WO2012143514A1 - Procédé de diagnostic d'une atteinte hépatique basé sur un profil métabolomique - Google Patents

Procédé de diagnostic d'une atteinte hépatique basé sur un profil métabolomique Download PDF

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WO2012143514A1
WO2012143514A1 PCT/EP2012/057275 EP2012057275W WO2012143514A1 WO 2012143514 A1 WO2012143514 A1 WO 2012143514A1 EP 2012057275 W EP2012057275 W EP 2012057275W WO 2012143514 A1 WO2012143514 A1 WO 2012143514A1
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levels
markers
sample
level
metabolic markers
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PCT/EP2012/057275
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Juan Manuel FALCÓN PÉREZ
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Asociación Centro De Investigación Cooperativa En Biociencias-Cic Biogune
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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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5038—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving detection of metabolites per se
    • 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
    • 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 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.
  • 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.
  • acute liver disease such as acute hepatitis and acute liver failure (ALF)
  • ALF acute liver failure
  • 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.
  • liver cells 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 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.
  • symptoms 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.
  • liver disease is often detected by the existence of elevated enzyme levels in the blood.
  • 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.
  • 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.
  • harmful stimulants and addictive substances narcotics, stimulants, alcohol
  • the invention in a first aspect, 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.
  • the invention in a second aspect, 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.
  • the invention in a third aspect, 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.
  • 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.
  • 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.
  • 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 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • subject is 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.
  • the subject is an animal which is typically used as preclinical model for hepatotoxicity or liver diseases such as rat, mouse or dog.
  • metabolic 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. 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 ⁇ -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 include 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.
  • isoprenoids including e.g. terpenes, sterols, steroids, carotenoids, xanthophylls
  • carbohydrates phenylpropanoids and derivatives
  • alcaloids benzenoids
  • indoles indole-sulfur compounds
  • porphyrines porphyrines
  • 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.
  • small molecule metabolites further comprise secondary metabolites having essential ecological function, e.g. metabolites which allow an organism to adapt to its environment.
  • 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.
  • artificial small molecule compounds may be metabolic products obtained from drugs by metabolic pathways of the animal.
  • metabolites further include peptides, oligopeptides, polypeptides, oligonucleotides and polynucleotides, such as RNA or DNA.
  • 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.
  • a metabolite has, however, a molecular weight of at least 50 Da.
  • a metabolite in accordance with the present invention has a molecular weight of 50 Da up to 1,500 Da.
  • the metabolic markers that can be used in the context of the present invention are those markers indicated in table 1.
  • 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 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
  • 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
  • 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
  • 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
  • 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:
  • 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.
  • 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:
  • 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:
  • 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:
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • the metabolite corresponds to a the lipid known as SM(dl 8: l/15:0) (pentadecanoyl)-sphing-4-enine-l-phosphocholine and having accession
  • 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
  • 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
  • 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
  • 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
  • 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.
  • 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).
  • CSF cerebral spinal fluid
  • the samples used for the determination of the metabolite profiles are samples which can be obtained using minimally invasive procedures.
  • the samples are serum samples.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 sample can be fractionated by passing the sample over a lectin chromatography column (which has a high affinity for sugars).
  • 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.
  • 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 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.
  • 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.
  • 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).
  • 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.
  • 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
  • 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.
  • LC Liquid Chromatography
  • IC Ion Chromatography
  • SEC Size-Exclusion Chromatography
  • SFC Supercritical-Fluid Chromatography
  • TLC Thin-Layer Chromatography
  • HPLC High Performance Liquid Chromatography
  • CE Capillary Electrophoresis
  • GC cyclopentadiol
  • LC cyclopentadiol
  • Suitable types of liquid chromatography 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 biological sample is fractionated by liquid chromatography prior to the determination of the levels of the metabolic marker or markers.
  • 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 ⁇ / ⁇ , 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 ⁇ ⁇ .
  • 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.
  • RI refractive index spectroscopy
  • UV Ultra- Violet spectroscopy
  • fluorescent analysis radiochemical analysis
  • ear-IR Near-InfraRed spectroscopy
  • NMR Nuclear Magnetic Resonance spectroscopy
  • LS
  • the determination of the metabolite levels 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 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
  • 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.
  • surrogate marker means a biological or clinical parameter that is measured in place of the biologically definitive or clinically most meaningful parameter.
  • 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.
  • M signifies the molecule of interest
  • 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.
  • 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.
  • 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 the mass spectrometer (for example, a 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 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 ⁇ / ⁇ 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.
  • 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.
  • the invention also provides a method for the determination of the efficacy of the therapy for liver damage.
  • 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 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.
  • 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.
  • 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.
  • liver damage 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;
  • 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.
  • LDH lactate dehydrogenase
  • ALP alkaline phosphatase
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • 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
  • 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.
  • 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).
  • ACT activated clotting time
  • PTT partial thromboplastin time
  • PCT prothrombin consumption time
  • fibrinogen coagulation factors
  • MCV mean corpuscular volume
  • platelet count alpha- fetoprotein
  • alpha- fetoprotein- L3 percent
  • 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 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.
  • 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.
  • the reference sample is a sample from a patient not suffering from liver damage.
  • 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.
  • 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.
  • 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).
  • 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.
  • the compound to be assayed for its suitability for the treatment of liver injury is a peptide or a peptide library
  • 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.
  • 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 ⁇ ) 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 ⁇ 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 ⁇ / ⁇ , 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 ⁇ .
  • 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.
  • 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 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 ⁇ 7 ⁇ 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.
  • 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.
  • parent e.g. cations in the positive ion mode, anions in the negative ion mode, adducts, multiple charges
  • product formed by spontaneous "in-source” CID
  • Serum metabolomic signature for liver apoptosis List of more significant serum metabolites correlating with in situ liver apoptosis.

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Abstract

La présente invention concerne des procédés de diagnostic d'atteinte hépatique. Le procédé repose sur la détermination de certains marqueurs métaboliques dans un échantillon biologique du sujet qui sont régulés à la hausse ou à la baisse dans le foie atteint.
PCT/EP2012/057275 2011-04-20 2012-04-20 Procédé de diagnostic d'une atteinte hépatique basé sur un profil métabolomique WO2012143514A1 (fr)

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EP3267199A1 (fr) 2016-07-06 2018-01-10 One Way Liver S.L. Procédés de diagnostic basés sur des profils lipidiques
WO2018007511A1 (fr) 2016-07-06 2018-01-11 One Way Liver,S.L. Méthodes diagnostiques basées sur des profils lipidiques
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WO2018112578A1 (fr) * 2016-12-21 2018-06-28 Universidade Estadual De Campinas- Unicamp Méthode d'identification de biomarqueurs pour cholestase néonatale, trousse d'identification des biomarqueurs et utilisation
CN109096326A (zh) * 2018-10-09 2018-12-28 江苏东南纳米材料有限公司 一种高纯溶血磷脂酰胆碱及其制备方法
CN109096326B (zh) * 2018-10-09 2021-04-30 江苏东南纳米材料有限公司 一种高纯溶血磷脂酰胆碱及其制备方法
CN110057955A (zh) * 2019-04-30 2019-07-26 中国医学科学院病原生物学研究所 乙型肝炎特异性血清标志物的筛选方法
CN110057955B (zh) * 2019-04-30 2021-06-11 中国医学科学院病原生物学研究所 乙型肝炎特异性血清标志物的筛选方法
CN112083110A (zh) * 2019-06-12 2020-12-15 中国人民解放军总医院第五医学中心 一种何首乌导致药物肝损伤标志物的筛选和应用
CN112083111A (zh) * 2019-06-12 2020-12-15 中国人民解放军总医院第五医学中心 一种慢性药物性肝损伤相关肝硬化非侵入性诊断标志物及其应用

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