohepatitis (NASH) and steatosis subjects. A set of metabolites showing different levels between NASH and steatosis has been assessed for subtypes M, non-M and I as profiled according to the first method of the invention in NAFLD subjects. Accordingly, the present invention also relates to diagnostic methods of NASH or steatosis in a subject suffering from NAFLD, wherein said subject has been profiled as M-subtype, non-M subtype or I-subtype according to the first method of the invention. Metabolites showing differential levels between NASH and steatosis in NAFLD patients profiled as M-subtype are shown in Table 2. Information concerning the platforms used for the analysis of metabolites upregulated (Table 13) and downregulated (Table 14) in the M-subtype NAFLD cluster of patients between groups of NASH vs. steatosis is shown in the Example.
Thus, in a further aspect, the invention relates to a method to diagnose nonalcoholic steatohepatitis (NASH) or steatosis in a subject suffering from NAFLD and profiled as M-subtype according to the first method of the invention (herein referred to as second method of the invention) that comprises determining in a biological sample from said subject the levels of one or more metabolic markers according to Table 2, wherein
(i) if the level of at least one metabolic marker according to Table 2(a) is increased with respect to the cut-off value according to Table 2(a) and/or the level of at least one metabolic marker according to Table 2(b) is decreased with respect to the cutoff value according to Table 2(b), then the subject is diagnosed with NASH, and
(ii) if the level of at least one metabolic marker according to Table 2(a) is decreased with respect to the cut-off value according to Table 2(a) and/or the level of at least one metabolic marker according to Table 2(b) is increased with respect to the cutoff value according to Table 2(b), then the subject is diagnosed with steatosis.
The terms "NAFLD", "subject", "biological sample", "metabolic marker", "level", "increased" and "decreased" have been previosusly described in the context of the first method of the invention.
In a particular embodiment of the second method of the invention, the subject who suffers from NAFLD is a mammal, preferably a human.
In a particular embodiment of the second method of the invention, the sample is a bio fluid from a subject. In a preferred embodiment, the sample is a serum sample.
Suitable methods to determine the levels of the metabolite markers of the invention have been previously described in the context of the first method of the invention. In a particular embodiment of the second method of the invention, the levels of the metabolic markers are determined by mass spectrometry. In a particular embodiment of the second method of the invention, the sample is fractionated by liquid chromatography prior to the determination of the level(s) of the metabolic marker(s). In a particular embodiment of the second method of the invention, the levels of metabolite markers according to Table 2 are determined by means of Platforms 1, 2 and/or 3 as described in the Example section.
The term "non-alcoholic steatohepatitis" or "NASH", as used herein, relates to a significant form of chronic liver disease characterized by inflammatory and fatty infiltration of the liver that is not associated with alcohol consumption.
The term "steatosis", as used herein, also known as fatty change, fatty degeneration or adipose degeneration, relates to the process describing the abnormal retention of lipids within a cell.
The term "diagnosis", as used herein, refers both to the process of attempting to determine and/or identify a possible disease in a subject, i.e. the diagnostic procedure, and to the opinion reached by this process, i.e. the diagnostic opinion. As such, it can also be regarded as an attempt at classification of an individual's condition into separate and distinct categories that allow medical decisions about treatment and prognosis to be made. It is to be understood that the method, in a preferred embodiment, is a method carried out in vitro, i.e. not practiced on the human or animal body. In particular, the diagnosis of non-alcoholic steatohepatitis (NASH) or steatosis is performed in a subject suffering from NAFLD, particularly in a subject suffering from NAFLD and profiled as M-subtype, non-M-subtype or I-subtype by means of the first method of the invention for profiling NAFLD subjects. This diagnosis, as it is understood by a person skilled in the art does not claim to be correct in 100% of the analyzed samples. However, it requires that a statistically significant amount of the analyzed samples are classified correctly. The amount that is statistically significant can be established by a person skilled in the art by means of using different statistical tools; illustrative, non-limiting examples of said statistical tools include determining confidence intervals, determining the p-value, the Student's t-test or Fisher's discriminating functions, etc. Preferred confidence intervals are at least 90%, at least 91%, at least 98%>, at least 99%>. The p- values are, preferably less than 0.1, less than 0.05, less than 0.01 , less than 0.005 or less than 0.0001. The teachings of the present invention preferably allow correctly diagnosing in at least 60%, in at least 70%, in at least 80%, or in at least 90% of the subjects of a determining group or population analyzed.
According to the second method of the invention, the levels of one or more metabolic markers according to Table 2 are determined in a biological sample from a subject suffering from NAFLD and profiled as M-subtype according to the first method of the invention. In a particular embodiment, one metabolic marker according to Table 2 is determined. In a more particular embodiment, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, at least twenty, at least twenty- one, at least twenty-two, at least twenty-three, at least twenty-four or at least twenty- five metabolic markers according to Table 2 are determined. In a particular embodiment, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the metabolic markers according to Table 2 are determined. In a more particular embodiment, all metabolic markers according to Table 2 are determined. Thus, it will be understood that the second method of the invention can be carried out by determining the levels of a variable number of metabolites as defined in table 2 in the biological sample of the subject under study. The determination of levels of combinations of the metabolic markers may allow greater sensitivity and specificity in the diagnosis.
According to the second method of the invention, if the level of at least one metabolic marker according to Table 2(a) is increased with respect to the cut-off value according to Table 2(a) and/or the level of at least one metabolic marker according to Table 2(b) is decreased with respect to the cut-off value according to Table 2(b), then the subject is diagnosed with NASH. Particularly, if the levels of at least 20%>, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, of the metabolic markers according to Table 2(a) are increased with respect to the cut-off value according to Table 2(a) and/or the levels of at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, of the metabolic markers according to Table 2(b) are decreased with respect to the cut-off value according to Table 2(b), then the subject is diagnosed with NASH. In a particular preferred embodiment, if the levels of all metabolic markers according to Table 2(a) are increased with respect to the cut-off value according to Table 2(a) and/or the levels of all metabolic markers according to Table 2(b) are decreased with respect to the cut-off value according to Table 2(b), then the subject is diagnosed with NASH.
According to the second method of the invention, if the level of at least one metabolic marker according to Table 2(a) is decreased with respect to the cut-off value according to Table 2(a) and/or the level of at least one metabolic marker according to Table 2(b) is increased with respect to the cut-off value according to Table 2(b), then the subject is diagnosed with steatosis. Particularly, if the levels of at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, of the metabolic markers according to Table 2(a) are decreased with respect to the cut-off value according to Table 2(a) and/or the levels of at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, of the metabolic markers according to Table 2(b) are increased with respect to the cut-off value according to Table 2(b), then the subject is diagnosed with steatosis. In a particular preferred embodiment, if the levels of all metabolic markers according to Table 2(a) are decreased with respect to the cut-off value according to Table 2(a) and/orthe levels of all metabolic markers according to Table 2(b) are increased with respect to the cut-off value according to Table 2(b), then the subject is diagnosed with steatosis.
In a particular embodiment of the second method of the invention, at least 20% of the metabolic markers according to Table 2 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as M-subtype according to the first method of the invention, wherein
(i) if the levels of at least 20% of the metabolic markers according to Table 2(a) are increased with respect to the cut-off value according to Table 2(a) and/or the level of at least 20% of the metabolic markers according to Table 2(b) are decreased with respect to the cut-off value according to Table 2(b), then the subject is diagnosed with NASH, and
(ii) if the levels of at least 20% of the metabolic markers according to Table 2(a) are decreased with respect to the cut-off value according to Table 2(a) and/or the levels of at least 20% of the metabolic markers according to Table 2(b) are increased with respect to the cut-off value according to Table 2(b), then the subject is diagnosed with steatosis.
In a particular embodiment of the second method of the invention, at least 40% of the metabolic markers according to Table 2 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as M-subtype according to the first method of the invention, wherein
(i) if the levels of at least 40% of the metabolic markers according to Table 2(a) are increased with respect to the cut-off value according to Table 2(a) and/or the level of at least 40% of the metabolic markers according to Table 2(b) are decreased with respect to the cut-off value according to Table 2(b), then the subject is diagnosed with NASH, and
(ii) if the levels of at least 40% of the metabolic markers according to Table 2(a) are decreased with respect to the cut-off value according to Table 2(a) and/or the levels of at least 40% of the metabolic markers according to Table 2(b) are increased with respect to the cut-off value according to Table 2(b), then the subject is diagnosed with steatosis.
In a particular embodiment of the second method of the invention, at least 60% of the metabolic markers according to Table 2 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as M-subtype according to the first method of the invention, wherein
(i) if the levels of at least 60% of the metabolic markers according to Table 2(a) are increased with respect to the cut-off value according to Table 2(a) and/or the level of at least 60% of the metabolic markers according to Table 2(b) are decreased with respect to the cut-off value according to Table 2(b), then the subject is diagnosed with NASH, and
(ii) if the levels of at least 60% of the metabolic markers according to Table 2(a) are decreased with respect to the cut-off value according to Table 2(a) and/or the levels of at least 60% of the metabolic markers according to Table 2(b) are increased with respect to the cut-off value according to Table 2(b), then the subject is diagnosed with steatosis. In a particular embodiment of the second method of the invention, at least 80% of the metabolic markers according to Table 2 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as M-subtype according to the first method of the invention, wherein
(i) if the levels of at least 80% of the metabolic markers according to Table
2(a) are increased with respect to the cut-off value according to Table 2(a) and/or the level of at least 80% of the metabolic markers according to Table 2(b) are decreased with respect to the cut-off value according to Table 2(b), then the subject is diagnosed with NASH, and
(ii) if the levels of at least 80% of the metabolic markers according to Table
2(a) are decreased with respect to the cut-off value according to Table 2(a) and/or the levels of at least 80% of the metabolic markers according to Table 2(b) are increased with respect to the cut-off value according to Table 2(b), then the subject is diagnosed with steatosis.
In a particular embodiment of the second method of the invention, all metabolic markers according to Table 2 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as M-subtype according to the first method of the invention, wherein
(i) if the levels of all metabolic markers according to Table 2(a) are increased with respect to the cut-off value according to Table 2(a) and the levels of all metabolic markers according to Table 2(b) are decreased with respect to the cut-off value according to Table 2(b), then the subject is diagnosed with NASH, and
(ii) if the levels of all metabolic markers according to Table 2(a) are decreased with respect to the cut-off value according to Table 2(a) and the levels of all metabolic markers according to Table 2(b) are increased with respect to the cut-off value according to Table 2(b), then the subject is diagnosed with steatosis.
In a particular embodiment of the second method of the invention, the method further comprises the administration of a therapeutically effective amount of a compound suitable for the treatment of NASH to the subject diagnosed with NASH, and the administration of a therapeutically effective amount of a compound suitable for the treatment of steatosis to the subject diagnosed with steatosis. The term "therapeutically effective amount", as used herein, relates to the sufficient amount of a compound according to the present invention, i.e. a compound for the treatment of NASH or a compound for the treatment of steatosis, to provide the desired effect, i.e. to achieve an appreciable prevention, cure, delay, reduction of severity or amelioration of one or more symptoms derived from a disease, and will generally be determined by, among other causes, the characteristics of the agent itself and the therapeutic effect to be achieved. It will also depend on the subject to be treated, the severity of the disease suffered by said subject, the chosen dosage form, etc. For this reason, any dose mentioned in this invention must be considered only as guides for the person skilled in the art, who must adjust the doses depending on the aforementioned variables. In an embodiment, the effective amount produces the amelioration of one or more symptoms of the disease that is being treated.
Metabolic markers according to second method of the invention are shown in Table 2. Cut-off values are indicated as percentage values, wherein 0% corresponds to the level of the metabolic marker as determined in a sample from one or more healthy subjects or in a sample from one or more subjects not suffering from NAFLD. Negative percentage cut-off values represent values of metabolites which are lower than the value as determined in a sample from one or more healthy subjects or in a sample from one or more subjects not suffering from NAFLD.
Table 2. Metabolic markers showing differential levels between NASH and steatosis in NAFLD patients profiled as M-subtype (RT: retention time)
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Figure imgf000036_0001
Figure imgf000037_0001
The term "X-HOl 3E" relates to HODE family isomers wherein the position of the double bond has not been identified; they can be distinguished by their retention time (RT) according to Table 13. The term "x-HETE/EET" relates to HETE/EET family isomers wherein the position of the double bond has not been identified; they can be distinguished by their RT according to Table 13. The term "x-DiHETrE" relates to DiHETrE family isomers wherein the position of the double bond has not been identified; they can be distinguished by their RT according to Table 13. The term "x- OxoODE" relates to OxoODE family isomers wherein the position of the double bond has not been identified; they can be distinguished by their RT according to Table 13. PE(22:5/0:0) can be distinguished by their retention time (RT) and mass-to-charge ratio (MZ) according to Table 14.
In a further aspect, the invention relates to a method to diagnose NASH or steatosis in a subject suffering from NAFLD and profiled as non-M-subtype according to the first method of the invention (herein referred to as third method of the invention) that comprises determining in a biological sample from said subject the levels of one or more metabolic markers according to Table 3, wherein (i) if the level of at least one metabolic marker according to Table 3(a) is increased with respect to the cut-off value according to Table 3(a) and/or the level of at least one metabolic marker according to Table 3(b) is decreased with respect to the cutoff value according to Table 3(b), then the subject is diagnosed with NASH, and
(ii) if the level of at least one metabolic marker according to Table 3(a) is decreased with respect to the cut-off value according to Table 3(a) and/or the level of at least one metabolic marker according to Table 3(b) is increased with respect to the cutoff value according to Table 3(b), then the subject is diagnosed with steatosis.
Metabolites showing differential levels between NASH and steatosis in NAFLD patients profiled as non-M-subtype are shown in Table 3. Information concerning the platforms used for the analysis of metabolites upregulated (Table 15) and downregulated (Table 16) in the non-M-subtype NAFLD cluster of patients between groups of NASH vs. steatosis is shown in the Example.
The terms "NAFLD", "subject", "biological sample", "metabolic marker", "level", "increased" and "decreased" have been previosusly described in the context of the first method of the invention. The terms "diagnosis", "NASH" and "steatosis" have been previously described as well in the context of the second method of the invention.
In a particular embodiment of the third method of the invention, the subject who suffers from NAFLD is a mammal, preferably a human.
In a particular embodiment of the third method of the invention, the sample is a biofluid from a subject. In a preferred embodiment, the sample is a serum sample.
Suitable methods to determine the levels of the metabolite markers of the invention have been previously described in the context of the first method of the invention. In a particular embodiment of the third method of the invention, the levels of the metabolic markers are determined by mass spectrometry. In a particular embodiment of the third method of the invention, the sample is fractionated by liquid chromatography prior to the determination of the level(s) of the metabolic marker(s). In a particular embodiment of the third method of the invention, the levels of metabolite markers according to Table 3 are determined by means of Platforms 1, 2 and/or 3 as described in the Example section.
According to the third method of the invention, if the level of at least one metabolic marker according to Table 3(a) is increased with respect to the cut-off value according to Table 3(a) and/or the level of at least one metabolic marker according to Table 3(b) is decreased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with NASH. Particularly, if the levels of at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, of the metabolic markers according to Table 3(a) are increased with respect to the cut-off value according to Table 3(a) and/or the levels of at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, of the metabolic markers according to Table 3(b) are decreased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with NASH. In a particular preferred embodiment, if the levels of all metabolic markers according to Table 3(a) are increased with respect to the cut-off value according to Table 3(a) and/or the levels of all metabolic markers according to Table 3(b) are decreased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with NASH.
According to the third method of the invention, if the level of at least one metabolic marker according to Table 3(a) is decreased with respect to the cut-off value according to Table 3(a) and/or the level of at least one metabolic marker according to Table 3(b) is increased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with steatosis. Particularly, if the levels of at least 20%>, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, of the metabolic markers according to Table 3(a) are decreased with respect to the cut-off value according to Table 3(a) and/or the levels of at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, of the metabolic markers according to Table 3(b) are increased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with steatosis. In a particular preferred embodiment, if the levels of all metabolic markers according to Table 3(a) are decreased with respect to the cut-off value according to Table 3(a) and/or the levels of all metabolic markers according to Table 3(b) are increased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with steatosis.
In a particular embodiment of the third method of the invention, at least 20% of the metabolic markers according to Table 3 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as non-M-subtype according to the first method of the invention, wherein (i) if the levels of at least 20% of the metabolic markers according to Table 3(a) are increased with respect to the cut-off value according to Table 3(a) and/or the levels of at least 20% of the metabolic markers according to Table 3(b) are decreased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with NASH, and
(ii) if the levels of at least 20% of the metabolic markers according to Table 3(a) are decreased with respect to the cut-off value according to Table 3(a) and/or the levels of at least 20% of the metabolic markers according to Table 3(b) are increased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with steatosis.
In a particular embodiment of the third method of the invention, at least 40% of the metabolic markers according to Table 3 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as non-M-subtype according to the first method of the invention, wherein
(i) if the levels of at least 40% of the metabolic markers according to Table
3(a) are increased with respect to the cut-off value according to Table 3(a) and/or the levels of at least 40% of the metabolic markers according to Table 3(b) are decreased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with NASH, and
(ii) if the levels of at least 40% of the metabolic markers according to Table
3(a) are decreased with respect to the cut-off value according to Table 3(a) and/or the levels of at least 40% of the metabolic markers according to Table 3(b) are increased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with steatosis.
In a particular embodiment of the third method of the invention, at least 60% of the metabolic markers according to Table 3 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as non-M-subtype according to the first method of the invention, wherein
(i) if the levels of at least 60% of the metabolic markers according to Table 3(a) are increased with respect to the cut-off value according to Table 3(a) and/or the levels of at least 60% of the metabolic markers according to Table 3(b) are decreased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with NASH, and
(ii) if the levels of at least 60% of the metabolic markers according to Table 3(a) are decreased with respect to the cut-off value according to Table 3(a) and/or the levels of at least 60% of the metabolic markers according to Table 3(b) are increased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with steatosis.
In a particular embodiment of the third method of the invention, at least 80% of the metabolic markers according to Table 3 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as non-M-subtype according to the first method of the invention, wherein
(i) if the levels of at least 80% of the metabolic markers according to Table 3(a) are increased with respect to the cut-off value according to Table 3(a) and/or the levels of at least 80% of the metabolic markers according to Table 3(b) are decreased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with NASH, and
(ii) if the levels of at least 80% of the metabolic markers according to Table 3(a) are decreased with respect to the cut-off value according to Table 3(a) and/or the levels of at least 80% of the metabolic markers according to Table 3(b) are increased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with steatosis.
In a particular embodiment of the third method of the invention, all metabolic markers according to Table 3 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as non-M-subtype according to the first method of the invention, wherein
(i) if the levels of all metabolic markers according to Table 3(a) are increased with respect to the cut-off value according to Table 3(a) and the levels of all metabolic markers according to Table 3(b) are decreased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with NASH, and
(ii) if the levels of all metabolic markers according to Table 3(a) are decreased with respect to the cut-off value according to Table 3(a) and the levels of all metabolic markers according to Table 3(b) are increased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with steatosis.
In a particular embodiment of the third method of the invention, the method further comprises the administration of a therapeutically effective amount of a compound suitable for the treatment of NASH to the subject diagnosed with NASH, and the administration of a therapeutically effective amount of a compound suitable for the treatment of steatosis to the subject diagnosed with steatosis. The term "therapeutically effective amount" has been described previously in the context of the second method of the invention.
Metabolic markers according to the third method of the invention are shown in
Table 3. Cut-off values are indicated as percentage values, wherein 0% corresponds to the level of the metabolic marker as determined in a sample from one or more healthy subjects or in a sample from one or more subjects not suffering from NAFLD. Negative percentage cut-off values represent values of metabolites which are lower than the value as determined in a sample from one or more healthy subjects or in a sample from one or more subjects not suffering from NAFLD.
Table 3. Metabolic markers showing differential levels between NASH and steatosis in
NAFLD patients profiled as non-M-subtype (RT: retention time)
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
The term "x-HETE/EET" relates to HETE/EET family isomers wherein the position of the double bond has not been identified; they can be distinguished by their RT according to Table 15. TG(54:6) relates to two isomers where the addition of the carbon atoms and double bonds coincides, they can be distinguished by their retention time (RT) and mass-to-charge ratio (MZ) according to Table 16.
In a further aspect, the invention relates to a method to diagnose NASH or steatosis in a subject suffering from NAFLD and profiled as I-subtype according to the first method of the invention (herein referred to as fourth method of the invention) that comprises determining in a biological sample from said subject the levels of one or more metabolic markers according to Table 4, wherein
(i) if the level of at least one metabolic marker according to Table 4(a) is increased with respect to the cut-off value according to Table 4(a) and/or the level of at least one metabolic marker according to Table 4(b) is decreased with respect to the cutoff value according to Table 4(b), then the subject is diagnosed with NASH, and
(ii) if the level of at least one metabolic marker according to Table 4(a) is decreased with respect to the cut-off value according to Table 4(a) and/or the level of at least one metabolic marker according to Table 4(b) is increased with respect to the cut- off value according to Table 4(b), then the subject is diagnosed with esteatosis.
Metabolites showing differential levels between NASH and steatosis in NAFLD patients profiled as I-subtype are shown in Table 4. Information concerning the platforms used for the analysis of metabolites upregulated (Table 17) and downregulated (Table 18) in the I-subtype NAFLD cluster of patients between groups of NASH vs. steatosis is shown in the Example.
The terms "NAFLD", "subject", "biological sample", "metabolic marker", "level", "increased" and "decreased" have been previosusly described in the context of the first method of the invention. The terms "diagnosis", "NASH" and "steatosis" have been previously described as well in the context of the second method of the invention.
In a particular embodiment of the fourth method of the invention, the subject who suffers from NAFLD is a mammal, preferably a human.
In a particular embodiment of the fourth method of the invention, the sample is a biofluid from a subject. In a preferred embodiment, the sample is a serum sample.
Suitable methods to determine the levels of the metabolite markers of the invention have been previously described in the context of the first method of the invention. In a particular embodiment of the fourth method of the invention, the levels of the metabolic markers are determined by mass spectrometry. In a particular embodiment of the fourth method of the invention, the sample is fractionated by liquid chromatography prior to the determination of the level(s) of the metabolic marker(s). In a particular embodiment of the fourth method of the invention, the levels of metabolite markers according to Table 4 are determined by means of Platforms 1, 2 and/or 3 as described in the Example section. According to the fourth method of the invention, if the level of at least one metabolic marker according to Table 4(a) is increased with respect to the cut-off value according to Table 4(a) and/or the level of at least one metabolic marker according to Table 4(b) is decreased with respect to the cut-off value according to Table 4(b), then the subject is diagnosed with NASH. Particularly, if the levels of at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, of the metabolic markers according to Table 4(a) are increased with respect to the cut-off value according to Table 4(a) and/or the levels of at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, of the metabolic markers according to Table 4(b) are decreased with respect to the cut-off value according to Table 4(b), then the subject is diagnosed with NASH. In a particular preferred embodiment, if the levels of all metabolic markers according to Table 4(a) are increased with respect to the cut-off value according to Table 4(a) and/or the levels of all metabolic markers according to Table 4(b) are decreased with respect to the cut-off value according to Table 4(b), then the subject is diagnosed with NASH.
According to the fourth method of the invention, if the level of at least one metabolic marker according to Table 4(a) is decreased with respect to the cut-off value according to Table 4(a) and/or the level of at least one metabolic marker according to Table 4(b) is increased with respect to the cut-off value according to Table 4(b), then the subject is diagnosed with steatosis. Particularly, if the levels of at least 20%>, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, of the metabolic markers according to Table 4(a) are decreased with respect to the cut-off value according to Table 4(a) and/or the levels of at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, of the metabolic markers according to Table 4(b) are increased with respect to the cut-off value according to Table 4(b), then the subject is diagnosed with steatosis. In a particular preferred embodiment, if the levels of all metabolic markers according to Table 4(a) are decreased with respect to the cut-off value according to Table 4(a) and/or the levels of all metabolic markers according to Table 4(b) are increased with respect to the cut-off value according to Table 4(b), then the subject is diagnosed with steatosis.
In a particular embodiment of the fourth method of the invention, at least 20% of the metabolic markers according to Table 4 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as I-subtype according to the first method of the invention, wherein
(i) if the levels of at least 20% of the metabolic markers according to Table 4(a) are increased with respect to the cut-off value according to Table 4(a) and/or the levels of at least 20% of the metabolic markers according to Table 4(b) are decreased with respect to the cut-off value according to Table 4(b), then the subject is diagnosed with NASH, and
(ii) if the levels of at least 20% of the metabolic markers according to Table 4(a) are decreased with respect to the cut-off value according to Table 4(a) and/or the levels of at least 20% of the metabolic markers according to Table 4(b) are increased with respect to the cut-off value according to Table 4(b), then the subject is diagnosed with esteatosis.
In a particular embodiment of the fourth method of the invention, at least 40% of the metabolic markers according to Table 4 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as I-subtype according to the first method of the invention, wherein
(i) if the levels of at least 40% of the metabolic markers according to Table 4(a) are increased with respect to the cut-off value according to Table 4(a) and/or the levels of at least 40% of the metabolic markers according to Table 4(b) are decreased with respect to the cut-off value according to Table 4(b), then the subject is diagnosed with NASH, and
(ii) if the levels of at least 40% of the metabolic markers according to Table 4(a) are decreased with respect to the cut-off value according to Table 4(a) and/or the levels of at least 40% of the metabolic markers according to Table 4(b) are increased with respect to the cut-off value according to Table 4(b), then the subject is diagnosed with esteatosis.
In a particular embodiment of the fourth method of the invention, at least 60% of the metabolic markers according to Table 4 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as I-subtype according to the first method of the invention, wherein
(i) if the levels of at least 60% of the metabolic markers according to Table 4(a) are increased with respect to the cut-off value according to Table 4(a) and/or the levels of at least 60% of the metabolic markers according to Table 4(b) are decreased with respect to the cut-off value according to Table 4(b), then the subject is diagnosed with NASH, and
(ii) if the levels of at least 60% of the metabolic markers according to Table 4(a) are decreased with respect to the cut-off value according to Table 4(a) and/or the levels of at least 60% of the metabolic markers according to Table 4(b) are increased with respect to the cut-off value according to Table 4(b), then the subject is diagnosed with esteatosis.
In a particular embodiment of the fourth method of the invention, at least 80% of the metabolic markers according to Table 4 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as I-subtype according to the first method of the invention, wherein
(i) if the levels of at least 80% of the metabolic markers according to Table 4(a) are increased with respect to the cut-off value according to Table 4(a) and/or the levels of at least 80% of the metabolic markers according to Table 4(b) are decreased with respect to the cut-off value according to Table 4(b), then the subject is diagnosed with NASH, and
(ii) if the levels of at least 80% of the metabolic markers according to Table 4(a) are decreased with respect to the cut-off value according to Table 4(a) and/or the levels of at least 80% of the metabolic markers according to Table 4(b) are increased with respect to the cut-off value according to Table 4(b), then the subject is diagnosed with esteatosis.
In a particular embodiment of the fourth method of the invention, all metabolic markers according to Table 4 are determined in a biological sample from a subject whose diagnosis of NASH or steatosis is to be determined and who has been profiled as I-subtype according to the first method of the invention, wherein
(i) if the levels of all metabolic markers according to Table 4(a) are increased with respect to the cut-off value according to Table 4(a) and the levels of all metabolic markers according to Table 4(b) are decreased with respect to the cut-off value according to Table 4(b), then the subject is diagnosed with NASH, and
(ii) if the levels of all metabolic markers according to Table 4(a) are decreased with respect to the cut-off value according to Table 4(a) and the levels of all metabolic markers according to Table 4(b) are increased with respect to the cut-off value according to Table 4(b), then the subject is diagnosed with esteatosis.
In a particular embodiment of the fourth method of the invention, the method further comprises the administration of a therapeutically effective amount of a compound suitable for the treatment of NASH to the subject diagnosed with NASH, and the administration of a therapeutically effective amount of a compound suitable for the treatment of steatosis to the subject diagnosed with steatosis. The term "therapeutically effective amount" has been described previously in the context of the second method of the invention.
Metabolic markers according to the fourth method of the invention are shown in
Table 4. Cut-off values are indicated as percentage values, wherein 0% corresponds to the level of the metabolic marker as determined in a sample from one or more healthy subjects or in a sample from one or more subjects not suffering from NAFLD. Negative percentage cut-off values represent values of metabolites which are lower than the value as determined in a sample from one or more healthy subjects or in a sample from one or more subjects not suffering from NAFLD.
Table 4. Metabolic markers showing differential levels between NASH and steatosis in NAFLD patients profiled as I-subtype (RT: retention time)
Figure imgf000050_0001
Figure imgf000051_0001
18 3n-6 7.9 Unsaturated fatty acids [FA0103] -23.9%
16 ln-x 8.57 Unsaturated fatty acids [FA0103] -37.0%
18 3n-x 8.12 Unsaturated fatty acids [FA0103] -51.1%
18 2n-6 trans 9.23 Unsaturated fatty acids [FA0103] -64.9%
18 0 11.49 Straight chain fatty acids [FA0101] -2.4%
18 ln-x 10.34 Unsaturated fatty acids [FA0103] -78.3% x-HODE 5.36 Octadecanoids [FA0200] -79.5%
13-HODE 5.07 Octadecanoids [FA0200] -94.6% x-HODE 4.55 Octadecanoids [FA0200] -96.7%
9-HODE 5.13 Octadecanoids [FA0200] -96.2%
The term "X-HODE" relates to HODE family isomers wherein the position of the double bond has not been identified; they can be distinguished by their retention time (RT) according to Table 18. The invention is detailed below by means of the following examples which are merely illustrative and by no means limiting for the scope of the invention.
EXAMPLE
Materials and methods
Animal experimentation
Eight-month old Matla (-/-) mice and age matched wild type animals were used. Animals were breed and housed in the CIC bioGUNE animal unit, accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Only Matla (-/-) mice showing elevated serum liver enzymes (ALT and AST) and testing positive for hepatic lipid accumulation, determined by high-frequency ultrasound imaging, were used in this study. At the end of the study, blood samples were collected.
Blood sampling in mice
Submandibular and retroorbital blood samples were collected at the beginning and at the end of the experiment. Blood samples were deposited in serum separator gel tubes (Microtainer, Becton-Dickinson, Franklin Park, NJ) and centrifuged (6000 rpm, 15 min, 4 °C) for serum separation.
Samples from subjects previously diagnosed with NAFLD Serum was prepared by incubation of venous blood of the patient in tubes with clot activator for 30 minutes before centrifugation (2500 g, 15 min); supematants were aliquoted into microtubes and stored at -80°C until metabolomic analysis.
Sample preparation
Serum samples were divided into aliquots and analyzed in three different metabolomic platforms:
Platform 1 - Methanol extract. Proteins were precipitated from the defrosted serum samples (75 μί) by adding 300 μΐ. of methanol in 1.5 mL microtubes on ice. The extraction solvent was spiked with the following compounds not detected in non-spiked human serum extracts: NEFA(19:0) (NEFA, non-esterified fatty acids), tryptophan- d5(indole-d5), dehydrocholic acid and PC(13 :0/0:0). After brief vortex mixing, the samples were incubated overnight at -20°C. Supematants (300 μί) were collected after centrifugation at 16000xg for 15 min and solvent removed. The dried extracts were then reconstituted in 120 μΐ. of methanol, centrifuged (16000xg 5 min), and transferred to vials for UPLC-MS analysis.
Platform 2 - Chloroform/Methanol extract. Proteins were precipitated from the defrosted serum samples (10 μί) by addinglO μΐ. of sodium chloride (50 mM) and 1 10 μί of chloroform/methanol (2: 1) in 1.5 mL microtubes on ice. The extraction solvent was spiked with the following compounds not detected in non-spiked human serum extracts: SM(dl 8:l/6:0), PE(17:0/17:0), PC(19:0/19:0), TG(13 :0/13 :0/13 :0), TG(17:0/17:0/17:0), Cer(dl 8:l/17:0), ChoE(12:0). After brief vortex mixing, the samples were incubated for 1 h at -20°C. After centrifugation at 16000xg for 15 min, 70 μΐ, of the lower organic phase was collected and the solvent removed. The dried extracts were then reconstituted in 100 μΐ, acetronitrile/isopropanol (50:50), centrifuged (16000xg 5 min), and transferred to vials for UPLC-MS analysis.
Platform 3 - Amino acids. 10 μί aliquots of the extracts prepared for platform 1 were transferred to microtubes and derivatized for amino acid analysis.
Liquid cromatography - mass spectrometry platforms
A UPLC-single quadrupole-MS amino acid analysis system was combined with two separate UPLC-time-of-flight (TOF)-MS based platforms analyzing methanol and chloroform/methanol serum extracts. Each platform involves the use of a different UPLC-MS method. Platform 1 : Chromatography was performed on a 1.0 mm i.d. x 100 mm ACQUITY 1.7 μιη CI 8 BEH column (Waters Corp., Milford, MA) using an ACQUITY UPLC system (Waters Corp., Milford, MA). The column was maintained at 40 °C and eluted with a 18 min gradient. The mobile phase, at a flow rate of 140 μΙ7ιηιη, initially consisted of 100% solvent A (0.05%> formic acid), with a linear increase of solvent B (acetonitrile containing 0.05% formic acid) up to 50% over two minutes, and a linear increase to 100% B over the next 11 min before returning to the initial composition in readiness for the subsequent injection which preceded a 45 s system recycle time. The volume of sample injected onto the column was 2 μΐ.. The eluent was introduced into the mass spectrometer (LCT-PremierXE, Waters Corp., Milford, MA) by electrospray ionization, with capillary and cone voltages set in the negative ion mode to 2800 and 50 V, respectively. The nebulization gas was set to 600 L/h at a temperature of 350 °C. The cone gas was set to 30 L/h, and the source temperature was set to 120 °C. Centroid data were acquired from m/z 50-1000 using an accumulation time of 0.2 s per spectrum.
Platform 2: Chromatography was performed on a 2.1mm i.d. x 100 mm
ACQUITY 1.7 μπι C18 BEH column (Waters Corp., Milford, MA) using an ACQUITY UPLC system (Waters Corp., Milford, MA). The column was maintained at 60 °C and eluted with a 17 min linear gradient of solvents A (water, acetonitrile and 10 mM ammonium formate), and B (acetonitrile, isopropanol and 10 mM ammonium formate). The mobile phase, at a flow rate of 400 μί/ιηίη, initially consisted of 40% solvent B, increasing up to 100% at 10 minutes. After 5 minutes the mobile phase was reset to the initial composition in readiness for the subsequent injection which preceded a 45 s system recycle time. The volume of sample injected onto the column was 3 μΐ,. The eluent was introduced into the mass spectrometer (Xevo G2 QTof, Waters Corp., Milford, MA) by electrospray ionization, with capillary and cone voltages set in the positive ion mode to 3200 and 30 V, respectively. The nebulization gas was set to 1000 L/h at a temperature of 500 °C. The cone gas was set to 30 L/h, and the source temperature was set to 120 °C. Centroid data were acquired from m/z 50-1200 using an accumulation time of 0.2 s per spectrum.
Platform 3: Analytes were separated by means of a gradient of solvents A (water
10 mM NH4HCO3, pH=8.8 adjusted with ammonium hydroxide) and B (acetonitrile). Flow rate was 140 μί/ιηίη. Gradient started with 98%> of solvent A that decreased linearly to 92% at min 6.5, 80% at min 10, 70% at min 11 and 0% at min 12. After 2 minutes the mobile phase was reset to the initial composition in readiness for the subsequent injection which preceded a 45 s system recycle time. The eluent was introduced into the mass spectrometer (SQD, Waters Corp., Milford, MA) by electrospray ionization, with capillary and cone voltages set in the positive ion mode to 3200 and 30 V, respectively. The nebulization gas was set to 600 L/h at a temperature of 350 °C. The cone gas was set to 10 L/h, and the source temperature was set to 120 °C. Selected ion recording (SIR) was used to analyze desired metabolites. An appropriate test mixture of standard compounds may be analyzed along the entire set of randomized sample injections in order to examine the retention time stability and sensitivity of the system throughout the course of the run which lasted a maximum of 48 h per batch of samples injected.
Online tandem mass spectrometry (MS/MS) experiments for metabolite identification were performed on an Acquity-SYNAPT G2 system and an Acquity- Xevo G2 QTof system (Waters Corp., Milford, MA), both instruments operating in positive and negative ion electrospray modes; source parameters were identical to those employed in the profiling experiments, except for the cone voltage which was increased (30-70 V) when pseudo MS/MS/MS data was required. During retention time windows corresponding to the elution of the compounds under investigation, the quadrupole was set to resolve and transmit ions with appropriate mass-to-charge values. The selected ions then traversed an argon-pressurized cell, with a collision energy voltage (typically between 5 and 50 V) applied in accordance with the extent of ion fragmentation required. Subsequent TOF analysis of the fragment ions generated accurate mass generally <3 ppm MS/MS or pseudo MS/MS/MS spectra corrected in real time by reference to leucine enkephalin, infused at 10 μΙ7ιηίη through an independent reference electrospray, sampled every 10 s.
Data Pre-Processing
All data were pre-processed using the TarkerLynx application manager for MassLynx 4.1 software (Waters Corp., Milford, MA). The complete set of predefined Pvt-m/z pairs was fed into the software, which generated associated extracted ion chromatograms (mass tolerance window = 0.05 Da), peak-detected and noise-reduced in both the LC and MS domains. A list of intensities (chromatographic peak areas) was then generated for each sample injection, using the Rt-m/z pairs (retention time tolerance = 6 s) as identifiers. Intra- and interbatch normalization followed the procedure summarized in the paper Martinez- Arranz et al (Martinez- Arranz et al 2015 J Proteomics 127 (Pt B): 275-288). This process involved (i) internal standard response correction (intrabatch normalization) and (ii) variable specific interbatch single point external calibration using repeat extracts of a commercial serum sample (interbatch normalization).
Data Processing
Data are represented as means ± standard deviation of the mean (SD). Univariate statistical analyses were performed calculating group fold-changes and unpaired Student's t-test (or Welch's t test where unequal variances were found) for the comparison among the different groups (Table 5). Significance was defined as p<0.05. The 'volcano plot' analysis was performed as an effective and easy-to-interpret graph that summarizes both fold-change and t-test criteria. It is a scatter-plot of the negative loglO-transformed p-values from the t-test against the log2 fold change. Nomenclature in volcano plots: AA, amino acids; SFA, MUFA, PUFA, saturated, monounsaturated and polyunsaturated fatty acids, respectively; oxFA, oxidized FA; DG, diglycerols; TG, triglycerides; ChoE, cholesteryl esters; BA, bile acids; PE, phosphatidylethanolamines; LPE, lyso-PE; PC, phosphatidylcholines; LPC, lyso-PC; PI, phosphatidylinositols; LPI, lyso-PI; Cer, ceramides; SM, sphingomyelins; CMH, monohexosylceramides; FSB, free sphingoid bases.
A hierarchical clustering algorithm based on metabolites ion intensity was used to visualize the differences in metabolite signatures between samples, as well as the ward's minimum variance method as agglomeration method. Biochemically related compounds were generally found to cluster together. The maximum of the average of the individual silhouette widths was calculated for the clusters as described in Rousseeuw PJ (1987 J. Comput. Appl. Math. 20: 53-65). The cluster analysis was calculated with the cluster R package as described in Maechler M, et al (2015, Cluster: Cluster Analysis Basics and Extensions. R package version 2.0.3). The heatmap was realized with the pheatmap R package as described in Kolde R (2015, pheatmap: Pretty Heatmaps. R package version 1.0.8. http://CRAN.R-project.org/package=pheatmap). Table 5. Serum metabolites significantly differentiated between Matl (-/-) mice and wildtype that are able to subclassif ing three clusters or profiles of NAFLD patients.
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Table 6.p-value (Student test) of upregulated serum metabolites in M-subtype of NAFLD patients.
Figure imgf000065_0002
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Table 7.p-value (Student test) for down regulated serum metabolites in M-sybtype NAFLD patients
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Table 8. p-value (Student test) for upregulated serum metabolites in non M-subtype NAFLD patients.
Figure imgf000077_0002
Figure imgf000078_0001
Figure imgf000079_0001
Table 9.p-value (Student test) for downregulated serum metabolites in non M-subtype NAFLD patients.
Figure imgf000079_0002
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Table 1 l .p-values (Student test) for down regulated serum metabolites in I-subtype NAFLD patients.
Figure imgf000083_0001
Table 12- Platforms used, retention time, mass-to charge, adduct and molecular formula for each metabolite significantly differentiated between Matl (-/-) mice and wildtype that are able to subclassifying three clusters or subtypes of NAFLD patients.
Figure imgf000084_0001
Figure imgf000085_0001
Table 13. Platform used, retention time, mass-to charge, adduct and molecular formula for each metabolite upregulated serum metabolites in th M-subtype NAFLD cluster of patients between groups of NASH vs. steatosis.
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Table 14. Platform used, retention time, mass-to charge, adduct and molecular formula for each metabolite downregulated serum metabolites in the M-subtype NAFLD cluster of patients between groups of NASH vs. steatosis.
Molecular
Metabolite PLATFORM RT MZ Adduct
Formula
18:3n-3 1 7.75 323.2223 [M+C02H]- C18H30O2
Methionine 3 9.46 320.1069 [Mderivatized+H]+ C5H11N02S
18: ln-x 1 10.34 327.2536 [M+C02H]- C18H3402
TG(54:5) 2 7.99 898.7864 [M+NH4J+ C57H100O6
18:2n-6 trans 1 9.23 325.2379 [M+C02H]- C18H3202
18:2n-x 1 9.04 325.2379 [M+C02H]- C18H3202
DG(36:4) 2 5.26 639.4965 [M+NH4J+ C39H6805
14:0 1 7.97 273.2066 [M+C02H]- C14H2802
18:3n-x 1 8.12 323.2223 [M+C02H]- C18H30O2
TG(54:6) 2 7.75 896.7707 [M+NH4J+ C57H9806
20:2n-6 1 10.46 353.2692 [M+C02H]- C20H36O2
18:3n-6 1 7.90 323.2223 [M+C02H]- C18H30O2
PE(22:5/0:0) 1 5.67 526.2934 [M-H]- C27H46N07P
PC( 18:2/20:4) 2 4.18 806.5700 [M+H]+ C46H80NO8P
PC( 1 8:2/1 :2) 2 4.29 782.5700 [M+H]+ C44H80NO8P
20: ln-6 1 11.61 355.2849 [M+C02H]- C20H38O2
18:2n-6 1 8.84 325.2379 [M+C02H]- C18H3202
PC(0:0/22:5) 1 5.49 614.3459 [M+C02H]- C30H52NO7P
TG(52:4) 2 7.99 872.7707 [M+NH4J+ C55H9806
PE(22:5/0:0) 1 5.45 526.2934 [M-H]- C27H46N07P
Glutamine 3 4.43 317.1249 [Mderivatized+H]+ C5H10N2O3
16:0 1 9.85 301.2379 [M+C02H]- C16H3202
TG(49:3) 2 7.82 832.7394 [M+NH4J+ C52H9406
PC(22:4/0:0) 1 6.02 616.3615 [M+C02H]- C30H54NO7P
18:0 1 11.49 329.2692 [M+C02H]- C18H3602
PE( 18:2/0:0) 1 5.18 476.2778 [M-H]- C23H44N07P
16: ln-7 1 8.35 299.2223 [M+C02H]- C16H30O2 PC(0:0/22:4) 1 5.87 616.3615 [M+C02H]- C30H54NO7P
16: ln-x 1 8.57 299.2223 [M+C02H]- C16H30O2
DG(36:3) 2 5.68 641.5121 [M+NH4J+ C39H70O5
PC(0-
2 5.85 824.6533 [M+H]+ C48H90NO7P 18:0/22:4)
TG(46:0) 2 8.23 796.7394 [M+NH4J+ C49H9406
TG(56:7) 2 7.90 922.7864 [M+NH4J+ C59H100O6
22:4n-6 1 9.95 377.2692 [M+C02H]- C22H3602
Table 15. Platform used, retention time, mass-to charge, adduct and molecular formula for each metabolite upregulated serum metabolites in the non-M-subtype NAFLD cluster of patients between groups of NASH vs. steatosis.
Molecular
Metabolite PLATFORM RT MZ Adduct
Formula
ChoE(16: l) 2 8.19 640.6033 [M+NH4J+ C43H7402 x-DiHETrE 1 4.37 337.2379 [M-H]- C20H34O4
5-HETE 1 5.91 319.2273 [M-H]- C20H32O3
Isoieucine 3 10.17 302.1504 [Mderivatized+H]+ C6H13N02
Aspartic Acid 3 1.49 304.0933 [Mderivatized+H]+ C4H7N04
15-HETE 1 5.26 319.2273 [M-H]- C20H32O3
Sphinganine 1-
1 4.33 380.2556 [M-H]- C18H40NO5P phosphate
PE(P- 18:0 0:0) 1 7.63 464.3142 [M-H]- C23H48N06P
Leucine 3 10.31 302.1504 [Mderivatized+H]+ C6H13N02
ChoE(18:3) 2 7.97 664.6033 [M+NH4J+ C45H7402 x-HETE/EET 1 4.95 319.2273 [M-H]- C20H32O3
Tyrosine 3 8.87 352.1297 [Mderivatized+H]+ C9H11N03
PE(P-18: l/0:0) 1 6.31 462.2985 [M-H]- C23H46N06P
9-HETE 1 5.77 319.2273 [M-H]- C20H32O3
12-HETE 1 5.61 319.2273 [M-H]- C20H32O3
Lysine 3 10.40 317.1613 [Mderivatized+H]+ C6H14N202 11-HETE 1 5.47 3 19.2273 [M-H]- C20H32O3
PC( 18:0/22:5 ) 2 5.19 836.6169 [M+H]+ C48H86N08P
Phenylalanine 3 10.70 336.1348 [Mderivatized+H]+ C9H11N02
ChoE(20:3) 2 8.27 692.6346 [M+NH4J+ C47H7802
Glutamic Acid 3 1 .90 318.1090 [Mderivatized+H]+ C5H9N04 x-HETE/EET 1 5.13 3 19.2273 [ M-H |- C20H32O3
ChoE(14:0) 2 8.19 614.5876 [M+NH4J+ C41 H7202
PC( 16:0/ 16:0) 2 5.16 734.5700 [M+H]+ C40H80 O8P x-HETE/EET 1 5.34 3 19.2273 [M-H]- C20H32O3
Table 16. Platform used, retention time, mass-to charge, adduct and molecular formula for each metabolite downregulated serum metabolites in the non-M-subtype NAFLD cluster of patients between groups of NASH vs. steatosis.
Metabolite PLATFORM RT MZ Adduct Molecular Formula
TG(54:5 ) 2 7.99 898.7864 [M+NH4J+ C57H 100O6
TG(54:6) 2 7.75 896.7707 [M+NH4J+ C57H9806
PC(20:0/20:4) 2 5.70 838.6326 [M+H]+ C48H88N08P
TG(52:4) 2 7.99 872.7707 [M+NH4J+ C55H9806
PC( 18:0/20:4 ) 2 5.19 810.6013 [M+H]+ C46H84N08P
TG(53:3) 2 8.34 888.8020 [M+NH4J+ C56H102O6
TG(5 1 :2) 2 8.33 862.7864 [M+NH4J+ C5411 10006
TG(52:3) 2 8.23 874.7864 [M+NH4J+ C55H 100O6
TG(51 :3) 2 8.09 860.7707 [M+NH4J+ C54H9806
PC( 18:2/20:4) 2 4.1 8 806.5700 [M+H]+ C46H80 O8P
TG(50:3) 2 7.96 846.755 1 [M+NH4J+ C53H9606
TG(56:7) 2 7.90 922.7864 [M+NH4J+ C59H 100O6
PC( 15:0/20:4 ) 2 4.33 768.5543 [M+H]+ C43H78N08P
PC( 15:0/22:6) 2 4.13 792.5543 [M+H]+ C45H78N08P
PC(16:0/18:0) 2 5.69 762.6013 [M+H]+ C42H84N08P
18:3n-3 1 7.75 323.2223 [M+C02H]- C18H30O2
16: ln-9 1 8.45 299.2223 [M+C02H]- C16H30O2
PC(0:0/22:5) 1 5.49 614.3459 [M+C02H]- C30H52 O7P
PC(O-16:0/22:4) 2 5.36 796.6220 [M+H]+ C46H86 07P
TG(54:6) 2 7.90 896.7707 [M+NH4J+ C57H9806
TG(52:2) 2 8.45 876.8020 [M+NH4J+ C55H 102O6
SM(38: 1) 2 5.71 759.6380 [M+H]+ C43H87N206P
SM(dl 8: 1/23:0) 2 6.42 801 .6850 [M+H]+ C46H93 206P
TG(53:2) 2 8.56 890.8177 [M+NH4J+ C56H 104O6
PE(22:5/0:0) 1 5.45 526.2934 [M-H]- C27H46 07P
18:2n-6 1 8.84 325.2379 [M+C02H]- C18H3202 TG(54:2) 2 8.67 904.8333 [M+NH4]+ C57H106O6
TG(54:5) 2 8.14 898.7864 [M+NH4J+ C57H100O6
DG(36:3) 2 5.68 641.5121 [M+NH4]+ C39H70O5
PC(0-18:020:4) 2 5.51 796.6220 [M+H]+ C46H86 07P
PC( 16:0/20:4) 2 4.63 782.5700 [M+H]+ C44H80 O8P
18:3n-x 1 8.12 323.2223 [M+C02H]- C18H30O2
TG(56:3) 2 8.67 930.8490 [M+NH4]+ C59H108O6
PC(0:0/20:0) 1 8.63 596.3928 [M+C02H]- C28H58 07P
TG(54:3) 2 8.43 902.8177 [M+NH4]+ C57H104O6
TG(56:8) 2 7.77 920.7707 [M+NH4]+ C59H9806
Table 17. Platform used, retention time, mass-to charge, adduct and molecular formula for each metabolite upregulated serum metabolites in the I-subtype NAFLD cluster of patients between groups of NASH vs. steatosis.
Molecular
Metabolite Platform RT MZ Adduct
Formula
12-1 IETF. 1 5.61 319.2273 [M-H]- C20H32O3
ChoE(18:l) 2 8.50 668.6346 [M+NH4J+ C45H7802
Aspartic Acid 3 1.49 304.0933 [Mderivatized+H] + C4H7N04
Taurine 3 5.50 296.0705 [Mderivatized+H] + C2H7N03S
Serine 3 3.85 276.0984 [Mderivatized+H]+ C3H7N03
Phenylalanine 3 10.70 336.1348 [Mderivatized+H] + C9H11N02
SM(d 18:0/16:0) 2 4.80 705.5911 [M+H]+ C39H81 206P
20:4n-6 1 8.65 349.2379 [M+C02H]- C20H32O2
CMH(d 18: 1/24:0) 2 6.74 812.6979 [M+H]+ C48H93 08
SM(dl8:l/18:0) 2 5.17 731.6067 [M+H]+ C41H83N206P
ChoE(20:3) 2 8.27 692.6346 [M+NH4]+ C47H7802
ChoE(18:3) 2 7.97 664.6033 [M+NH4J+ C45H7402
ChoE(18:2) 2 8.22 666.6189 [M+NH4]+ C45H7602
Glycine 3 4.27 246.0878 [Mderivatized+H] + C2H5N02
PC(0-18:l/22:4) 2 5.37 822.6377 [M+H]+ C48H88 07P
SM(42:3) 2 5.71 811.6693 [M+H]+ C47H91 206P
ChoE(16:l) 2 8.19 640.6033 [M+NH4]+ C43H7402
Lysine 3 10.40 317.1613 [Mderivatized+H] + C6H14N202
SM(d 18:1 22:0) 2 6.20 787.6693 [M+H]+ C45H91 206P
PC( 16:0/20:4) 2 4.63 782.5700 [M+H]+ C44H80 O8P
ChoE(22:6) 2 7.90 714.6189 [M+NH4]+ C49H7602
PC(18:0/20:4) 2 5.19 810.6013 [M+H]+ C46H84N08P
LPC(16:1) 1 4.71 538.3146 [M+C02H]- C24H48N07P
Leucine 3 10.31 302.1504 [Mderivatized+H] + C6H13N02
SM(dl8:0/18:0) 2 5.39 733.6224 [M+H]+ C41H85N206P
SM(dl8:0/15:0) 2 4.48 691.5754 [M+H]+ C38H79 206P Table 18. Platform used, retention time, mass-to charge, adduct and molecular formula for each metabolite downregulated serum metabolites in the I-subtype NAFLD cluster of patients between groups of NASH vs. steatosis.
Metabolite Platform RT MZ Adduct Molecular Formula
18: ln-x 1 10.34 327.2536 [M+C02H]- C18H3402
18:2n-6 trans 1 9.23 325.2379 [M+C02H]- C18H3202
20:4n-x 1 8.34 349.2379 [M+C02H]- C20H32O2
18:3n-3 1 7.75 323.2223 [M+C02H]- C18H30O2
18:3n-x 1 8.12 323.2223 [M+C02H]- C18H30O2
18:2n-x 1 9.04 325.2379 [M+C02H]- C18H3202
18:2n-6 1 8.84 325.2379 [M+C02H]- C18H3202 x-HODE 1 5.36 295.2273 [M-H ]- C18H3203
18:3n-6 1 7.90 323.2223 [M+C02H]- C18H30O2
13-HODE 1 5.07 295.2273 [M-H]- C18H3203
16: l n-9 1 8.45 299.2223 [M+C02H]- C16H30O2
22:5n-3 1 8.93 375.2536 [M+C02H]- C22H3402
TG(54:5) 7.99 898.7864 [M+NH4J+ C57H100O6
16: ln-x 1 8.57 299.2223 [M+C02H]- C16H30O2
14:0 1 7.97 273.2066 [M+C02H]- C14H2802
DG(36:4) 5.26 639.4965 [M+NH4J+ C39H6805 x-HODE 1 4.55 295.2273 [M-H]- C18H3203
18:0 1 11.49 329.2692 [M+C02H]- C18H3602
9-HODE 1 5.13 295.2273 [M-H]- C18H3203
TG(54:6) 2 7.75 896.7707 [M+NH4J+ C57H9806
Table 19. Analysis of expression of metabolites characterizing M-subtype according to Table 2
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
The cutoff is the best threshold value for discriminate between Steatosis and NASH.
The cutoff(%) is the best threshold in percentage between Steatosis increase compared to QC and NASH increase compared to QC. Last column shows the NASH value (mean) minus the Steatosis value (mean),
(sd): standard deviation.
Table 20. Analysis of expression of metabolites characterizing non-M-subtype according to Table 3
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Table 21. Analysis of expression of metabolites profiling subtypes M, non-M and I according to Table 4
Figure imgf000110_0002
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0001

Claims

1. Method to profile a subject suffering from non-alcoholic fatty liver disease (NAFLD) as M-subtype, non-M-subtype or I-subtype that comprises determining in a biological sample from said subject the levels of one or more metabolic markers as defined in Table 1 , wherein
(i) if the level of at least one metabolic marker according to Table 1(a) is increased with respect to the cut-off value according to Table 1(a) and/or the level of at least one metabolic marker according to Table 1(b) is decreased with respect to the cut-off value according to Table 1(b), then the subject is profiled as M-subtype,
(ii) if the level of at least one metabolic marker according to Table 1(c) is increased with respect to the cut-off value according to Table 1(c), then the subject is profiled as non-M-subtype, and
(iii) if the subject is not profiled as M-subtype according to step (i) and is not profiled as non-M-subtype according to step (ii), then the subject is profiled as I-subtype.
2. Method to diagnose non-alcoholic steatohepatitis (NASH) or steatosis in a subject suffering from NAFLD and profiled as M-subtype according to the method of claim 1 that comprises determining in a biological sample from said subject the levels of one or more metabolic markers according to Table 2, wherein
(i) if the level of at least one metabolic marker according to Table 2(a) is increased with respect to the cut-off value according to Table 2(a) and/or the level of at least one metabolic marker according to Table 2(b) is decreased with respect to the cut-off value according to Table 2(b), then the subject is diagnosed with NASH, and
(ii) if the level of at least one metabolic marker according to Table 2(a) is decreased with respect to the cut-off value according to Table 2(a) and/or the level of at least one metabolic marker according to Table 2(b) is increased with respect to the cut-off value according to Table 2(b), then the subject is diagnosed with steatosis.
Method to diagnose non-alcoholic steatohepatitis (NASH) or steatosis in a subject suffering from NAFLD and profiled as non-M-subtype according to the method of claim 1 that comprises determining in a biological sample from said subject the levels of one or more metabolic markers according to Table 3, wherein
(i) if the level of at least one metabolic marker according to Table 3(a) is increased with respect to the cut-off value according to Table 3(a) and/or the level of at least one metabolic marker according to Table 3(b) is decreased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with NASH, and
(ii) if the level of at least one metabolic marker according to Table 3(a) is decreased with respect to the cut-off value according to Table 3(a) and/or the level of at least one metabolic marker according to Table 3(b) is increased with respect to the cut-off value according to Table 3(b), then the subject is diagnosed with steatosis.
Method to diagnose non-alcoholic steatohepatitis (NASH) or steatosis in a subject suffering from NAFLD and profiled as I-subtype according to the method of claim 1 that comprises determining in a biological sample from said subject the levels of one or more metabolic markers according to Table 4, wherein
(i) if the level of at least one metabolic marker according to Table 4(a) is increased with respect to the cut-off value according to Table 4(a) and/or the level of at least one metabolic marker according to Table 4(b) is decreased with respect to the cut-off value according to Table 4(b), then the subject is diagnosed with NASH, and
(ii) if the level of at least one metabolic marker according to Table 4(a) is decreased with respect to the cut-off value according to Table 4(a) and/or the level of at least one metabolic marker according to Table 4(b) is increased with respect to the cut-off value according to Table 4(b), then the subject is diagnosed with esteatosis.
5. Method according to any of the preceding claims, wherein the sample is a serum sample.
6. Method according to any of the preceding claims, wherein the levels of the metabolic markers are determined by mass spectrometry.
7. Method according to any of the preceding claims, wherein the sample is fractionated by liquid chromatography prior to the determination of the level(s) of the metabolic marker(s).
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