Novel biomarkers predict liver fibrosis in hepatitis C patients: alpha 2 macroglobulin, vitamin D binding protein and apolipoprotein AI
- Ai-Sheng Ho†1,
- Chun-Chia Cheng†2, 3,
- Shui-Cheng Lee3,
- Meng-Lun Liu1,
- Jing-Ying Lee1,
- Wen-Ming Wang4, 5, 6 and
- Chia-Chi Wang7Email author
© Ho et al; licensee BioMed Central Ltd. 2010
Received: 24 August 2009
Accepted: 15 July 2010
Published: 15 July 2010
The gold standard of assessing liver fibrosis is liver biopsy, which is invasive and not without risk. Therefore, searching for noninvasive serologic biomarkers for liver fibrosis is an importantly clinical issue.
A total of 16 healthy volunteers and 45 patients with chronic hepatitis C virus (HCV) were enrolled (F0: n = 16, F1: n = 7, F2: n = 17, F3: n = 8 and F4: n = 13, according to the METAVIR classification). Three serum samples of each fibrotic stage were analyzed by two-dimension difference gel electrophoresis (2D-DIGE). The differential proteins were identified by the cooperation of MALDI-TOF/TOF and MASCOT; then western blotting and Bio-Plex Suspension Array were used to quantify the protein levels.
Three prominent candidate biomarkers were identified: alpha 2 macroglobulin (A2M) is up regulated; vitamin D binding protein (VDBP) and apolipoprotein AI (ApoAI) are down regulated. The serum concentration of A2M was significantly different among normal, mild (F1/F2) and advanced fibrosis (F3/F4) (p < 0.01). The protein levels of VDBP and ApoAI were significantly higher in normal/mild fibrosis, when compared to those in advanced fibrosis (both p < 0.01).
This study not only reveals three putative biomarkers of liver fibrosis (A2M, VDBP and ApoAI) but also proves the differential expressions of those markers in different stages of fibrosis. We expect that combination of these novel biomarkers could be applied clinically to predict the stage of liver fibrosis without the need of liver biopsy.
In hepatitis C virus (HCV)-infected patients, liver fibrosis is a noticeable disease and could progress to liver cirrhosis or hepatocellular carcinoma gradually. Although the pathogenesis of HCV-infected fibrosis is poorly understood, liver fibrosis may be a response of repair when liver is injured or inflamed . In addition, the detection of early stage of liver fibrosis is very important before the irreversible damage occurs. Liver biopsy followed by histological examination is still the gold standard for the assessment of liver fibrosis . However, it has several limits and disadvantages such as invasive character and sampling error [3–5]. Therefore, it is necessary to have a reliable and noninvasive assessment for liver fibrosis.
Two-dimension difference gel electrophoresis (2D-DIGE), first proposed by Unlu et al , allow different samples to be labeled with cy3 or cy5 flour in one gel. It used cy2-labeled internal standard to tell the differences and found out the reliable biomarkers among different gels. Furthermore, multi-analyte profiling (xMAP) technique was used to quantify the concentration of putative biomarkers. By using these methods, we could not only identify but also quantify the candidate biomarkers, which could be a serologic predictor for the severity of liver fibrosis. In this study, we enrolled patients of chronic HCV infection and healthy controls, and used proteomic technique to analyze their sera. The aim is to search for noninvasive serological biomarkers of liver fibrosis, which could predict the stage of fibrosis without the need of liver biopsy.
Materials and methods
Totally 61 serum samples from 45 patients of chronic HCV infection and 16 healthy volunteers were obtained from Cheng Hsin General Hospital in Taiwan (approval No. 97016). The assessment of liver fibrosis was based on liver biopsy and subsequent histological examination. The stage was determined according to METAVIR classification . The stages of liver fibrosis were distributed as following in the chronic hepatitis C patients: F1, n = 7, F2, n = 17, F3, n = 8 and F4, n = 13. The serum samples of healthy control (F0 stage, n = 16) were obtained from healthy volunteers who had no evidence of known hepatitis. Liver biopsy was not performed in these subjects due to ethical issues.
Two dimension-difference gel electrophoresis
Arrangement for protein samples labeled with three CyDye flours
Pool of samples
Pool of samples
Pool of samples
Pool of samples
Pool of samples
Pool of samples
Pool of samples
Pool of samples
In-gel tryptic digestion
The gels were stained with Sybro Ruby (sigma) for at least four hours and then destained with 10% methanol/7% acetic acid for exactly 30 min. The interesting proteins in the gels were picked up manually on UV transilluminator (Spectroline). Those gel particles were washed with 10% methanol/7% acetic acid overnight to remove Sybro Ruby chemicals thoroughly; 100 μL of 25 mM ammonium bicarbonate in 50% acetonitrile for 15 min; 200 μL of 25 mM ammonium bicarbonate in deionized water for 15 min twice. The saturated gel particles were added enough acetonitrile to shrink for 5 min. After drying down, the gel particles were added 3 μL of 20 ng/μL trypsin in 25 mM ammonium bicarbonate at 4°C for 1 hour and subsequently added 3 μL of 25 mM ammonium bicarbonate to keep the gels wet at 56°C for 1 hour. After In-gel digestion the solution were added 2 μL of 100% acetonitrile with 1% trifluoracetic acid and sonicated for 10 min to release peptides from gel particles.
Mass spectrometric analysis for protein identification
Each trypsin-digested solution was mixed 1:1 with 10 mg/mL α-cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% trifluoracetic acid and spotted on AnchorChip MALDI target (Bruker Daltonics GmbH, Bremen, Germany). Peptides were analyzed with MALDI-TOF/TOF UltraflexIII (Bruker Daltonics) by peptide mass fingerprinting after calibration in positive reflection mode under 20 KV and calculated the molecular weight with FlexAnalysis™ 3.0 software (Bruker Daltonics). MASCOT 2.2 (Matrix Science) was used to match the peptides with NCBI or Swiss-Prot database for protein identification. The calculation was restricted to human taxonomy, allowing carbamidomethyl cysteine as a fixed modification and oxidized methionine as a variable modification. The probability was based on Mowse score calculated from -10 × Log (P), where P was the probability that the observed match was a random event. Protein scores greater than 56 were significant (p < 0.05). Moreover, one of the major peptide peaks appeared on the spectrum was used to confirm the searching result by peptide fragment fingerprinting method.
Each serum sample was diluted 1:10 with sodium dodecyl sulfate buffer (50 mM Tris-Cl, 8 M urea, 30% glycerol, 2% sodium dodecyl sulfate, 20 mM dithiothreitol and 0.1% bromophenol blue). The sample solutions were heated at 100°C for 5 min; and then 2 μL of samples (approximately 20-30 μg) were loaded to 4-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Invitrogen). The iblot (Invitrogen) was used for transforming the proteins to polyvinylidene fluoride (PVDF). After using 0.5% milk to blot the PVDF for 30 min, the A2M, ApoAI and VDBP were detected by rabbit anti-A2M antibody (AbD serotec), chicken anti-ApoAI antibody (CHEMICON) and rabbit anti-VDBP antibody (AbD serotec) for two hour at room temperature. After washing three times in PBS buffer (10 mM sodium phosphate, pH7.4 and 0.9% sodium chloride), the second antibody conjugated with horseradish peroxidase was added to incubate for one hour. The ECL detection system (Millipore) was used and the images were acquired by Imaging System (Gel Doc XR System, Bio-Rad) depending on the moderate exploring time.
Bio-plex suspension array system
Bio-Plex 200 Suspension Array System (Bio-Rad) was based on flexible multi-analyte profiling (xMAP) technique developed by Luminex Corporation using the principle of sandwich immunoassay. Individual serum sample was diluted 100 thousands fold with Bio-Plex human serum diluent. For A2M measurement, Bio-Plex Pro Human Acute Phase 4-Plex Panel (cat. 171-A4009M, Bio-Rad) was performed. For ApoAI and VDBP measurement, there are many processes need to complete, including antibody labeled with microsphere and biotin individually. These experiments were completed by following the manual of Amine Coupling Kit (Bio-Rad) and Lynx Rapid Biotin Antibody Conjugation Kit (AbD Serotec) respectively. After pre-wet of 96-well plate, each 50 μL of diluted sample was incubated with 1.25 × 106 microspheres, which labeled with primary antibody, at 300 rpm for exactly 1 hour. Subsequently each 50 μL of biotin-conjugated secondary antibody (2 μg/mL) was added to incubate at 300 rpm for 30 min. Finally 50 μL of streptavidin-Phycoerythrin was added to each well and mixed at 300 rpm for 10 min. After drying up and washing, 125 μL of assay buffer was allowed to suspend each well of microspheres. The protein concentration was measured by Bio-Plex 200 Suspension Array System. SPSS software (Ver.14.0; SPSS Inc.) was used to calculate the p value and to present the curve of expressional trend with Box-and-Whisker Plot.
The serum samples labeled with cy2, cy3 or cy5 individually are presented in Table 1. From F0 to F4 fibrotic stages, we selected three samples of each stage to do 2D-DIGE experiments. For this purpose, 15 samples needed to separate into eight gels, in which F0-1 was used in gel 1 and gel 8 for fitting the arranging design. This design would not influence the data calculation. In this study we did not use albumin/IgG removal kit to remove high abundant albumin and IgG because we wanted to simplify the experimental process. Moreover removing albumin protein would remove albumin-binding proteins in the same time and influence the reproducibility.
Protein spots identified by MALDI/TOF-TOF-MS
We used Bio-Plex Suspension Array System to measure the absolute protein concentration of A2M, VDBP and ApoAI according to protein standard curve. We found that the serum concentration of A2M from F0 to F4 was increased significantly (F0: 4.3 ± 2.8, F1/F2: 7.2 ± 4.3, F3/F4: 13.0 ± 6.8 mg/mL, p < 0.01) (Fig. 3B). The correlation coefficient of A2M was 0.98. The result suggests that A2M protein could distinguish the stages among normal (F0), mild (F1/F2) and advanced fibrosis (F3/F4). The serum concentration of VDBP was decreased from F0/F1 to F2-F4 stage (F0/F1: 1.2 ± 0.3 mg/mL, F2-F4: 0.6 ± 0.2 mg/mL, p < 0.01) (Fig. 3C). The result indicates that VDBP protein could differentiate F0/F1 from F2-F4 stage. The protein concentration of ApoAI was decreased in F3/F4 stage (F0-F2: 2.0 ± 0.7 mg/mL, F3/F4: 1.1 ± 0.5 mg/mL, p < 0.01) (Fig. 3D). This result implies that ApoAI could be a biomarker to differentiate normal/mild (F0-F2) from advanced fibrosis (F3/F4).
Searching for novel serological biomarkers of HCV-infected fibrosis is to avoid the use of invasive liver biopsy. Developing an efficient and noninvasive method for liver fibrosis is important for prognosis and treatment plan in patients with chronic hepatitis C virus. A noninvasive diagnosis of liver fibrosis could also enhance the development of antifibrotic therapies. There are several non-invasive methods to assess liver fibrosis in patients with chronic hepatitis C, including FibroScan (FS) [8, 9], Fibrotest (FT) . However, FS could not assess liver fibrosis properly when patients were overweight or morbid obese; besides, stiffness measurement is hard to acquire in ascitic patients . Ziol M et al.  indicated that FS appeared as a reliable tool to detect significant fibrosis or cirrhosis rather than early liver fibrosis. Castera L et al.  suggested that the combined use of FS and FT to assess liver fibrosis could avoid liver biopsy in most patients with chronic hepatitis C. In our study, although two of the three identified biomarkers, A2M and ApoAI, are the same as that in FT, ApoAI is decreased significantly only in advanced fibrosis (F3/F4). This result suggests ApoAI could only be an indictor for advanced fibrosis or cirrhosis. In addition, this is the first report that VDBP could be a biomarker of liver fibrosis in patients with chronic hepatitis C.
A2M is a well-known biomarker of hepatic fibrosis  and a significant component of measuring liver fibrosis in FibroTest, FIBROSpect II, Fibrometer or Hepascore [10, 14–16]. A2M is able to inactivate an enormous variety of proteinases and inhibit fibrinolysis by reducing plasmin and kallikrein. In inflammatory or injured liver, the increase of A2M inhibit catabolism of matrix proteins and thus cause liver fibrosis [17–20]. Gangadharan B et al. indicated that thioester cleavage of A2M may increase gradually with the development of fibrosis . In our study, we consistently confirmed that serum concentration of A2M may be an indicator to predict liver fibrosis. However, serum A2M level is increased in patients with depression or nephrotic syndrome [22, 23]. Therefore, while using A2M as liver fibrosis biomarker, other biomarkers are needed to decrease the interference of other diseases such as nephrotic syndrome or depression.
VDBP is also known as Gc-globulin (group-specific component globulin) which binds and transports vitamin D metabolites [24, 25]. The significant function of VDBP is involved in actin scavenger system, thus to protect the organism from the toxic effect of intravascular actin polymerization . Moreover, VDBP can also be converted into a macrophage-activator factor, and actin-free VDBP is associated with organ dysfunction in acute liver failure [27, 28]. However, this is the first study to validate that the level of VDBP is negatively associated with the development of liver fibrosis. ApoAI, applied also in FibroTest and Fibrometer test [29, 30], was a putative biomarker of HCV-infected fibrosis which was also verified to be a protein with down regulation in liver hepatic fibrosis in this study. Moreover, we found that the level of ApoAI was changeable between F0-F2 and F3/F4 stage. Our result demonstrates that ApoAI is a candidate biomarker of advanced fibrosis (F3/F4).
2D-DIGE technologies, a useful tool in proteomics analysis recently, were used to search for reliable biomarker of liver fibrosis in our study. Cy2-labeled internal standard can correct the analytical error among gels so that more than two samples can be analyzed. Furthermore, the samples, which were labeled with high sensitive CyDye, could pour together in one gel. Thus, thousands of serum proteins could be easily analyzed and identified as reliable biomarkers [31, 32]. In addition, there were several advantages for 2D-DIGE technique such as reducing variation among gels and increasing the reproducibility of proteins . The sensitivity of CyDye flours (<0.05 ng) was better than silver staining or coomassine blue staining [34, 35]. Therefore 2D-DIGE was more powerful than traditional two-dimension electrophoresis (2DE) in biomarker discovery. However, it still had some limitations in detecting the hydrophobic proteins, proteins bigger than 200 kDa, or those smaller than 10 kDa. Many proteins of extreme acidity or basicity were also not presented in the gels . Moreover, because CyDye flours needed to conjugate with lysine residue of proteins, the high abundant proteins with few or no lysine residues were difficult to be detected.
Cut-off value of A2M, VDBP and ApoAI to predict liver fibrosis§
In summary, this study not only reveals three putative biomarkers of liver fibrosis (A2M, VDBP and ApoAI) but also proves the differential expressions in different stages of fibrosis. Furthermore, we discovered a novel biomarker, VDBP, which is decreased in liver fibrosis (F2-F4). In addition, the algorithm combining three biomarkers could be used clinically to predict the stage of liver fibrosis and to reduce the use of liver biopsy.
This project was supported by the grant NSC96-3111-P-042A-004-Y from National Science Council of Republic of China and the founding of Cheng Hsin Rehabilitation Medical Center.
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