Patent Application
20080090223
This invention is included within the field of the detection of oxidised forms of apolipoprotein AI.
Hepatocellular carcinoma (HCC) is the neoplastic disease with the fifth highest incidence and the third cause of cancer death, with over 500,000 new cases diagnosed every year. Although the main causes of HCC are known, from them, infection by hepatitis B virus (HBV) or C virus (HCV), the consumption of food contaminated with aflatoxin, or abusive alcohol consumption, the prognosis for HCC patients is bad due to the aggressiveness of the lesion at the time of diagnosis and the lack of effective therapies. The identification of biomarkers which make it possible to effectively describe the stage of this disease is, therefore, of great interest.
There is evidence that many pathological processes are associated with quantitative and functional changes in the molecular constituents of body fluids. Excluding cellular components, the comparative analysis of body fluid samples from healthy and ill donors is only possible at the proteomic level and not at the transcriptional level. Although cerebrospinal fluid and urine are used in diagnostic medicine, there is increasing interest in the human serum proteome due to the fact that the serum constantly penetrates the tissues and, therefore, the beginning or the presence of a disease could be determined by measuring and characterising the thousands of individual circulating proteins and peptides. Moreover, serum determination techniques are bloodless and non-invasive, have good sample availability and are easy to perform, quick and inexpensive.
Traditional techniques which make it possible to diagnose HCC, such as the increase in alpha-fetoprotein (AFP) levels, are not effective in all cases and are positive when the stage of the disease is too advanced. Studies have been conducted to identify HCC markers using high-performance proteomic techniques, both in the liver (Zeindl-Eberhart E. et al. Hepatology (2004) 39:540-549), which would entail using invasive techniques to perform the diagnosis, and in serum (Steel F. L. et al. Proteomics (2003) 3:601-609; Quina Yu He et al. Proteomics (2003) 3:666-674). However, although this technology has proven effective in the detection of molecular targets related to the development of various diseases, its methodological complexity makes its use in clinical practise unthinkable. None of the above-mentioned studies provide information which may serve as the basis for the development of diagnostic methodologies applicable to clinical practise. On the other hand, various studies, including the above-mentioned ones, have established a relationship between the decrease in Apo AI levels and the development of liver diseases. However, the isoform of Apo AI, and its increase in the serum of patients suffering from hepatocellular carcinoma, which is the subject of this invention, has not been previously described.
The subject of this invention is the detection in a biological sample, preferably serum, of an increase in an acidic apolipoprotein AI isoform, oxidised in at least a tryptophan or methionine residue. Specifically, an apolipoprotein AI isoform oxidised in residues W50 and W108.
The present invention relates to a procedure to detect the increase in oxidised apolipoprotein AI isoforms in at least one tryptophan or methionine residue, or fragments thereof which contain said oxidised residues. In a preferred embodiment of the invention, it comprises the detection of the increase in at least one oxidised apolipoprotein AI isoform in at least one residue selected from W50, W108, and M112, or fragments thereof which contain said residues. Residues W50 and W108 may be oxidised to kynurenine, formylkynurenine, hydroxytryptophan, or 3-OH-kynurenine. On the other hand, residue M112 may be oxidised to M112 sulfoxide, or methionine sulfone.
Moreover, the procedure object of this invention is characterised in that said detection is performed on isolated biological samples. With these samples being selected from: serum, plasma, blood, urine, saliva, cerebrospinal fluid, tears, amniotic fluid, tissue wash, tissue homogenate and cell lysate. In a preferred embodiment of the invention, said biological sample is serum.
In a specific embodiment of this invention, the detection of oxidised apolipoprotein AI isoforms is performed on samples from individuals with hepatitis, and preferably individuals with hepatitis B (HBV).
As used in this invention, the term “individual” relates to any animal, preferably a mammal. In a preferred embodiment of the invention, the individual is a man or a woman.
In the procedure disclosed in this invention, the detection of said increase may be conducted by comparing with purified Apo AI standards in known quantities, or by comparing with the quantities of said isoform present in the same type of biological sample obtained from healthy control individuals.
In a preferred embodiment, the increase of the oxidised form in the serum with respect to the control level observed by means of said procedure is at least 1.5-fold, and preferably at least 2.5-fold.
In addition, the present invention is characterised in that it comprises the use of methods to detect and quantify the presence of oxidised Apo AI isoforms selected from: mass spectrometry, immunoassays, chemical assays, liquid chromatography, direct and indirect photometric methods, and combinations thereof. In a preferred embodiment, the mass spectrometry methods are selected from: tandem mass spectrometry coupled with liquid chromatography (LC-MS) and MALDI-TOF-MS (matrix-assisted laser desorption/ionization mass spectrometry time-of-flight MS). Moreover, in a preferred embodiment of the invention, the immunoassays are selected from: homogeneous assays, heterogeneous assays, enzyme immunoassays (EIA, ELISA), competition assays, immunometric assays (sandwich), turbidimetric assays, nephelometric assays and combinations thereof; which are extensively described in “The Immunoassay Book”, edited by David Wild, 2nd Edition 2001, Nature Publishing Group, which is included as a reference. In a preferred embodiment, the methods of detection and quantification of oxidised forms of apolipoprotein AI comprise the use of antibodies, aptamers and/or lecithins which specifically recognise said Apo AI isoforms and fragments thereof.
This invention also makes it possible to relate said increase in apolipoprotein AI isoforms to the presence of hepatocellular carcinoma in the individual from whom the biological sample has been obtained. In a specific embodiment, said hepatocellular carcinoma is at an early stage.
Moreover, this invention relates to a kit for the determination of the increase in apolipoprotein AI isoforms described above, characterised in that it comprises reagents to conduct the methods used in this invention. In a preferred embodiment, said reagents are selected from specific ligands of Apo AI isoforms, marker components to detect Apo AI isoforms, buffers, diluents, standards and controls. In a preferred embodiment, the kit additionally comprises bottles, phials, tubes, needles, solid substrates and instructions.
In another respect, the present invention relates to a procedure for in vitro diagnosis of hepatocellular carcinoma, characterised in that it comprises the detection of the increase in oxidised apolipoprotein AI isoforms in at least one tryptophan or methionine residue, or fragments thereof which contain at least one of said oxidised residues. Said diagnostic procedure is characterised in that it detects said oxidised apolipoprotein AI isoforms as previously described in this section.
In this study, MATIA−/− mice were used, characterised by a chronic reduction in hepatic AdoMet enzyme, which induces the spontaneous development of non-alcoholic steatohepatitis (NASH) and cellular hepatocarcinoma at 18 months of age.
Serum samples were taken from control and MATIA−/− mice, 3, 5, 12, and 18 months of age, and were subsequently analysed by means of two-dimensional electrophoresis (2DE), followed by mass spectrometry. In this way, an average resolution of 500 proteins was obtained, and a differential comparison was conducted by means of PDQUEST, in which increases or decreases of at least two-fold were accepted as differences. Using this criterion, only two proteic bands were obtained from the serum of MATIA−/− mice, whose increase was consistent in all assays. Furthermore, these differences were observed starting with mice 3 months of age.
Analysis by means of PMF made it possible to identify these two increased proteic bands as apolipoprotein AIV and apolipoprotein AI (
Table 1. Analysis of Apo AI isoforms in murine serum by means of LC/MS/MS. Tryptic peptides from Apo A-I isoforms from MATIA−/− serum were resolved by means of reverse-phase nanoflow liquid chromatography connected to an ESI/MS/MS mass spectrometer. The sequences of the Apo A-I peptides were deduced by means of de novo synthesis analysis. The oxidised methionine residues are highlighted.
The levels and the post-translational state of Apo AI were studied in serum samples obtained from patients with different liver pathologies, including non-alcoholic steatohepatitis, alcoholic cirrhosis, HBV, HCV, and HCC. Four bands were identified as Apo AI by means of PMF (Peptide Mass Fingerprint) and de novo peptide sequencing in all the samples and 2D Western blot analysis. Through this study, pathology-specific alterations in the relative amount of Apo AI isoforms were detected. All the serum samples from patients with HBV and HCC showed a 2.5-fold increase in isoform 1, with respect to the controls (relative amount 41.93±3.00% as compared to 18.23±1.08% in control individuals) (
Subsequently, the decision was made to analyse whether the increase in the acidic isoform of Apo AI was the result of an increase in the protein's oxidation state. The products of the tryptic digestion of Apo AI isoforms from three different patients suffering from HBV+HCC were analysed by means of nano-liquid chromatography (nano LC) coupled to a Q TOF Micro-mass spectrometer by means of an electrospray ionisation source (ESI/MS/MS). Three specific oxidations were selectively detected in isoform 1. Oxidation of methionine 112 to methionine sulfoxide was evidenced by the +16 Da deviation of the ion's molecular mass (
Table 2. Analysis of Apo AI isoforms in human serum by means of LC/MS/MS. Tryptic peptides of Apo AI isoforms from human serum were resolved by means of a C18 reverse-phase nanoflow column connected to a Q TOF Micro-mass spectrometer (ESI/MS/MS). Apo AI peptide sequences were deduced from their fragmentation spectra. The oxidised methionine and tryptophan residues are highlighted.
| Number | Date | Country | Kind |
|---|---|---|---|
| P 200402989 | Dec 2004 | ES | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/ES05/00074 | 2/15/2005 | WO | 10/10/2007 |
