WO2014013051A1

WO2014013051A1 - Method of predicting or diagnosing an hepatic encephalopathy in patients with cirrhosis treated with transjugular intra-hepatic porto-systemic shunt (tips)

Info

Publication number: WO2014013051A1
Application number: PCT/EP2013/065277
Authority: WO
Inventors: Christophe Bureau; Frédéric LOPEZ
Original assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm); Universite Paul Sabatier Toulouse Iii
Priority date: 2012-07-19
Filing date: 2013-07-19
Publication date: 2014-01-23

Abstract

The invention relates to a method of predicting or diagnosing an hepatic encephalopathy in patients with cirrhosis treated with transjugular intra-hepatic porto-systemic shunt (TIPS) and to kits for performing these methods.

Description

METHOD OF PREDICTING OR DIAGNOSING AN HEPATIC ENCEPHALOPATHY IN PATIENTS WITH CIRRHOSIS TREATED WITH TRANSJUGULAR INTRA-HEPATIC

PORTO-SYSTEMIC SHUNT (TIPS) Field of the invention

The invention relates to a method of predicting or diagnosing an hepatic encephalopathy in patients with cirrhosis treated with transjugular intra-hepatic portosystemic shunt (TIPS). The invention also relates to kits for performing these methods. Background to the invention

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome resulting from acute or chronic liver failure. Minimal and overt hepatic encephalopathy (MHE and OHE) are constituents of the spectrum of neurocognitive impairment in cirrhosis. MHE is defined by impaired performance on psychometric or neurophysiologic testing (PHES Score) (Ferenci P et al., Hepatic encephalopathy-definition, nomenclature, diagnosis, and quantification: final report of the working party at the 11 th World Congresses of Gastroenterology, Vienna, 1998. Hepatology 2002, 35(3)716-721 ; Bajaj JS et al., Review article: the design of clinical trials in hepatic encephalopathy-an International Society for Hepatic Encephalopathy and Nitrogen Metabolism (ISHEN) consensus statement. Aliment Pharmacol Ther 2011 , 33(7):739-747; Schomerus H et al., Neuropsychological aspects of portal-systemic encephalopathy. Metab Brain Dis 1998, 13(4):361 -377; Weissenborn K et al., Neuropsychological characterization of hepatic encephalopathy. J Hepatol 2001 , 34(5):768-773.) while it is not perceived by the physician. There is no biological marker of this status which is known to be associated with a poor prognosis. Actually, MHE is associated with poor quality of life, an increased risk of driving accidents and with an increased risk of falls.

Furthermore, patients with MHE are at increased risk of developing OHE characterized by a progression of symptoms from deficits in attentiveness that may progress to lethargy, asterixis, disorientation, stupor and coma with a risk of persistence of cognitive impairment after resolution of OHE (Hartmann et a/.,The prognostic significance of subclinical hepatic encephalopathy. Am J Gastroenterol 2000, 95(8):2029-2034; Romero-Gomez et al., Subclinical hepatic encephalopathy predicts the development of overt hepatic encephalopathy. Am J Gastroenterol 2001 , 96(9):2718-2723).

OHE is closely related to severity of liver function and fibrosis and is considered as a criterion in assessing severity of cirrhosis (Child Pugh score). OHE occurs in 30%-45% of patients with cirrhosis and 10%-50% of patients after transjugular intra-hepatic porto- systemic shunt (TIPS) (Sanyal et at., Portosystemic encephalopathy after transjugular intrahepatic portosystemic shunt: results of a prospective controlled study. Hepatology 1994, 20(1 Pt 1 ):46-55; Jalan et at., Analysis of prognostic variables in the prediction of mortality, shunt failure, variceal rebleeding and encephalopathy following the transjugular intrahepatic portosystemic stent-shunt for variceal haemorrhage. J Hepatol 1995, 23(2):123-128; Bai et at., Predictors of hepatic encephalopathy after transjugular intrahepatic portosystemic shunt in cirrhotic patients: a systematic review. J Gastroenterol Hepatol 2011 ).

Since its first introduction, TIPS has been increasingly used as a means of reducing portal pressure in patients with complications related to portal hypertension such as refractory variceal bleeding or refractory ascites (Peron et a/.,The role of TIPS in the treatment of refractory ascites. Gastroenterol Clin Biol 2007, 31 (3):341 -346; de Franchis R: Evolving consensus in portal hypertension. Report of the Baveno IV consensus workshop on methodology of diagnosis and therapy in portal hypertension. J Hepatol 2005, 43(1 ):167-176; Papatheodoridis et at., Transjugular intrahepatic portosystemic shunt compared with endoscopic treatment for prevention of variceal rebleeding: A metaanalysis. Hepatology 1999, 30(3):612-622; Salerno et at., Transjugular intrahepatic portosystemic shunt for refractory ascites: a meta-analysis of individual patient data. Gastroenterology 2007, 133(3):825-834).

Created now with covered stents under radiologic guidance (Garcia-Pagan et at.,

Improved clinical outcome using polytetrafluoroethylene-coated stents for TIPS: results of a randomized study. Gastroenterology 200 , 126(2):469-475; Pagan JC et at., Patency of stents covered with polytetrafluoroethylene in patients treated by transjugular intrahepatic portosystemic shunts: long-term results of a randomized multicentre study. Liver Int 2007, 27(6):742-747), TIPS intrinsically functions as a side-to-side porta-cava shunt.

The main drawback of the TIPS procedure is progressive hepatic encephalopathy caused partly by direct portosystemic shunting. The pathogenesis of HE in general but also in patients treated by TIPS is still not well understood and new prognostic indicators are clearly warranted. The current standard of care for patients with HE are based on treatment with nonabsorbable dissacharrides to decrease the absorption of ammonia (Sharma et at., Secondary prophylaxis of hepatic encephalopathy: an open-label randomized controlled trial of lactulose versus placebo. Gastroenterology. 2009 Sep;137(3):885-91 , 891 ) or antibiotics to reduce ammonia-producing enteric bacteria (Bass NM et at., Rifaximin treatment in hepatic encephalopathy. N Engl J Med. 2010 Mar 25;362(12):1071 -81 ) in patients not treated by TIPS. Few data are available in patients treated by TIPS and these therapeutics seem to be less effective in this group of patients. Therefore, there is a real challenge in discovering new molecular mechanisms involved in pathogenesis of HE as well as new molecular markers of HE to predict major risks of HE in patients treated by TIPS.

Description of the invention

The invention provides a method of predicting or diagnosing an hepatic encephalopathy in a patient with cirrhosis treated with transjugular intra-hepatic portosystemic shunt (TIPS), said method comprising:

a) measuring the level of expression of at least one protein selected from the group consisting of IgA constant heavy chain, paraoxonase, fibuline, prothrombin, complement C3, apolipoprotein, ceruloplasmin, complement factor H-related protein, and, fibrinogen in a blood, serum or plasma sample from said patient;

b) comparing the level of expression of said at least one protein measured in step a) with a reference level of expression for said at least one protein;

c) deducing therefrom if the patient is at risk of having or has hepatic encephalopathy.

The invention further relates to kits for performing these methods.

The term "hepatic encephalopathy" (HE) refers to or describes the physiological condition in mammals of a serious neuropsychiatric complication of both acute and chronic liver failure with the potential to affect heath-related quality of life, clinical management strategies, liver transplant priority and patient survival.

In some embodiments, said hepatic encephalopathy is "minimal hepatic encephalopathy" (MHE) or "overt hepatic encephalopathy" (OHE).

"Minimal hepatic encephalopathy" presents no clinical characteristics but is associated with reduced quality of life, increased frequency of falls and driving accidents. It is also a risk factor for the development of OHE.

"Overt hepatic encephalopathy", is characterized by a progression of symptoms from deficits in attentiveness that may progress to lethargy, asterixis, disorientation, stupor and coma. It may ultimately lead to death.

The term "Transjugular Intrahepatic Portosystemic Shunt" (TIPS) refers to an artificial channel within the liver that establishes communication between the inflow portal vein and the outflow hepatic vein. It is used to treat portal hypertension (which is often due to liver cirrhosis).

The term "cirrhosis" refers to a consequence of chronic liver disease characterized by replacement of liver tissue by fibrosis, scar tissue and regenerative nodules (lumps that occur as a result of a process in which damaged tissue is regenerated), leading to loss of liver function. Cirrhosis is most commonly caused by alcoholism, hepatitis B and C, and fatty liver disease, but has many other possible causes.

The terms "biomarker" or "marker" can be used herein interchangeably. They refer to a substance that is a distinctive indicator of a biological process, biological event, and/or pathologic condition. In clinical proteomics, biomarkers could be defined as biomolecules easily accessible, detectable, measurable across populations and whose variations of expression can provide useful information about diseases from early detection to prediction of recurrence and monitoring of drug treatments.

As used herein , the term "patient" refers to a human that is afflicted with cirrhosis treated with transjugular intra-hepatic porto-systemic shunt (TIPS), but may or may not have an hepatic encephalopathy.

The term "sample" is used herein in its broadest sense. A blood, serum or plasma sample is generally obtained from a patient. In one embodiment, it can be peripheral blood, serum or plasma sample. The blood, serum or plasma sample can be portal vein or hepatic vein or inferior vena cava or peripheral blood, serum or plasma sample, an inferior vena cava blood, serum or plasma sample having the same composition as a peripheral blood, serum or plasma sample. In the scope of the present invention, a sample will be a "clinical sample", i.e., a sample derived from a patient.

The term "sample" also encompasses any material derived by processing a sample. Derived materials include, but are not limited to cells (or their progeny) isolated from the sample or proteins extracted from the sample. Processing of a biological sample may involve one or more of : filtration , distillation , extraction , concentration, inactivation of interfering components, addition of reagents, and the like.

The term "control patient" refers to a patient with cirrhosis treated with transjugular intra-hepatic porto-systemic shunt (TIPS) that has not shown any hepatic encephalopathy symptoms and has not been diagnosed for this disease.

The term "reference level" denotes a level of expression of a marker protein in a control patient or group of control patients, or in a patient or a group of patients diagnosed for an hepatic encephalopathy. The reference level(s) may be determined as a single value or a range of values which is determined based on the level of expression of said marker protein measured in a population of control patients.

Typically, the analysed population could be divided into quantiles based on the measured level of expression of said marker protein. The reference level could be defined as the median, or the second tertile, or the second or third quartile, or the third or fourth quintile etc...

Comparison with a reference level may also be performed by comparing the level of expression of said marker protein with the level of expression of said marker protein, as appropriate, measured in a standard sample obtained from patients having hepatic encephalopathy.

The reference level for a given marker may vary depending on the method used for measuring.

By "quantifying" is meant quantifying the level of expression of said protein, or detecting a decrease or increase of the level of expression of a protein.

By "measuring" is meant measuring the level of expression of said protein, or detecting a decrease or increase of the level of expression of said protein.

By "decrease in the level of expression" is meant a decrease of expression level of said protein in comparison to a reference or to a predetermined threshold value.

By "increase in the level of expression" is meant an increase of expression level of said protein in comparison to a reference or to a predetermined threshold value.

The term "predetermined threshold" for one protein may refer to the median value of the expression level of said protein in biological samples of a control patient, or to the median value of the expression level of said protein in patients having an hepatic encephalopathy. The skilled person can easily determine such a predetermined threshold using methods well-known in the art.

Detailed description of the invention

The inventors have conducted pioneering studies to identify circulating protein biomarkers to refine hepatic encephalopathy diagnosis in a population of patients with cirrhosis treated with TIPS.

For the first time, the inventors have found that IgA constant heavy chain, paraoxonase, fibuline, prothrombin, complement C3, apolipoprotein, ceruloplasmin, complement factor H-related protein, and fibrinogen are differentially expressed in patients with cirrhosis treated with TIPS suffering from hepatic encephalopathy. IgA constant heavy chain is the large polypeptide subunit of immunoglobulin A which plays a critical role in mucosal immunity.

Complement C3 is a protein of the immune system. It plays a central role in the complement system and contributes to innate immunity.

Ceruloplasmin is a major copper-carrying protein in blood and was demonstrated for its implication in Parkinson's (Jin et al., Decreased serum ceruloplasmin levels characteristically aggravate nigral iron deposition in Parkinson's disease. Brain. 201 1 Jan;134(Pt 1 ):50-8. Epub 2010 Nov 24.) and Alzheimer's (Squitti et al., 'Free' copper in serum of Alzheimer's disease patients correlates with markers of liver function. J Neural Transm. 2007;1 14(12):1589-94. Epub 2007 Jul 4, Duce et al., Iron-export ferroxidase activity of β-amyloid precursor protein is inhibited by zinc in Alzheimer's disease. Cell. 2010 Sep 17;142(6):857-67.) diseases, and in a case-report of Wilson disease, a genetic disease associated with liver and neurological disorders (Honma and al., Late diagnosed Wilson disease with hepatic and neurological manifestations. Hepatol Res. 201 1 Mar;41 (3):270-6. doi: 10.1 1 1 1 /j.1872-034X.2010.00754.).

Apolipoproteins are constituents of high density lipoproteins in plasma. Among them, ApoA1 and ApoA4 over-expression has been shown in relation with advanced stages of cirrhosis in patients with non-alcoholic fatty liver disease (NAFLD) (Gray et al., A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease. BMC Cancer. 2009 Aug 5;9:271 ).

According to one embodiment of the present invention, apolipoproteins can be apolipoprotein A1 (ApoA1 ) and/or apolipoprotein A4 (ApoA4).

Prothrombin is a precursor of thrombin and a key molecule in the coagulation cascade. Beyond its role in dynamic process of thrombus formation, this protein has pro- inflammatory character and could be also involved in neurodegenerative processes (Sokolova et al., Prothrombin/thrombin and the thrombin receptors PAR-1 and PAR-4 in the brain: localization, expression and participation in neurodegenerative diseases. Thromb Haemost. 2008 Oct;100(4):576-81 ).

Paraoxonase is an enzyme involved in the hydrolysis of organophosphates. Its isoform 1 is synthesized in liver and transported by HDL (high density lipoprotein). The PON1 (serum paraoxonase/acyl esterase 1 ) polymorphism may be responsible for neurodegeneration and a risk factor for Parkinson's disease.

Fibuline is a secreted glycoprotein, known to be associated with fibrinogen and extracellular matrix components. Complement factor H-related protein is a soluble glycoprotein that regulates the Alternative Pathway of the complement system, ensuring that the complement system is directed towards pathogens and does not damage host tissue.

Fibrinogen is a soluble glycoprotein synthesized by hepatocytes in liver; it is a key regulator of inflammation in several diseases (Dimitrios et al., Fibrinogen as a key regulator of inflammation in disease. Semin Immunopathol (2012) 34:43-62.)

According to one embodiment of the present invention, fibrinogen can be fibrinogen B or fibrinogen G.

According to a further embodiment of the invention, said at least one protein is selected from the group consisting of IgA constant heavy chain, prothrombin, complement C3, apolipoprotein, ceruloplasmin, complement factor H-related protein and fibrinogen.

According to another embodiment of the invention, said at least one protein is selected from the group consisting of IgA constant heavy chain, prothrombin, complement C3, apolipoprotein, ceruloplasmin, fibuline and fibrinogen.

According to another embodiment of the invention, said at least one protein is selected from the group consisting of IgA constant heavy chain, prothrombin, complement C3, apolipoprotein and fibrinogen.

According to still another embodiment of the invention, said at least one protein is selected from the group consisting of IgA constant heavy chain, prothrombin and complement C3.

In a particular embodiment of the invention, the method of predicting or diagnosing an hepatic encephalopathy in a patient with cirrhosis treated with TIPS comprises:

(a) measuring the level of expression of each of IgA constant heavy chain, prothrombin and complement C3 in a blood, serum or plasma sample from said patient;

(b) comparing the level of expression of each of IgA constant heavy chain, prothrombin and complement C3 measured in step a) with respectively a reference level expression for IgA constant heavy chain, prothrombin and complement C3;

(c) deducing therefrom if the patient is at risk of having or has hepatic encephalopathy.

In another particular embodiment of the invention, the method of predicting or diagnosing an hepatic encephalopathy in a patient with cirrhosis treated with TIPS comprises:

(a) measuring the level of expression of each of IgA constant heavy chain, prothrombin, complement C3, apolipoprotein and fibrinogen in a blood, serum or plasma sample from said patient; (b) comparing the level of expression of each of IgA constant heavy chain, prothrombin, complement C3, apolipoprotein and fibrinogen measured in step a) with respectively a reference level expression for IgA constant heavy chain, prothrombin, complement C3, apolipoprotein and fibrinogen;

In still another particular embodiment of the invention, the method of predicting or diagnosing an hepatic encephalopathy in a patient with cirrhosis treated with TIPS comprises:

(a) measuring the level of expression of each of IgA constant heavy chain, prothrombin, complement C3, apolipoprotein, ceruloplasmin, complement factor H-related protein and fibrinogen in a blood, serum or plasma sample from said patient;

(b) comparing the level of expression of each of IgA constant heavy chain, prothrombin, complement C3, apolipoprotein, ceruloplasmin, complement factor H-related protein and fibrinogen measured in step a) with respectively a reference level expression for IgA constant heavy chain, prothrombin, complement C3, apolipoprotein, ceruloplasmin, complement factor H-related protein and fibrinogen;

(a) measuring the level of expression of each of IgA constant heavy chain, prothrombin, complement C3, apolipoprotein, ceruloplasmin, fibuline and fibrinogen in a blood, serum or plasma sample from said patient;

(b) comparing the level of expression of each of IgA constant heavy chain, prothrombin, complement C3, apolipoprotein, ceruloplasmin, fibuline and fibrinogen measured in step a) with respectively a reference level expression for IgA constant heavy chain, prothrombin, complement C3, apolipoprotein, ceruloplasmin, fibuline and fibrinogen;

The inventors have also reported the first evidence that when the level of expression of fibuline and/or prothrombin and/or complement factor H-related protein and/or fibrinogen is decreased as compared with the reference level of expression for fibuline and/or prothrombin and/or complement factor H-related protein and/or fibrinogen, it indicates that the patient is at risk of having or has hepatic encephalopathy.

The inventors have further reported the first evidence that when the level of expression of IgA constant heavy chain and/or paraoxonase and/or complement C3 and/or apolipoprotein and/or ceruloplasmin is increased as compared with the reference level of expression for IgA constant heavy chain and/or paraoxonase and/or complement C3 and/or apolipoprotein and/or ceruloplasmin, it indicates that the patient is at risk of having or has hepatic encephalopathy.

According to a further embodiment of the present invention said blood, serum or plasma sample is a portal or hepatic vein blood serum or plasma sample and said at least one protein is selected from the group consisting of paraoxonase, fibrinogen and apolipoprotein.

In another embodiment, said blood, serum or plasma sample is a peripheral blood, serum or plasma sample and said at least one protein is selected from the group consisting of IgA constant heavy chain, fibuline, prothrombin, complement C3, apolipoprotein, ceruloplasmin, complement factor H-related protein, and fibrinogen.

In the methods according to the present invention, the measure of the level of expression of proteins can be performed by immunoassay or immunoblots or by analytical methods, like for example mass spectrometry (MS), capillary electrophoresis- mass spectrometry (CE-MS), liquid chromatography coupled to mass spectrometry (LC- MS, LC-MS/MS), quantitative methods with isotopic labeling (stable isotope labeling by amino acids in cell culture (SILAC), isotope coded affinity tags (ICAT), isobaric tag for relative and absolute quantitation (ITRAQ),...), label-free methods like selective reaction monitoring (SRM) or multiple reaction monitoring (MRM) assays, or bio-molecular interaction analysis/surface plasmon resonance (BIA/SPR) technologies encompassing methods with calibration and without calibration as calibration free concentration analysis for example.

The term "immunoassay" as used according to the present invention includes competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, agglutination test, enzyme-labeled and mediated immunoassays, such as ELISA, biotin/avidin type assay, radioimmunoassay, Immunoelectrophoresis, and immunoprecipitation.

Mass spectrometry (MS), capillary electrophoresis-mass spectrometry (CE-MS), liquid chromatography coupled to mass spectrometry (LC-MS/MS), stable isotope labeling by amino acids in cell culture (SILAC), isotope coded affinity tags (ICAT), isobaric tag for relative and absolute quantitation (ITRAQ), selective reaction monitoring (SRM) assays, multiple reaction monitoring (MRM) assays, bio-molecular interaction analysis/surface plasmon resonance (BIA/SPR) technologies, calibration free concentration analysis, are all analytical methods very well know by the man skilled in the art which are suitable to carry out the method according to the invention.

In one embodiment of the invention, the measure of the level of expression of proteins is performed by mass spectrometry (MS) and comprises the following steps:

- providing a known amount of one or more protein calibration standard peptide(s) having a sequence of amino acids which is identical with a sequence to be found in the at least one protein and optionally labeling said protein calibration standard peptides(s);

- degrading the blood, serum or plasma sample to obtain a mixture of peptides, and optionally labeling said peptides with one or more labeling agent(s);

wherein at least the peptides in the degraded blood, serum or plasma sample or the calibration standard peptide(s) are labeled, and if both the peptides in the degraded sample and the protein calibration standard peptide(s) are labeled, the labeling agent(s) used for labeling the protein calibration standard peptide(s) are different from the labeling agent(s) used for labeling the peptides of the degraded blood, serum or plasma sample;

- quantifying the absolute amount of the at least one protein by correlating the amount of the protein calibration standard peptide(s) with the amount of the corresponding peptide(s) of the degraded blood, serum or plasma sample by mass analysis.

According to the invention, the sample can be degraded by treatment with one or more enzymes in order to obtain a mixture of peptides. The enzyme can be chosen to have a very predictable degradation pattern enabling that peptides obtained may be identified and quantified by comparison with the protein calibration standard peptide. The enzyme can be one or more protease(s) such as e.g. two proteases or one or more enzyme(s). Examples of proteolytic enzymes include trypsin, papain, pepsin, ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin and carboxypeptidase C. For example, the proteolytic enzyme trypsin is a serine protease that cleaves peptide bonds between lysine and arginine and an unspecific amino acid thereby produce peptides that comprise an amine terminus (N-terminus) and lysine or arginine carboxyl terminal amino acid (C- terminus). In this way, the peptides from the cleavage of the protein are predictable and their quantity, in a sample from a trypsin digest, is indicative of the quantity of the protein of their origin. Moreover, the free amine termini of a peptide can be a good nucleophile that facilitates its labelling. Because activity of the enzymes is predictable, the sequence of peptides that are produced from degradation of a protein of a known sequence can be predicted. With this information, "theoretical" peptide information can be generated. A determination of the "theoretical" peptide fragments in e.g. computer assisted analysis of daughter fragment ions from mass spectrometry analysis of an actual sample can therefore be used to identify one or more peptides.

In the present context, the terms "protein calibration standard peptides having a sequence of amino acids which is identical with a sequence to be found in the at least one protein" refers to an amino acid sequence region which is constant i.e. identical of the one of the protein to quantify.

The protein calibration standard peptide(s) to be used according to the invention depend on the label and on the quantification method to be used in order to give a detectable signal and fragmentation when analysed in a mass-spectrometry (MS) instrument.

Peptides which can be used can be made using protein and/or nucleotide databases and cleavage analyses program(s) and/or performing in vitro mass fingerprints experiment(s).

For the man skilled in the art, it will be obvious that there are numerous possibilities for labeling the sample and the protein calibration standard peptides in order to introduce, in a predetermined manner, different mass modifying functionalities that makes quantification of the protein peptides possible. Labeling can e.g. be performed as described in WO2004/070352, US6,864,089, Stemman O et al. Cell 2001 ; 107(6):715-26, and Gerber SA et al. Proc Natl Acad Sci USA 2033; 100(12):6940-5.

For example, the labeling can be performed with ITRAQTM chemistry (Applied Biosystems, Foster Cityy, CA, USA) or with ICPL (Isotope Coded Protein Labelling) method described by Kellemrann et al., ProteomicsA A .

According to a still further embodiment of the present invention, an antibody directed against a protein selected from the group consisting of IgA constant heavy chain, paraoxonase, fibuline, prothrombin, complement C3, apolipoprotein, ceruloplasmin, complement factor H-related protein, and fibrinogen can be used for in vivo or in vitro predicting or diagnosing an hepatic encephalopathy in a patient with cirrhosis treated with transjugular intra-hepatic porto-systemic shunt (TIPS).

The antibody may be polyclonal or monoclonal, preferably monoclonal.

Polyclonal antibodies of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.

Monoclonal antibodies of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV- hybridoma technique (Cole et al., 1985).

Alternatively, techniques described for the production of single chain antibodies (see e.g. U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies directed against biomarkers of the invention. Antibodies useful in practicing the present invention also include anti-biomarkers fragments including but not limited to F(ab')₂ fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to biomarkers of the invention.

For example, phage display of antibodies may be used. In such a method, single- chain Fv (scFv) or Fab fragments are expressed on the surface of a suitable bacteriophage, e. g., M13. Briefly, spleen cells of a suitable host, e.g., mouse that has been immunized with a protein are removed. The coding regions of the VL and VH chains are obtained from those cells that are producing the desired antibody against the protein. These coding regions are then fused to a terminus of a phage sequence. Once the phage is inserted into a suitable carrier, e. g., bacteria, the phage displays the antibody fragment. Phage display of antibodies may also be provided by combinatorial methods known to those skilled in the art. Antibody fragments displayed by a phage may then be used as part of an immunoassay. Examples of commercially available monoclonal antibodies are given in Table 1 below:

Table 1 . Examples of commercially available monoclonal antibodies

Proteins References Supplier Applications

Apo A4 3109-1 Epitomics WB

Apo A1 1920-1 Epitomics WB

FGG Sc- 133226 Santa Cruz WB+ELISA

FGG Sc-133157 Santa Cruz WB+ELISA

FGG Sc-133156 Santa Cruz WB+ELISA

FGG Sc-81620 Santa Cruz WB

FGG 2743-S Epitomics WB Proteins References Supplier Applications

FGG MA5- 15906 Thermoscientific WB

FGG MA5- 15902 Thermoscientific WB

FGB Sc-271035 Santa Cruz WB+ELISA

FGB Sc-271017 Santa Cruz WB+ELISA

FGB Sc-271018 Santa Cruz WB+ELISA

FGB 2736-1 Epitomics WB

FGB MA5-15519 Thermoscientific WB

Ceruloplasmin Sc- 135866 Santa Cruz WB

Ceruloplasmin Sc-365206 Santa Cruz WB+ELISA

Ceruloplasmin Sc-365205 Santa Cruz WB+ELISA

Ceruloplasmin 5989-1 Epitomics WB

Prothrombin Sc-69769 Santa Cruz WB

Prothrombin Sc- 136449 Santa Cruz WB

Prothrombin Sc-73470 Santa Cruz WB

Prothrombin 3571 -1 Epitomics WB

Prothrombin MA1 -43020 Thermoscientific WB+ELISA

Complement C3 Sc-55458 Santa Cruz WB+ELISA

Complement C3 Sc-52629 Santa Cruz WB

Complement C3 Sc-25298 Santa Cruz WB+ELISA

Complement C3 Sc-28294 Santa Cruz WB+ELISA

Complement C3 HYB 005-01 -02 Thermoscientific WB+ELISA

Complement factor H- Sc-47686 Santa Cruz WB

related protein

Complement factor H- Sc-47685 Santa Cruz WB

related protein

Complement factor H- Sc-166613 Santa Cruz WB+ELISA related protein

Complement factor H- Sc166608 Santa Cruz WB+ELISA related protein

Ig A Sc-69785 Santa Cruz WB+ELISA ig A Sc- 166334 Santa Cruz WB+ELISA

Ig A Sc-73307 Santa Cruz WB+ELISA

Ig A 5205-1 Epitomics WB+ELISA

Paraoxonase Sc-374158 Santa Cruz WB+ELISA

Paraoxonase Sc-373981 Santa Cruz WB+ELISA

Fibuline Sc-374539 Santa Cruz WB+ELISA

Fibuline Sc-55470 Santa Cruz WB+ELISA

In the above Table 1 , "WB" means "Western Blot", "E LISA" means "enzyme-linked immunosorbent assay", "FGG" and "FGB" respectively relates to "fibrinogen G" and "fibrinogen B".

The antibody can be labeled. The term "labeling", is intended to encompass direct labeling by coupling (i.e., physically linking) of a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5)) to the antibody, as well as indirect labeling of antibody by reactivity with a detectable substance. The labeling with a radioactive molecule can be done by any method known in the art. For example radioactive molecules include, but are not limited to, radioactive atom for scintigraphic studies such as 1123, 1124, In1 1 1 , Re186, Re 188.

The present invention also relates to kit for performing the method herein described. Said kit comprises means for measuring the level of expression of at least one protein selected from the group consisting of IgA constant heavy chain, paraoxonase, fibuline, prothrombin, complement C3, apolipoprotein, ceruloplasmin, complement factor H-related protein, and fibrinogen in a blood, serum or plasma sample from the patient, and a standard for determination of the reference level of expression of said at least one protein in a patient having an hepatic encephalopathy.

According to an embodiment of the present invention, said means can be a specific antibody directed against one of the proteins herein cited or protein calibration standard peptide(s) having a sequence of amino acids which is identical with a sequence to be found in one of the proteins herein cited.

Means for measuring the level of expression of said at least one protein include antibodies specifically binding to said at least one protein. Such means can be labeled with detectable compound such as fluorophores or radioactive compounds. For example, the antibody specifically binding to said at least one protein may be labeled with a detectable compound. Alternatively, when the kit comprises an antibody, the kit may further comprise a secondary antibody, labeled with a detectable compound, which binds to an unlabeled antibody specifically binding to said at least one protein

The kits according to the invention may for example comprise, in addition to the means for measuring the level of expression of said at least one protein and the standard for determination of the reference level of expression of said at least one protein in a patient having an hepatic encephalopathy, instructions for the use of said kit in a method of predicting or diagnosing an hepatic encephalopathy in a patient with cirrhosis treated with transjugular intra-hepatic porto-systemic shunt (TIPS).

Instructions for using the kit according to methods of the invention may comprise instructions for processing the biological sample obtained from the patient and/or for performing the test, or instructions for interpreting the results. A kit may also contain a notice in the form prescribed by a governmental agency regu lating the man ufacture, use or sale of pharmaceuticals or biological products.

In addition, a kit of the invention generally also comprises at least one reagent for the detection of a complex between the means for measuring the expression level of expression of said at least one protein included in the kit and said at least one protein cited above.

Depending on the procedure, the kit may further comprise one or more of: extraction buffer and/or reagents, western blotting buffer and/or reagents, and detection means.

Protocols for using these buffers and reagents for performing different steps of the procedure may be included in the kit.

The different reagents included in a kit of the invention may be supplied in a solid (e.g. lyophilized) or liquid form.

The kits of the present invention may optionally comprise different containers (e.g., vial, ampoule, test tube, flask or bottle) for each individual buffer and/or reagent. Each component will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Other containers suitable for conducting certain steps of the disclosed methods may also be provided. The individual containers of the kit are preferably maintained in close confinement for commercial sale.

The invention will be further illustrated by the following examples. EXAMPLE 1 Material and methods

Human Plasma preparation.

At the time of TIPS insertion under radiological guidance, blood was collected on EDTA-coated tubes from portal vein (PV), hepatic vein (HV) and inferior vena cava (IVC). Immediately after collection, plasma were prepared at 4°C with a 20 minutes, 2500 rpm centrifugation, aliquoted and stored at -80 °C.

2 Dimension-Differential in Gel Electrophoresis (2D-DIGE).

Sample preparation and labeling: To decrease the amount of the high-abundance proteins and simultaneously concentrate low-abundance proteins, the samples were prepared using the ProteoMiner protein enrichment kit, according to Bio-Rad's instructions.

After elution (Urea 8M, chaps 2%, acetic acid 5%) and clean up precipitation (2D clean up kit, GE-Healthcare), the pellet was resuspended in 2D-DIGE sample buffer (Urea 8M, Thiourea 2M, chaps 4%, pH 8.5). Protein concentration was determined with 2D Quant kit (GE-Healthcare).

50 μg of proteins were labeled with 400 pmol of CyDye DIGE Fluor Minimal Dyes

(GE Healthcare) and incubated on ice in the dark for 30 minutes according to the manufacturer's instructions. Samples were reversed labeled in order to prevent potential dye labeling bias. The reaction was stopped by the addition of 1 μΙ of 10mM lysine and incubated on ice for 10 minutes.

The differentially Cy-3 and Cy-5 labeled samples were then mixed with the Cy-2 labeled internal standard (sample composed of equal aliquots of each sample in the experiment) and IEF rehydratation buffer (Urea 8M, Thiourea 2M, Chaps 2%, 10mM DTT, 1 ,2% IPG buffer, pH4-7, GE Healthcare) was added up to 350 μΙ.

2D-DIGE electrophoresis: Isoelectric focusing (IEF) was performed using an IPGPhor 2 apparatus (GE Healthcare) according to the manufacturer's recommendations with immobilized pH gradient (IPG) pH 4-7, 18 cm. Upon IEF, strips were incubated in equilibration buffer (Urea 6M, SDS 2%, Tris 50mM pH8.6, glycerol 30%, bromophenol blue trace) containing 1 % DTT (dithiothreitol) for 15 minutes and thereafter in the same buffer with 4.5% iodoacetamide, for 15 minutes, in the dark.

The strips were then loaded on the top of 12.5% acrylamide gels for electrophoresis.

Gel image Analysis: Gels were scanned using a Typhoon trio Imager (GE Healthcare) at 100 μηι resolution. Image analysis was performed using DeCyder 6,5 software (GE Healthcare).

Relative protein quantification was performed using Differential In gel Analysis (DIA) and Biological Variance Analysis (BVA) from DeCyder software.

Features detected from non protein sources were filtered out. Spots displaying an increase superior or equal to 1 ,5 or decrease in abundance with a p-value inferior to 0,05 were selected for protein identification.

Spot were excised using an Ettan Spot Picker (GE Healthcare).

Protein identification: Gel plugs were washed twice for 20 minutes using 50 mM ammonium bicarbonate/50% (vol/vol) methanol in water and one more wash with 75% (vol/vol) acetonitrile in distilled water for 30 minutes. The gel plugs were dried in a SpeedVac.

Proteins from each gel spot have been digested with trypsin and resulting peptides have been extracted from gel. Peptide mixtures were analyzed by nanoHPLC-Chip- MS/MS system consisting of nanoHPLC-microfluidic chip cube (Agilent) interfaced to an Amazon ETD mass spectrometer (Bruker Daltonics). Scan of mass spectrometry (MS) will have been acquired on the 300-1500 m/z range and most intense ions per scan will be selected for CID (collision induced dissociation) fragmentation. A peaklist was created and imported in Protein Scape (Bruker Daltonics) for each gel spot analyzed. Mascot searches have been performed for each gel spot for protein identification.

Results

Protein profiles from PV and HV plasma of two groups of 8 patients who were positive or negative for OHE were established.

Before intra-hepatic shunting, all the patients were tested for OHE according to West-Haven criteria (Ferenci et al.\ Hepatic encephalopathy-definition, nomenclature, diagnosis, and quantification: final report of the working party at the 11 th World Congresses of Gastroenterology, Vienna, 1998. Hepatology 2002, 35(3)716-721 ., Cash et at., Current concepts in the assessment and treatment of hepatic encephalopathy. OJM. 2010 Jan;103(1 ):9-16. Epub 2009 Nov 10), without deciphering between MHE and negative OHE.

All the patients were negative for OHE at the moment of TIPS procedure according to clinical tests and observations. Eight of them were diagnosed for OHE during follow-up, with apparition of neurocognitive impairments varying in time (see Table 2).

Table 2. Baseline clinical characteristics of patient treated with TI PS.

Child- Delay for

Age OHE TIPS

Patients OHE Gender Aetiology Pugh OHE after

(years) history** Indication

class * TIPS (days)

1 Yes M^*** 69 Alcohol B Yes Ascites 155

2 Yes M 72 Alcohol B No Ascites 7

3 Yes M 55 Alcohol B Yes Ascites 1

4 Yes M 72 Alcohol C No Ascites 5

5 Yes M 53 Alcohol B No Ascites 1

6 Yes M 69 Alcohol C No Ascites 1

Hepatitis

7 Yes 71 Virus B No Ascites 18

8 Yes M 54 Alcohol B Yes Ascites 103

9 No F 61 Alcohol B No Ascites

10 No M 50 Alcohol B No Ascites

Hepatitis

11 No M 36 Virus A No Bleeding

12 No M 72 Alcohol B No Bleeding 13 No F 61 Alcohol A No Ascites

14 No M 44 Alcohol A No Bleeding

15 No F 57 Alcohol B No Bleeding

Hepatitis

16 No M 45 Virus B No Ascites

^* According to Chil-Pugh classification of liver disease, class A (5 to 6 points) indicates the least severe disease, class B (7 to 9 points) moderately severe disease, class C (10 to 15 points) the most severe disease

*^* OHE history means OHE antecedents

^*** M and F respectively means male and female

Therefore, the 16 patients were retrospectively split into two groups of 8 patients that were positive (OHE+) or negative (OHE-) for hepatic encephalopathy.

Protein samples from plasma were labeled with fluorescent dyes for 2D-DIGE analysis with samples from the same venous origin migrating in the same gel. After fluorescent imaging, images were subjected to bio-informatic analyses with Decyder software, allowing detection of proteins spots differentially expressed between the two groups. A total of 23 spots and 34 spots were significantly up or down-regulated in patients with hepatic encephalopathy in PV and HV territories respectively.

Among these 23 and 34 proteins, nine are of great interest as they are the same in PV and HV plasma samples, meaning that their differential expression between patients having an hepatic encephalopathy and control patients is strictly linked to the hepatic encephalopathy disorder, independently of the venous origin.

Among these nine proteins, 7 were clearly over-expressed and 2 under-expressed in patients having an hepatic encephalopathy, with a level of expression of variation ranging from 2 to 20 fold.

These nine protein spots were picked up from the gel and analyzed in mass spectrometry as described in material and methods. The table 3 summarizes the biological variation analysis of differentially expressed proteins, their identity, score in mass spectrometry and major biochemical features. Table 3. Summary of biological variations analyisis (De Cyder) and identity of differentially expressed proteins between OHE+ and OHE- plasma independently of venous origin (portal vein or hepatic vein).

"PM" refers to molecular weight, "PI" to isoelectric point, "MS" to mass spectrometry and "Seq Cov" to sequence coverage.

All the proteins identified are synthesized in the liver and most of these protein spots were identified as constituents of fibrinogen.

The difference in protein profiles between OHE+ and OHE- patients was established and compared with 2D-DIGE, for plasma protein profiles from inferior vena cava. These blood samples are of interest because they correspond to peripheral blood; inferior vena cava being downstream hepatic veins on the way back of blood to the general circulation. As previously described, IVC plasmas from the two groups were labeled, separated in 2D gel, analyzed for differential expression pattern with excision and identification of spots of interest. Most of the proteins identified are related to the liver function and inflammatory disorders (see Table 4), reinforcing previous results on PV and

HV plasma samples. Table 4. Summary of biological variation analysis (DeCyder) and identity of differentially expressed proteins between OHE+ and OHE- plasma from IVC.

"PM" refers to molecular weight and "PI" to isoelectric point. EXAMPLE 2

Material and methods

Human plasma preparation

The samples used are the same as those mentioned in example 1 .

Nano-HPLC-Chip-MS/MS technology

Sample preparation: To decrease the amount of the high-abundance proteins and simultaneously concentrate low-abundance proteins, the samples were prepared using the ProteoMiner protein enrichment kit, according to Bio-Rad's instructions, until elution with Laemmli Buffer (Tris 80mM, pH 6.8, SDS 3%, glycerol 10%, DTT 80mM). The samples were then reduced (10min-95°C) and alkylated (lodoacetamide 90mM, 30min, room temperature) before running on a 7.5% SDS-PAGE.

Proteins were stained by Coomassie Blue (Instant Blue-Euromedex). Each lane was cut and each gel piece was washed four times in ACN 100%, ammonium bicarbonate 100mM (15min, 37°C), ACN-ammonium bicarbonate 10OmM (vol/vol) (15min, 37°C) and dried in vacuum. In gel digestion and peptides extraction: Gel pieces were rehydrated with 50μΙ modified trypsin 20ng^l (sequencing grade, Promega) prepared in ammonium bicarbonate ^"l OOmM, and digested overnight at 37°C.

Digestion was stopped by acidification with 50μΙ of 1% formic acid and 40% ACN. Samples were then sonicated and supernatants collected (S1 ). Gel pieces were dried and 20μΙ of ACN 25% with 1 % formic acid were added, before sonication. Finally, 25μΙ of ACN 60% were added, the samples were sonicated and supernatants collected (S2), S1 and S2, containing the peptides mixture. Mass Spectrometry:

Mass Spectrometry: Proteins from each gel spot were digested with trypsin and resulting peptides were extracted from gel. Peptide mixtures were analyzed by nanoHPLC-Chip-MS/MS system consisting of nanoHPLC-microfluidic chip cube (Agilent) interfaced to an Amazon ETD mass spectrometer (Bruker Daltonics). Scan MS were acquired on the 300-1500 m/z range and most intense ions per scan were selected for CID fragmentation. A peaklist was created and imported in Protein Scape (Bruker Daltonics) for each gel spot analyzed. Mascot searches were performed for each gel spot for protein identification.

Peptide Profiling and sequencing: For the profiling experiment, the Amazon was operated in full scan MS. Scan MS were acquired on the 300-1500 m/z range in the enhanced resolution mode. A set of target peptides was obtained after data analysis and an inclusion list was built to fragment preferentially relevant peptides. MS/MS spectra were acquired using the scheduled precursor list but the mass spectrometer was allowed to acquire spectra in empty time intervals. The most intense ions per scan were selected for CID fragmentation and dynamic exclusion was employed within 12 s to prevent repetitive selection of the same peptide. The fragmentation amplitude was set to 0.6 V.

Data analysis: The Bruker data files (.d folder) generated with the nanoHPLC- Chip-MS/MS technology were loaded to Progenesis LC-MS version 4.0 (Nonlinear Dynamics, UK). In order to select features of interest, statistical filters (p-value < 5%, max fold change > 1 .5, power > 80%, max coefficient of variation (CV) within condition < 20% and q-value<1 %) were set and only features that match all filters were kept. Descriptive statistics and Principal Component Analysis (PCA) were performed using mixOmics (15) R Package.

Statistical treatment using PLSDA (Partial Least Squares Discriminant Analysis) is a step forward for patient classification. This classification tool is based on the PLS model in which the dependent variable is chosen to represent the class membership. In the present study, the known variable was the diagnosis of HE, meaning that the analysis was performed after the study population had been split into 2 groups according to whether or not they experienced HE (HE + and HE -, respectively). The aim of this analysis was to define a set of peptides predictive of post-TIPS HE.

Results

This experiment allows identifying the following proteins: fibrinogen, prothrombin, complement C3, apolipoprotein, ceruloplasmin, complement factor H-related protein, and IgA constant heavy chain.

CONCLUSION

Differences in protein profiles between patients who underwent or not OHE after TIPS was shown. Fibrinogen, paraoxonase, fibuline, prothrombin, complement C3, apolipoprotein, ceruloplasmin, complement factor H-related protein, and IgA constant heavy chain were identified as molecular biomarkers of OHE after TIPS.

Claims

1 . Method of predicting or diagnosing an hepatic encephalopathy in a patient with cirrhosis treated with transjugular intra-hepatic porto-systemic shunt (TIPS), said method comprising:

a. measuring the level of expression of at least one protein selected from the group consisting of IgA constant heavy chain, paraoxonase, fibuline, prothrombin, complement C3, apolipoprotein, ceruloplasmin, complement factor H-related protein, and, fibrinogen in a blood, serum or plasma sample from said patient;

b. comparing the level of expression of said at least one protein measured in step a) with a reference level of expression for said at least one protein; c. deducing therefrom if the patient is at risk of having or has hepatic encephalopathy.

2. Method according to claim 1 , wherein said at least one protein is selected from the group consisting of fibuline, complement factor H-related protein, fibrinogen, and prothrombin, and wherein a level of expression of said at least one protein which is decreased as compared with the reference level of expression of said at least one protein indicates that the patient is at risk of having or has hepatic encephalopathy.

3. Method according to claim 1 or 2, wherein fibrinogen is fibrinogen B or fibrinogen

G.

4. Method according to claim 1 , wherein said at least one protein is selected from the group consisting of IgA constant heavy chain, complement C3, apolipoprotein, ceruloplasmin, and, paraoxonase and wherein a level of expression of said at least one protein which is increased as compared with the reference level of expression of said at least one protein indicates that the patient is at risk of having or has hepatic encephalopathy.

5. Method according to any one of claims 1 or 4, wherein said apolipoprotein is apolipoprotein A1 (ApoA1 ) and/or apolipoprotein A4 (ApoA4).

6. Method according to any one of claims 1 to 5, wherein said blood, serum or plasma sample is a portal or hepatic vein blood, serum or plasma sample and said at least one protein is selected from the group consisting of paraoxonase, fibrinogen and apolipoprotein.

7. Method according to any one of claims 1 to 4, wherein said blood, serum or plasma sample is a peripheral blood, serum or plasma sample, and wherein said at least one protein is selected from the group consisting of IgA constant heavy chain, fibrinogen, fibuline, prothrombin, complement C3, apolipoprotein, ceruloplasmin and complement factor H-related protein.

8. Method according to any one of claims 1 to 7, wherein the measure of the level of expression of said at least one protein is performed by immunoassay or by immunoblot.

9. Method according to any one of claims 1 to 7, wherein the measure of the level of expression of said at least one protein is performed by an analytical method.

10. Method according to claim 9, wherein said analytical method is chosen from mass spectrometry (MS), capillary electrophoresis-mass spectrometry (CE-MS), liquid chromatography coupled to mass spectrometry (LC-MS/MS), stable isotope labeling by amino acids in cell culture (SILAC), isotope coded affinity tags (ICAT), isobaric tag for relative and absolute quantitation (ITRAQ), selective reaction monitoring (SRM) assays, multiple reaction monitoring (MRM) assays, bio-molecular interaction analysis/surface plasmon resonance (BIA SPR) technologies, calibration free concentration analysis.

1 1 . Method according to claim 10, wherein said analytical method is mass spectrometry (MS) and comprises the following steps:

a. providing a known amount of one or more protein calibration standard peptide(s) having a sequence of amino acids which is identical with a sequence to be found in the at least one protein and optionally labeling said protein calibration standard peptides(s);

b. degrading the blood, serum or plasma sample to obtain a mixture of peptides, and optionally labeling said peptides with one or more labeling agent(s);

wherein at least the peptides in the degraded blood, serum or plasma sample or the calibration standard peptide(s) are labeled, and if both the peptides in the degraded sample and the protein calibration standard peptide(s) are labeled, the labeling agent(s) used for labeling the protein calibration standard peptide(s) are different from the labeling agent(s) used for labeling the peptides of the degraded blood, serum or plasma sample; c. quantifying the absolute amount of the at least one protein by correlating the amount of the protein calibration standard peptide(s) with the amount of the corresponding peptide(s) of the degraded blood, serum or plasma sample by mass spectrometry analysis.

12. Use of at least one antibody directed against a protein selected from the group consisting of IgA constant heavy chain, paraoxonase, fibuline, prothrombin, complement C3, apolipoprotein, ceruloplasmin, complement factor H-related protein, and fibrinogen for in vitro predicting or diagnosing an hepatic encephalopathy in a patient with cirrhosis treated with transjugular intra-hepatic porto-systemic shunt (TIPS).

13. Kit for performing a method according to any one of claims 1 to 1 1 , wherein said kit comprises means for measuring the level of expression of at least one protein selected from the group consisting of IgA constant heavy chain, paraoxonase, fibuline, prothrombin, complement C3, apolipoprotein, ceruloplasmin, complement factor H-related protein, and fibrinogen in a blood, serum or plasma sample from the patient, and a standard for determination of the reference level of expression of said at least one protein in a patient having an hepatic encephalopathy.

14. Kit according to claim 13 wherein said means are a specific antibody of the at least one protein or protein calibration standard peptide(s) having a sequence of amino acids which is identical with a sequence to be found in the at least one protein.