WO2012143514A1 - Method for the diagnosis of liver injury based on a metabolomic profile - Google Patents

Abstract

The invention relates to methods for the diagnosis of liver damage. The method relies on the determination of certain metabolic markers in a biological sample of the subject which are up- or down-regulated in injured liver.

Description

METHOD FOR THE DIAGNOSIS OF LIVER INJURY BASED ON A

METABOLOMIC PROFILE

FIELD OF THE INVENTION

The invention relates to the field of diagnostic methods and, more in particular, to a method for the diagnosis of liver injury based on the determination of the levels of a series of metabolic markers which are altered in patients suffering liver injury with respect to control patients.

BACKGROUND OF THE INVENTION

Liver disease is an acute or chronic damage to the liver, usually caused by infection, injury, exposure to drugs or toxic compounds, alcohol, impurities in foods, and the abnormal build-up of normal substances in the blood, an autoimmune process, or by a genetic defect (such as haemochromatosis). Sometimes the exact cause of the injury may not be known. Liver disease can be classified as acute or chronic liver disease based in the duration of the disease. In acute liver disease, such as acute hepatitis and acute liver failure (ALF), the history of the disease does not exceed six months. Liver diseases of longer duration are classified as chronic liver disease.

The common liver diseases include cirrhosis, liver fibrosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatic ischemia reperfusion injury, primary biliary cirrhosis (PBC), and hepatitis, including viral and alcoholic hepatitis. Most common forms of viral hepatitis are hepatitis B and C (HBV and HCV, respectively). Chronic hepatitis may result in cirrhosis. Cirrhosis caused by chronic hepatitis C infection accounts for 8,000-12,000 deaths per year in the United States, and HCV infection is the leading indication for liver transplantation. The death of liver cells through a process known as apoptosis is common in all forms of liver disease. Apoptosis of liver cells is linked to liver fibrosis and other liver disease. Prevention of excessive apoptosis liver cells is an important component in the treatment of acute and chronic liver disease (see, Guicciardi et al. Gut, 2005: 54, 1024-1033 and Ghavami et al, Med. Sci. Monit., 2005: 11(11): RA337-345).

Drug-induced liver injury (DILI) remains a major cause of worldwide mortality (Boelsterli and Lim, Toxicol. Appl. Pharmacol. 2007, 220: 92-107) and represent a serious clinical and financial problem because is the single greatest cause of attrition in drug development and withdrawal of approved drugs from the market (Kaplowitz, Drug Saf, 2001, 24: 483-90). The cost of introducing a new drug to the market (estimated at hundred of millions of dollars) generates a high socio-economical pressure to generate new tools and approaches for the identification of highly sensitive biomarkers of liver toxicity not only for human diagnostic but also for model organisms such as rats that are used in the preclinical studies of the development of new drugs. Such drugs also designated as "potentially liver-damaging drugs" in the present application are to be found in a very wide range of active substance classes with applications for virtually all clinical indications, it being known that the risk is particularly high in the case of some drugs.

Liver disease and, in particular, liver disease caused by drugs, manifests itself clinically in a variety of symptoms which as such are not particular informative. For example, loss of appetite, exhaustion, giddiness, weight loss, nausea, vomiting, fever, pain in the upper right abdominal region, arthralgias, myalgias, itching, rashes and discoloration of excretions may be mentioned. The most striking symptom, which however occurs only in a relatively far advanced state, is yellowing of the eyes and even of the skin, which must then be a reason for an immediate gastroenterological diagnosis since severe and possibly irreversible damage to the liver is to be feared.

The presence of active liver disease is often detected by the existence of elevated enzyme levels in the blood. Specifically, blood levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase), above clinically accepted normal ranges, are known to be indicative of on-going liver damage. Routine monitoring of liver disease patients for blood levels of ALT and AST is used clinically to measure progress of the liver disease while on medical treatment. Reduction of elevated ALT and AST to within the accepted normal range is taken as clinical evidence reflecting a reduction in the severity of the patients on-going liver damage.

Since possible liver damage due to the side effects of drugs should be detected as early as possible in the interest of the patient, of public health and of avoidance of subsequent negative costs to the economy, but the diagnostic means available to date are not suitable or suitable only to an insufficient extent for such an early identification, there is a considerable need for novel high sensitive, reliable and readily determinable diagnostic parameters for the early identification of liver damage which is attributable to the external supply of liver-damaging substances, such as, in particular, of drugs, but also of harmful stimulants and addictive substances (narcotics, stimulants, alcohol) or other substances to which the persons and groups of persons are exposed.

Cleary there is a need for non-invasive, highly sensitive methods as alternatives to existing diagnosis techniques, reducing patient discomfort and hospital- stay costs whilst providing a more robust, standardised assessment. Importantly, a need for these biomarkers in the pharmaceutical industry is also desired in order to contribute to reduce cost and time of the development of new drugs, and remarkably to launch safer drugs to the market.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a method for the diagnosis of liver damage in a subject comprising determining in a biological sample of said subject the levels of the metabolic markers at positions 1 to 6 in table 1 and comparing the levels of said markers with the levels of the same markers in control samples wherein the subject is diagnosed as having liver damage when the levels of the markers at positions 1 to 5 are increased with respect to the level of the same metabolic markers in a reference sample and wherein the level of the marker at position 6 is decreased with respect to the level of the same metabolic markers in a reference sample. In a second aspect, the invention relates to a method for the determination of the efficacy of a therapy for liver damage determining in a biological sample of a subject suffering from liver damage and having been treated with said therapy the levels of the metabolic markers at positions 1 to 6 in table 1 wherein the therapy is considered as effective for the treatment of liver damage when the levels of the metabolic marker or markers at positions 1 to 5 in table 1 are decreased with respect to the level of the same metabolic markers in a reference sample and when the level of the metabolic markers at position 6 in table 1 is increased with respect to the level of the same metabolic markers in a reference sample.

In a third aspect, the invention relates to a method for the identification of compounds causing liver damage comprising determining in a biological sample of a subject having been treated with a test compound the levels of the metabolic markers at positions 1 to 6 as defined in table 1 wherein the test compound is considered as causing liver damage when the levels of the metabolic markers at positions 1 to 5 are increased with respect to the level of the same metabolic markers in reference sample and when the level of the metabolic marker at position 6 is decreased with respect to the level of the same metabolic markers in a reference sample. DETAILED DESCRIPTION OF THE INVENTION

I. Diagnosis of liver damage

The authors of the present invention have taken a significant step to addressing the need for non-invasive methods for the diagnosis of liver damage by performing metabolic profiling of serum samples. The authors of the present invention have identified a series of metabolic markers present in the serum of experimental models suffering from liver damage, which are present at different levels with respect to the serum of control samples. These metabolic markers can then be used in a rapid non-invasive diagnostic method for liver damage. Thus, in a first aspect, the invention relates to a method (hereinafter first method of the invention) for the diagnosis of liver damage in a subject comprising determining in a biological sample of said subject the levels of the metabolic markers at positions 1 to 6 in table 1 and comparing the levels of said markers with the levels of the same markers in control samples wherein the subject is diagnosed as having liver damage when the levels of the markers at positions 1 to 5 are increased with respect to the level of the same metabolic markers in a reference sample and wherein the level of the marker at position 6 is decreased with respect to the level of the same metabolic markers in a reference sample.

Although the invention is preferably carried out by considering the levels of metabolites 1 to 6, the invention also comprises methods for the diagnosis of liver damage based on the determination of the expression level of each individual metabolite and comparing said level to the level in a reference sample. Thus, the invention relates to:

- a method for the diagnosis of liver damage in a subject comprising determining in a biological sample of said subject the levels of the metabolic markers at position 1 in table 1 and comparing the level of said marker with the level of the same markers in control samples wherein the subject is diagnosed as having liver damage when the levels of said marker is increased with respect to the level of the same metabolic markers in a reference sample;

a method for the diagnosis of liver damage in a subject comprising determining in a biological sample of said subject the levels of the metabolic markers at positions 2 in table 1 and comparing the level of said marker with the level of the same marker in a reference control sample wherein the subject is diagnosed as having liver damage when the levels of said marker is increased with respect to the level of the same metabolic markers in said reference sample;

a method for the diagnosis of liver damage in a subject comprising determining in a biological sample of said subject the levels of the metabolic markers at position 3 in table 1 and comparing the level of said marker with the level of the same marker in a reference sample wherein the subject is diagnosed as having liver damage when the level of said marker is increased with respect to the level of the same metabolic marker in said reference sample; a method for the diagnosis of liver damage in a subject comprising determining in a biological sample of said subject the levels of the metabolic markers at position 4 in table 1 and comparing the level of said marker with the level of the same markers in a reference sample wherein the subject is diagnosed as having liver damage when the levels of said marker is increased with respect to the level of the same metabolic markers in said reference sample;

a method for the diagnosis of liver damage in a subject comprising determining in a biological sample of said subject the levels of the metabolic markers at position 5 in table 1 and comparing the level of said marker with the level of the same markers in a reference sample wherein the subject is diagnosed as having liver damage when the levels of said marker is increased with respect to the level of the same metabolic markers in said reference sample;

a method for the diagnosis of liver damage in a subject comprising determining in a biological sample of said subject the levels of the metabolic markers at position 6 in table 1 and comparing the level of said marker with the level of the same marker in a reference sample wherein the subject is diagnosed as having liver damage when the levels of said marker is decreased with respect to the level of the same metabolic markers in said reference sample. The expression "method for diagnosing" as referred to in accordance with the present invention means that the method may essentially consist of the aforementioned steps or may include further steps. However, 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. Diagnosing as used herein refers to assessing the probability according to which a subject is suffering from a disease. As will be understood by those skilled in the art, such an assessment, although preferred to be, may usually not be correct for 100% of the subjects to be diagnosed. The term, however, requires that a statistically significant portion of subjects can be identified as suffering from the disease or as having a predisposition therefore. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test, etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90% at least 95%. The p-values are, preferably, 0.05, 0.025, 0.001 or lower. The term "liver damage", as used herein, is used to denote any type of hepatic trauma (injury), including chronic and acute trauma as well as pathological change present in liver cell or tissue. The clinical conditions of liver damage may include, without being limited thereto, degeneration of live cells, vasculitis of liver, spotty necrosis or focal necrosis present in liver, inflammatory cell infiltration or fibroblast proliferation in liver and portal area, or hepatomegaly, and hepatocirrhosis, hepatoma resulted from severe liver damage, and the like. Liver disease results from an injury to the liver. In one embodiment, injury to the liver is caused by toxins, including alcohol, some drugs, impurities in foods, the abnormal build-up of normal substances in the blood, by infection or by an autoimmune disorder. In some cases, the liver damage resulting from an injury to the liver include, but is not limited to fatty liver, cirrhosis, primary biliary cirrhosis, primary sclerosing cholangitis, and alphal- antitrypsin deficiency. The liver damage includes, but is not limited to cirrhosis, liver fibrosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatic ischemia reperfusion injury, hepatitis, including viral and alcoholic hepatitis and primary biliary cirrhosis (PBC).

The terms "subject", "patient" or "individual"' are used herein interchangeably to refer to any member of the animal kingdom and can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian, including a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. Preferably, the subject is an animal which is typically used as preclinical model for hepatotoxicity or liver diseases such as rat, mouse or dog.

The term "metabolic marker", 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. Metabolic pathways are well known in the art and may vary between species. Preferably, said pathways include at least citric acid cycle, respiratory chain, photosynthesis, photorespiration, glycolysis, gluconeogenesis, hexose monophosphate pathway, oxidative pentose phosphate pathway, production and β-oxidation of fatty acids, urea cycle, amino acid biosynthesis pathways, protein degradation pathways such as proteasomal degradation, amino acid degrading pathways, biosynthesis or degradation of: lipids, polyketides (including e.g. flavonoids and isoflavonoids), isoprenoids (including e.g. terpenes, sterols, steroids, carotenoids, xanthophylls), carbohydrates, phenylpropanoids and derivatives, alcaloids, benzenoids, indoles, indole-sulfur compounds, porphyrines, anthocyans, hormones, vitamins, cofactors such as prosthetic groups or electron carriers, lignin, glucosinolates, purines, pyrimidines, nucleosides, nucleotides and related molecules such as tRNAs, microRNAs (miRNA) or mRNAs. Accordingly, small molecule compound metabolites are preferably composed of the following classes of compounds: alcohols, alkanes, alkenes, alkines, aromatic compounds, ketones, aldehydes, carboxylic acids, esters, amines, imines, amides, cyanides, amino acids, peptides, thiols, thioesters, phosphate esters, sulfate esters, thioethers, sulfoxides, ethers, or combinations or derivatives of the aforementioned compounds. The small molecules among the metabolites may be primary metabolites which are required for normal cellular function, organ function or animal growth, development or health. Moreover, small molecule metabolites further comprise secondary metabolites having essential ecological function, e.g. metabolites which allow an organism to adapt to its environment. Furthermore, metabolites are not limited to said primary and secondary metabolites and further encompass artificial small molecule compounds. Said artificial small molecule compounds are derived from exogenously provided small molecules which are administered or taken up by an organism but are not primary or secondary metabolites as defined above. For instance, artificial small molecule compounds may be metabolic products obtained from drugs by metabolic pathways of the animal. Moreover, metabolites further include peptides, oligopeptides, polypeptides, oligonucleotides and polynucleotides, such as RNA or DNA. More preferably, a metabolite has a molecular weight of 50 Da (Dalton) to 30,000 Da, most preferably less than 30,000 Da, less than 20,000 Da, less than 15,000 Da, less than 10,000 Da, less than 8,000 Da, less than 7,000 Da, less than 6,000 Da, less than 5,000, Da, less than 4,000 Da, less than 3,000 Da, less than 2,000 Da, less than 1,000 Da, less than 500 Da, less than 300 Da, less than 200 Da, less than 100 Da. Preferably, a metabolite has, however, a molecular weight of at least 50 Da. Most preferably, a metabolite in accordance with the present invention has a molecular weight of 50 Da up to 1,500 Da. In preferred embodiments, the metabolic markers that can be used in the context of the present invention are those markers indicated in table 1.

The markers suitable for use in the method of the present invention are those defined at positions 1 to 6 in table 1 and corresponding to: Metabolite 1 :

The metabolite corresponding to the lipid known as PE(16:0/18:2) or l-hexadecanoyl-2- (9Z, 12Z-octadecadienoyl)-sn-glycero-3-phosphoethanolamine, having accession number HMDB08928 in the human metabolome database and having the structure:

Metabolite 2

The metabolite corresponds to a the lipid known as LysoPE(22:6) or 1- docosahexaenoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine having accession number HMDB11526 in the human metabolome database and the structure

Metabolite 3

The metabolite corresponds to a the lipid known as LysoPE(18:2) or l-linoleoyl-2- hydroxy-sn-glycero-3-phosphoethanolamine and having accession number HMDB11507 in the human metabolome database and the structure

Metabolite 4

The metabolite corresponds to a the lipid known as PC(15:0/18:2) or 1-pentadecanoyl- 2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine and having accession number HMDB07940 in the human metabolome database and the structure

Metabolite 5

The metabolite corresponds to a the lipid known as PC(17:0/18:2) or 1-heptadecanoyl- 2-(9Z,12Z-octadecadienoyl)-glycero-3-phosphocholine and having accession number LMGPOIOI 1505 in the LIPIDOMICS GATEWAY database and the structure

Metabolite 6:

The metabolite corresponds to the lipid known as LysoPC(19:0) or 1-nonadecanoyl- glycero-3-phosphocholine, having accession number LMGPO 1050041 in LIPIDOMICS GATEWAY database and having the structure:

In a preferred embodiment, the first method of the invention further comprises determining in said sample the levels of the metabolic markers at positions 7 to 23 in table 1 wherein the subject is diagnosed as having liver damage when the levels of the metabolic marker or markers at positions 7, 10, 17 and 23 in table 1 are increased with respect to the level of the same metabolic markers in a reference sample and when the levels of the metabolic markers at positions 8, 9, 11 to 16 and 18 to 22 in table 1 are decreased with respect to the level of the same metabolic markers in a reference sample.

Metabolite 7:

The metabolite corresponds to the lipid known as PC( 14:0/16:0) or l-tetradecanoyl-2- hexadecanoyl-sn-glycero-3-phosphocholine, having accession number HMDB07869 in the human metabolome database and having the structure:

Metabolite 8:

The metabolite corresponds to the lipid known as LysoPC(18:3) or 1-gamma- linolenoyl-glycero-3-phosphocholine, having accession number HMDB 10387 in the human metabolome database and having the structure:

Metabolite 9:

The metabolite corresponds to the lipid known as PC(20: 1/0:0) or l-(9Z-eicosenoyl) glycero-3-phosphocholine, having accession number LMGPO 1050047 in LIPIDOMICS GATEWAY database and having the structure:

Metabolite 10:

The metabolite corresponds to a the lipid known as PC( 16:0/16:0) or 1 ,2-Dipalmitoyl- sn-3-glycerophosphocholine having accession number HMDB00564 in the human metabolome database and the structure

Metabolite 11

The metabolite corresponds to a the lipid known as PC(0:0/18:0) or 1-stearoyl-glycero- 3-phosphocholine having accession number HMDB10384 in the human metabolome database and the structure

Metabolite 12

The metabolite corresponds to a the lipid known as PC(O-18:0/0:0) or 1-Octadecyl-sn- glycero-3-phosphocholine having accession number HMDB11149 in the human metabolome database and the structure

Metabolite 13

The metabolite corresponds to a the lipid known as PC(17:0/0:0) or 1-heptadecanoyl- glycero-3-phosphocholine having accession number HMDB12108 in the human metabolome database and the structure

Metabolite 14

The metabolite corresponds to a the lipid known as PC(20:4/0:0) or 1-arachidonoyl- glycero-3-phosphocholine having accession number HMDB 10395 in the human metabolome database and the structure

Metabolite 15

The metabolite corresponds to a the lipid known as PC(22: 1/0:0) or 1-erucoyl-glycero- 3-phosphocholine having accession number HMDB 10399 in the human metabolome database and the structure

Metabolite 16

The metabolite corresponds to a the lipid known as PC(24: 1/0:0) or 1-nervonoyl- glycero-3-phosphocholinehaving accession number HMDBl 0406 in the human metabolome database and the structure

Metabolite 17

The metabolite corresponds to a the lipid known as LysoPE(18:0) or l-Octadecanoyl-2- hydroxy-sn-glycero-3-phosphoethanolamine and having accession number HMDBl 1130 in the human metabolome database and the structure

Metabolite 18

The metabolite corresponds to a the lipid known as SM(dl 8: l/15:0) (pentadecanoyl)-sphing-4-enine-l-phosphocholine and having accession

LMSP03010038 in the LIPIDOMICS GATEWAY database and the structure

Metabolite 19

The metabolite corresponds to a the lipid known as SM(dl 8: 1/16:0) or N- (hexadecanoyl)-sphing-4-enine-l-phosphocholine and having accession number LMSP03010003 in the LIPIDOMICS GATEWAY database and the structure

Metabolite 20

The metabolite corresponds to a the lipid known as LysoPC(22:0) or 1-docosanoyl-sn- glycero-3-phosphocholine having accession number HMDBl 0398 in the human metabolome database and the structure

Metabolite 21

The metabolite corresponds to a the lipid known as LysoPC(20:0/0:0)) or 1-eicosanoyl- sn-glycero-3-phosphocholine having accession number HMDB10390 in the human metabolome database and the structure

Metabolite 22

The metabolite corresponds to a the lipid known as PC(P- 18:0/20:4) or 1 -f 17- octaclecenyl )-2-( 5Z,8Z.11Z, 14Z-eicosatetraenoyl)-sn-glycero-3-phosphocholine having accession number HMDB 11253 in the human metabolome database and the structure

Metabolite 23

The metabolite corresponds to a the lipid known as PE(38:6) corresponding to phosphatidic acid with a total of 38 carbons in fatty acyl substituents at snl and sn2 combined and six insaturations.

As used herein, "sample" or "biological sample" means 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, prostate tissue, blood, blood plasma, serum, urine or cerebral spinal fluid (CSF). Preferably, the samples used for the determination of the metabolite profiles are samples which can be obtained using minimally invasive procedures. In a preferred embodiment, the samples are serum samples.

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 then 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 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 or serum sample, serum albumin or other proteins abundant in serum such as apolipoproteins, glycoproteins, inmunoglobulins 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 albumin in order to detect the metabolites or minor proteins. For this purpose, the blood serum 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 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 samples. An example of one other type of affinity chromatography available to pre- fractionate 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 bio molecules from a sample.

The method of the invention includes the step of determining the levels of the metabolic markers in a sample and comparing said levels to the levels of the same markers in a reference sample. The reference sample may be a sample of a subject which does not show symptoms of liver diseases. The control sample may result from the pooling of samples from one individual or a population of two or more healthy individuals. The population, for example, may comprise three, four, five, ten, 15, 20, 30, 40, 50 or more individuals. The levels of the metabolite or metabolites under study in the "reference sample" may be an absolute or relative amount or concentration of the biomarker, a presence or absence of the biomarker, a range of amount or concentration of the biomarker, a minimum and/or maximum amount or concentration of the biomarker, a mean amount or concentration of the biomarker, and/or a median amount or concentration of the biomarker; and, in addition, "reference levels" of combinations of biomarkers may also be ratios of absolute or relative amounts or concentrations of two or more biomarkers with respect to each other. Appropriate positive and negative reference levels of biomarkers for a particular disease state, phenotype, or lack thereof may be determined by measuring levels of desired biomarkers in one or more appropriate subjects, and such reference levels may be tailored to specific populations of subjects (e.g., a reference level may be age-matched so that comparisons may be made between biomarker levels in samples from subjects of a certain age and reference levels for a particular disease state, phenotype, or lack thereof in a certain age group). Such reference levels may also be tailored to specific techniques that are used to measure levels of biomarkers in biological samples (e.g., LC-MS, GC-MS, etc.), where the levels of biomarkers may differ based on the specific technique that is used. In a preferred embodiment, the reference sample is obtained from a healthy subject or from a subject without previous history of NAFLD. A metabolic marker is considered to be increased in a sample from the subject under study when the levels are increased with respect to the reference sample by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%: by at least 85%, by at least 90%, by at least 95%, by at least 100%, by at least 110%, by at least 120%, by at least 130%, by at least 140% by at least 150%, or more. Similarly, the metabolic marker is considered to be decreased when its levels are decreased with respect to a reference sample by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%. by at least 5094, by at least 55%. by at least 60%, by at least 65%, by at least 70%. by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by 100% (i.e., absent).

Moreover, the determination of the metabolites in the methods according to the present invention, comprises, preferably, a step of separation of the metabolites present in the sample prior to the analysis step. Preferably, said compound separation step yields a time resolved separation of the metabolites comprised by the sample. Suitable techniques for separation to be used preferably in accordance with the present invention, therefore, include all chromatographic separation techniques

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") and Capillary Electrophoresis ("CE"). Gas Chromatography, can be used to separate volatile compounds. Liquid chromatography ("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 (vapor 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 compounds. 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.

In a still more preferred embodiment, the biological sample is fractionated by liquid chromatography prior to the determination of the levels of the metabolic marker or markers. In a preferred embodiment, the liquid chromatography is performed on a C8 column at 40 °C. The column may be eluted with a 10 minute linear gradient using a mobile phase at a flow rate of 140 μί/ηιίη, consisting of 100% solvent A (typically 0.05% formic acid) for 1 minute followed by an incremental increase of solvent B (typically acetonitrile containing 0.05% formic acid) up to 50% over a further minute, increasing to 100% B over the next 6 minutes before returning to the initial composition in readiness for the subsequent injection which proceeded a 45 s system re-cycle time. The volume of sample injected onto the column may be of 1 μΐ_^.

Once the sample has been processed, the first method of the invention involves the determination of the levels of the metabolite in the sample. The expression "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, Near-InfraRed spectroscopy (ear-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, MALDI combined with mass spectroscopy, ion spray spectroscopy combined with mass spectroscopy, capillary electrophoresis, NMR and IR detection.

In a preferred embodiment, the determination of the metabolite levels 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 mass spectrometry (GC-MS), liquid chromatography mass spectrometry (LC-MS), direct infusion mass spectrometry or Fourier transform ion-cyclotrone-resonance mass spectrometry (FT-ICR-MS), capillary electrophoresis mass spectrometry (CE-MS), high-performance liquid chromatography coupled mass spectrometry (HPLC-MS), quadrupole mass spectrometry, any sequentially coupled mass spectrometry, such as MS-MS or MS-MS-MS, inductively coupled plasma mass spectrometry (ICP-MS), pyrolysis mass spectrometry (Py-MS), ion mobility mass spectrometry or time of flight mass spectrometry (TOF), of electrospray ionization mass spectrometry (ESI-MS), ESI- MSMS, ESI-MS/(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- MSIMS, APCI-(MS)ⁿ, atmospheric pressure photoionization mass spectrometry (APPI- MS), APPI-MSIMS, and APPI-(MS)ⁿ, quadrupole mass spectrometry, fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry, where n is an integer greater than zero. Most preferably, LC-MS 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 disclosure content of which is hereby incorporated by reference.

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 result from the addition of a proton or a hydrogen nucleus, [M+H]⁺ where M signifies the molecule of interest, and H signifies the hydrogen ion, 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]⁺ 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, 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 the mass spectrometer (for example, a LCT Premier™, Waters Corp., Milford, USA) by electrospray ionisation, 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. The nebulization gas may be set to 500 L/h at a temperature of 200 ° C. The cone gas may be set to 50 L/h and the source temperature to 120 ° C. Centroid data may be acquired from m/z 50-1000 using an accumulation time of 0.2 s per spectrum. The spectra may be mass corrected in real time by reference to leucine enkephalin, infused at 50 μί/ηιίη through an independent reference electrospray, sampled every 10 s. An appropriate test mixture of standard compounds may be analysed before and after the entire set of randomized, duplicated sample injections in order to examine the retention time stability, mass accuracy and sensitivity of the system throughout the course of the run which lasted a maximum of 3 6 h per batch of samples injected.

In a still more preferred embodiment, the biological sample is fractionated by liquid chromatography prior to the determination of the levels of the metabolic marker or markers using the methods defined above.

II. Method for the determination of the efficacy of a therapy for liver damage The invention also provides a method for the determination of the efficacy of the therapy for liver damage. Thus, in another aspect, the invention relates to a method (hereinafter "the second method of the invention") for the determination of the efficacy of a therapy for liver damage determining in a biological sample of a subject suffering from liver damage and having been treated with said therapy the levels of the metabolic markers at positions 1 to 6 in table 1 wherein the therapy is considered as effective for the treatment of liver damage when the levels of the metabolic marker or markers at positions 1 to 5 in table 1 are decreased with respect to the level of the same metabolic markers in a reference sample and when the level of the metabolic markers at position 6 in table 1 is increased with respect to the level of the same metabolic markers in a reference sample.

The different aspects of the second method of the invention (the methods used for the determination of the levels of the markers, the nature of the sample which is to be studied, the thresholds for consideration of a marker as having been increased or decreased) are essentially as defined previously in respect of the first method of the invention. In a preferred embodiment, the second method of the invention further comprises determining in said biological sample the levels of the metabolic markers at positions 7 to 23 in table 1 wherein the therapy is considered as effective for the treatment of liver damage when the levels of the metabolic marker or markers at positions 7, 10, 17 and 23 in table 1 are decreased with respect to the level of the same metabolic markers in a reference sample and when the levels of the metabolic markers at positions 8, 9, 11 to 16 and 18 to 22 in table 1 are increased with respect to the level of the same metabolic markers in a reference sample.

The term "reference sample", as used in respect of the second method of the invention for the determination of the efficacy of a therapy for liver damage, relates to either a sample derived from the patient wherein the efficacy of the therapy is being tested but obtained from the patient suffering liver damage prior to the administration of the therapy. In another embodiment, the reference sample is a sample from a patient suffering from liver damage which has either been left untreated or which has been treated with a control therapy, preferably, the same excipient, carrier or vehicle which is used in the therapy whose efficacy for the treatment of liver damage is to be assessed.

The term "therapy" as used herein, encompasses the treatment of existing liver damage as well as preventative treatment (i.e., prophylaxis). Therapy includes, but is not limited to, administering an agent for treating liver damage, treating associated metabolic conditions such as diabetes and hyperlipidemia, improving insulin resistance, following a balanced and healthy diet, avoiding alcohol, and avoiding unnecessary medications. Tests than can be used to determine whether there exists liver damage include, without limitation, the following;

(i) Assays to determine the levels of serum enzymes such as lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT), where an increase in enzyme levels indicates liver disease.

(ii) Assays to determine serum bilirubin levels. Serum bilirubin levels are reported as total bilirubin and direct bilirubin. Normal values of total serum bilirubin are 0.1 - 1.0 mg.dl (e.g., about 2 - 18 mmol/L). Normal values of direct bilirubin are

0.0 - 0.2 mg/dl (0 - 4 mmol/L). Increases in serum bilirubin are indicative of liver disease.

(iii) Assays to determine serum protein levels, for example, albumin and the globulins (e.g., alpha, beta, gamma). Normal values for total serum proteins are 6.0- 8.0 g/dl (60-80 g/L). A decrease in serum albumin is indicative of liver disease. An increase in globulin is indicative of liver disease.

Other tests include prothrombin time, international normalized ratio, activated clotting time (ACT), partial thromboplastin time (PTT), prothrombin consumption time (PCT), fibrinogen, coagulation factors; mean corpuscular volume (MCV), platelet count, alpha- fetoprotein, and alpha- fetoprotein- L3 (percent).

III. Method for the identification of compounds causing liver damage The authors of the present invention have also developed a method for the identification of a compound causing liver damage. Thus, in another aspect, the invention relates to a method (hereinafter "the third method of the invention") for the identification of compounds causing liver damage comprising determining in a biological sample of a subject having been treated with a test compound the levels of the metabolic markers at positions 1 to 6 as defined in table 1 wherein the test compound is considered as causing liver damage when the levels of the metabolic markers at positions 1 to 5 are increased with respect to the level of the same metabolic markers in reference sample and when the level of the metabolic marker at position 6 is decreased with respect to the level of the same metabolic markers in a reference sample.

The different aspects of the third method of the invention (the methods used for the determination of the levels of the markers, the nature of the sample which is to be studied, the thresholds for consideration of a marker as having been increased or decreased) are essentially as defined previously in respect of the first method of the invention. In a preferred embodiment, the third method of the invention further comprises determining in said sample the levels of the metabolic markers at positions 7 to 23 in table 1 wherein the test compound is considered as causing liver damage when the levels of the metabolic markers at positions 7, 10, 17 and 23 in table 1 are increased with respect to the level of the same metabolic markers in reference sample and when the levels of the metabolic markers at positions 8, 9, 11 to 16 and 18 to 22 in table 1 are decreased with respect to the level of the same metabolic markers in a reference sample.

The term "reference sample", as used in respect of the third method of the invention, relates to either a sample derived from the patient before being contacted with the test compound. In another embodiment, the reference sample is a sample from a patient not suffering from liver damage.

Examples of suitable animals for use in the screening method of the invention include, but are not limited to, mice, rats, rabbits, monkeys, guinea pigs, dogs and cats. In accordance with this embodiment, the test compound or a control compound is administered (e.g., orally, rectally or parenterally such as intraperitoneally or intravenously) to a suitable animal and the effect on the levels of one or more of the metabolites shown in tables 2 or 3 is determined. Examples of agents include, but are not limited to, nucleic acids (e.g., DNA and R A), carbohydrates, lipids, proteins, peptides, peptidomimetics, small molecules and other drugs. Agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art. Test compounds further include, for example, antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab') 2, Fab expression library fragments, and epitope-binding fragments of antibodies). Further, agents or libraries of compounds may be presented, for example, in solution, on beads, chips, bacteria, spores, plasmids or phage. If the compound is a low-molecular weight compound, then this can be generated by various methods known to the art, preferably synthetically, in particular by combinatorial chemistry, or by biochemical methods, in particular by recombinant expression or purification from biological probes. The compound is of low molecular weight ("small molecules") or the library is composed of molecules with low molecular weight ("small molecule library"). A "small molecule" is defined as a complex collection of compounds, which are produced in a non-biological way, that means which are not produced by recombinant expression, like for instance most protein or peptide libraries. "Small molecules" can be generated by various methods known to the art, but are preferably produced by synthetically, more preferably by combinatory chemistry, to generate a compound library with a maximum chemical diversity within the constraints of predicted attractive drug characteristics. If the compound to be assayed for its suitability for the treatment of liver injury is a peptide or a peptide library, then these can be generated by various methods known to the art for their use as candidate compounds, but they are preferably produced by biochemical methods, more preferably by recombinant expression in prokaryotic or eukaryotic cells.

The compound to be tested for its ability for causing liver damage can be formulated with a pharmaceutically acceptable carrier to produce a pharmaceutical composition, which can be administered to a human or other animal. A pharmaceutically-acceptable carrier can be, for example, water, sodium phosphate buffer, phosphate-buffered saline, normal saline or Ringer's solution or other physiologically-buffered saline, or other solvent or vehicle such as a glycol, glycerol, an oil such as olive oil or an injectable organic ester. A pharmaceutically acceptable carrier can also contain physiologically acceptable compounds that act, for example, to stabilize or increase the absorption of the modulatory compound. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the composition. The invention is described herein by way of the following examples which are to be construed as merely illustrative and not limitative of the invention. EXAMPLES Method

A global metabolite profiling UPLC®-MS methodology was employed where all endogenous metabolite related features, characterized by their mass-to-charge ratio m/z and retention time Rt, are included in a subsequent multivariate analysis procedure used to study metabolic differences between the different groups of samples. Where possible, Rt-m/z features corresponding to putative biomarkers were identified. The analytical methodology was designed to provide maximum coverage over classes of compounds involved in key hepatic metabolic pathways, such as major phospholipids, fatty acids, and organic acids, whilst offering relatively high-throughput with minimal injection-to- injection carryover effects.

Sample Details. A group of rats were intraperitoneally injected with galactosamine Gal (1 g / kg / 5 ml) whilst a separate group received a similar volume of saline vehicle solution. Serum samples were taken from each animal just before treatment and 24 hours later. Liver cell apoptosis was quantify in liver sections of each animal by histology on completion of the treatment time period. Sample Preparation. Proteins were precipitated from the defrosted serum samples (50 μί) by adding four volumes of methanol in 1.5 mL microtubes at room temperature. After brief vortex mixing the samples were incubated overnight at -20 °C. Supernatants were collected after centrifugation at 13,000 rpm for 10 minutes, and transferred to vials for UPLC®-MS analysis.

Chromatography. Chromatography was performed on a 1 mm i.d. x 100 mm ACQUITY 1.7 μιη C8 BEH column (Waters Corp., Milford, USA) using an ACQUITY UPLC® system (Waters Corp., Milford, USA). The column was maintained at 40 °C and eluted with a 10 minute linear gradient. The mobile phase, at a flow rate of 140 μΕ/ηιίη, consisted of 100% solvent A (0.05% formic acid) for 1 minute followed by an incremental increase of solvent B (acetonitrile containing 0.05% formic acid) up to 50% over a further minute, increasing to 100% B over the next 6 minutes before returning to the initial composition in readiness for the subsequent injection which proceeded a 45 s system re-cycle time. The volume of sample injected onto the column was 1 μί.

Mass spectrometry. The eluent was introduced into the mass spectrometer (LCT PremierTM, Waters Corp., Milford, USA) by electrospray ionisation, 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. The nebulisation gas was set to 600 L/h at a temperature of 350 °C. The cone gas was set to 50 L/h and the source temperature set to 150 °C. Centroid data were acquired from m/z 50-1000 using an accumulation time of 0.2 s per spectrum. All spectra were mass corrected in real time by reference to leucine enkephalin, infused at 50 μΐνιηίη through an independent reference electrospray, sampled every 10 s. A test mixture of standard compounds (Acetaminophen, Sulfaguanidine, Sulfadimethoxine, Val-Tyr-Val, Terfenadine, Leucine-Enkephaline, Reserpine and Erythromicyn - all 5nM in water) was analyzed before and after the entire set of randomized, duplicated sample injections in order to examine the retention time stability (generally < 6 s variation, injection-to-injection), mass accuracy (generally < 3 ppm for m/z 400-1000, and < 1.2 mDa for m/z 50-400) and sensitivity of the system throughout the course of the run which lasted a maximum of 28 h per batch of samples injected. For each injection batch, the overall quality of the analysis procedure was monitored using ten repeat extracts of a pooled serum sample.

Corresponding root mean square coefficients of variation were 15.3% over all variables detected in the positive ion mode, and 11.7% in the negative ion mode. Online tandem mass spectrometry (MS/MS) experiments for metabolite identification were performed on a Waters QTOF PremierTM (Waters Corp., Milford, USA) instrument operating in both the 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 for m/z 400-1000, and < 1.2 mDa for m/z 50-400) MS/MS or pseudo MS/MS/MS spectra corrected in real time by reference to leucine enkephalin, infused at 50 μΙ7ιηίη through an independent reference electrospray, sampled every 10 s. Centroid data were acquired between m/z 50-1000 using an accumulation time of 0.2 s per spectrum.

Data processing. All data were processed using the MarkerLynx application manager for MassLynx 4.1 software (Waters Corp., Milford, USA). The LC/MS data are peak detected and noise-reduced in both the LC and MS domains such that only true analytical peaks are further processed by the software (e.g. noise spikes are rejected). A list of intensities (chromatographic peak areas) of the peaks detected is then generated for the first chromatogram, using the Rt-m/z data pairs as identifiers. This process is repeated for each LC-MS analysis and the data sorted such that the correct peak intensity data for each Rt-m/z pair are aligned. Detected variables which were found to be zero in both injections of any one serum extract corresponding to an untreated animal were then removed from the dataset. Although this process would be expected to remove variables associated with xenobiotic metabolites, the influence of these species still has a strong impact on the analysis by way of ion suppression effects. In order to remove this interference, all variables co-eluting with xenobiotic metabolites, found in the retention time region 2.75-3.55 minutes, were eliminated. In the case of the positive ion mode, the resulting variable ion intensities were then normalized within each injection, to the sum of peak intensities in that injection. In the negative ion mode the normalization factor for each injection was calculated from the ratio of the median intensity to the median intensity of a reference serum injection, as obtained from all variables contained in the retention time bin 6-8 minutes (Wang, W. et al., Anal. Chem 2003, 75 :4818-4826). There was no significant correlation (F < Fcrit) between the normalization factors used and the sample groups being compared in the study in either ion mode. All zeros contained within the normalized dataset were substituted with missing values to form a single matrix with Rt-m/z pairs for each injection. Finally, the Rt-m/z pairs were associated with metabolite identifiers: ID (compound identification), ION (ion detected) and MSMS (level of metabolite identification confidence). The final dataset was mean centered and pareto scaled during multivariate data analysis.

Metabolite Biomarker Identification. Exact molecular mass data from redundant m/z peaks corresponding to the formation of different parent (e.g. cations in the positive ion mode, anions in the negative ion mode, adducts, multiple charges) and product (formed by spontaneous "in-source" CID) ions were first used to help confirm the metabolite molecular mass. This information was then submitted for database searching, either inhouse or using the online ChemSpider database where the Kegg, Human Metabolome Database and Lipid Maps data source options were selected. MS/MS data analysis highlights neutral losses or product ions, which are characteristic of metabolite groups and can serve to discriminate between database hits.

Table 1. Serum metabolomic signature for liver apoptosis. List of more significant serum metabolites correlating with in situ liver apoptosis.

Spearman Fold

RT M/Z ID OTHER IDs

Coef.^a change^b

1 7.6088 716 .5239 PE(16:0/18:2) HMDB08928 0. .79 13.9

2 3.97 526. .2932 LysoPE(22:6) HMDB1 1526 0. .75 6.7

3 3.9317 478 .2927 LysoPE(18:2) HMDB1 1507 0. .79 5.77

4 7.3798 744 .5561 PC(15:0/18:2) HMDB07940 0. .70 2.4

5 7.77 772 .5880 PC(17:0/18:2) LMGP01011505 0. .67 2.8

6 4.961 1 538 .3838 LysoPC(19:0) LMGP01050041 -0. .88 0.3

7 7.3883 706 .5384 PC(14:0/16:0) HMDB07869 0. .71 2.0

8 3.98 518. .3219 LysoPC(18:3) HMDB10387 -0. .85 0.3

9 4.81 550. .3853 PC(20: 1/0:0) LMGP01050047 -0. .96 0.2

10 7.80 734. .5724 PC(16:0/16:0) HMDB00564 0. .79 1 .9

1 1 4.54 524. .3718 PC(0:0/18:0) HMDB10384 -0. .95 0.2

12 4.80 510. .3917 PC(0-18:0/0:0) HMDB1 1 149 -0. .96 0.3

13 4.3676 510 .3551 PC(17:0/0:0) HMDB12108 -0. .90 0.3

14 3.99 544. .3407 PC(20:4/0:0) HMDB10395 -0. .86 0.6

15 5.38 578. .4156 PC(22: 1/0:0) HMDB10399 -0. .92 0.4

16 5.96 606. .4473 PC(24: 1/0:0) HMDB10406 -0. .96 0.4

17 4.6361 482 .3237 LysoPE(18:0) HMDB1 1 130 0. .75 2.4

18 6.7493 689 .5582 SM (d18:1/15:0) LMSP03010001 -0. .93 0.5

19 6.9934 703 .5782 SM (d18:1/16:0) LMSP03010003 -0. .92 0.5

20 5.8642 580 .4316 LysoPC(22:0) HMDB10398 -0. .92 0.4

21 5.2609 552 .3993 LysoPC(20:0/0:0 HMDB10390 -0. .92 0.4 22 7.7899 794.6074 PC(P-18:0/20:4) HMDB1 1253 -0.96 0.6

23 7.46 764.5215 PE (38:6) 0.80 8 ^aCorrelation: negative value indicates that higher liver injury results in less metabolite in serum, positive value means that higher degree of liver injury results in higher levels of the metabolite.

bMedian Fold change relative to normal conditions.

Claims

1. A method for the diagnosis of liver damage in a subject comprising determining in a biological sample of said subject the levels of the metabolic markers at positions 1 to 6 in table 1 and comparing the levels of said markers with the levels of the same markers in control samples wherein the subject is diagnosed as having liver damage when the levels of the markers at positions 1 to 5 are increased with respect to the level of the same metabolic markers in a reference sample and wherein the level of the marker at position 6 is decreased with respect to the level of the same metabolic markers in a reference sample.

2. A method as defined in claim 1 further comprising determining in said sample the levels of the metabolic markers at positions 7 to 23 in table 1 wherein the subject is diagnosed as having liver damage when the levels of the metabolic marker or markers at positions 7, 10, 17 and 23 in table 1 are increased with respect to the level of the same metabolic markers in a reference sample and when the levels of the metabolic markers at positions 8, 9, 11 to 16 and 18 to 22 in table 1 are decreased with respect to the level of the same metabolic markers in a reference sample.

3. A method for the determination of the efficacy of a therapy for liver damage comprising determining in a biological sample of a subject suffering from liver damage and having been treated with said therapy the levels of the metabolic markers at positions 1 to 6 in table 1 wherein the therapy is considered as effective for the treatment of liver damage when the levels of the metabolic marker or markers at positions 1 to 5 in table 1 are decreased with respect to the level of the same metabolic markers in a reference sample and when the level of the metabolic markers at position 6 in table 1 is increased with respect to the level of the same metabolic markers in a reference sample.

4. A method according to claim 3 further comprising determining in said biological sample the levels of the metabolic markers at positions 7 to 23 in table 1 wherein the therapy is considered as effective for the treatment of liver damage when the levels of the metabolic marker or markers at positions 7, 10, 17 and 23 in table 1 are decreased with respect to the level of the same metabolic markers in a reference sample and when the levels of the metabolic markers at positions 8, 9, 11 to 16 and 18 to 22 in table 1 are increased with respect to the level of the same metabolic markers in a reference sample.

5. A method as defined in claim 4 wherein the reference sample is selected from the group of

a. a sample of the patient before being contacted with the therapy and b. a sample from a patient suffering from liver damage either left untreated or having been treated with a control therapy.

A method for the identification of compounds causing liver damage comprising determining in a biological sample of a subject having been treated with a test compound the levels of the metabolic markers at positions 1 to 6 as defined in table 1 wherein the test compound is considered as causing liver damage when the levels of the metabolic markers at positions 1 to 5 are increased with respect to the level of the same metabolic markers in reference sample and when the level of the metabolic marker at position 6 is decreased with respect to the level of the same metabolic markers in a reference sample.

7. A method according to claim 6 further comprising determining in said sample the levels of the metabolic markers at positions 7 to 23 in table 1 wherein the test compound is considered as causing liver damage when the levels of the metabolic markers at positions 7, 10, 17 and 23 in table 1 are increased with respect to the level of the same metabolic markers in reference sample and when the levels of the metabolic markers at positions 8, 9, 11 to 16 and 18 to 22 in table 1 are decreased with respect to the level of the same metabolic markers in a reference sample.

8. A method as defined in claims 6 or 7 wherein the reference sample is selected from the group of

(i) a sample of the subject before being contacted with the test compound and

> (ii) a sample from a subject not suffering liver damage.

A method as defined in any of claims 1 to 8 wherein the biological sample is blood, plasma or serum.

A method as defined in any of claims 1 to 9 wherein the determination of the level of the one or more biological markers is carried out by mass spectrometry (MS).

11. A method as defined in claim 1 to 10 wherein the biological sample is fractionated by liquid chromatography prior to the determination of the levels of the metabolic markers.