Limits...
Discovery of novel biomarker candidates for liver fibrosis in hepatitis C patients: a preliminary study.

Gangadharan B, Bapat M, Rossa J, Antrobus R, Chittenden D, Kampa B, Barnes E, Klenerman P, Dwek RA, Zitzmann N - PLoS ONE (2012)

Bottom Line: Identified markers were validated across all Ishak fibrosis stages and compared to the markers used in FibroTest, Enhanced Liver Fibrosis (ELF) test, Hepascore and FIBROSpect by Western blotting.These five novel fibrosis markers which are secreted in blood showed a promising consistent change with increasing fibrosis stage when compared to the markers used for the FibroTest, ELF test, Hepascore and FIBROSpect.This study identifies 20 novel fibrosis biomarker candidates.

View Article: PubMed Central - PubMed

Affiliation: Oxford Antiviral Drug Discovery Unit, Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, Oxford, United Kingdom. Bevin.Gangadharan@bioch.ox.ac.uk

ABSTRACT

Background: Liver biopsy is the reference standard for assessing liver fibrosis and no reliable non-invasive diagnostic approach is available to discriminate between the intermediate stages of fibrosis. Therefore suitable serological biomarkers of liver fibrosis are urgently needed. We used proteomics to identify novel fibrosis biomarkers in hepatitis C patients with different degrees of liver fibrosis.

Methodology/principal findings: Proteins in plasma samples from healthy control individuals and patients with hepatitis C virus (HCV) induced cirrhosis were analysed using a proteomics technique: two dimensional gel electrophoresis (2-DE). This technique separated the proteins in plasma samples of control and cirrhotic patients and by visualizing the separated proteins we were able to identify proteins which were increasing or decreasing in hepatic cirrhosis. Identified markers were validated across all Ishak fibrosis stages and compared to the markers used in FibroTest, Enhanced Liver Fibrosis (ELF) test, Hepascore and FIBROSpect by Western blotting. Forty four candidate biomarkers for hepatic fibrosis were identified of which 20 were novel biomarkers of liver fibrosis. Western blot validation of all candidate markers using plasma samples from patients across all Ishak fibrosis scores showed that the markers which changed with increasing fibrosis most consistently included lipid transfer inhibitor protein, complement C3d, corticosteroid-binding globulin, apolipoprotein J and apolipoprotein L1. These five novel fibrosis markers which are secreted in blood showed a promising consistent change with increasing fibrosis stage when compared to the markers used for the FibroTest, ELF test, Hepascore and FIBROSpect. These markers will be further validated using a large clinical cohort.

Conclusions/significance: This study identifies 20 novel fibrosis biomarker candidates. The proteins identified may help to assess hepatic fibrosis and eliminate the need for invasive liver biopsies.

Show MeSH

Related in: MedlinePlus

Western blot band densitometry.The five plots on the left show densitometry data for our five markers; from top to bottom: LTIP, complement C3d, apolipoprotein L1, apolipoprotein J and corticosteroid-binding globulin. The five plots on the right show densitometry data for all the markers that were blotted for in the ELF test (TIMP1 and PIIIP), FibroTest (apolipoprotein A1, alpha 2 macroglobulin and haptoglobin), Hepascore (alpha 2 macroglobulin) and FIBROSpect (TIMP-1, and alpha 2 macroglobulin). Each point represents the average band intensity for four patient samples. Error bars show +/− standard error.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3383672&req=5

pone-0039603-g005: Western blot band densitometry.The five plots on the left show densitometry data for our five markers; from top to bottom: LTIP, complement C3d, apolipoprotein L1, apolipoprotein J and corticosteroid-binding globulin. The five plots on the right show densitometry data for all the markers that were blotted for in the ELF test (TIMP1 and PIIIP), FibroTest (apolipoprotein A1, alpha 2 macroglobulin and haptoglobin), Hepascore (alpha 2 macroglobulin) and FIBROSpect (TIMP-1, and alpha 2 macroglobulin). Each point represents the average band intensity for four patient samples. Error bars show +/− standard error.

Mentions: Complement C3 has several cleavage products within its sequence and was chosen for further peptide sequence analysis to identify which cleavage product was higher in cirrhotic patient plasma. A fragment of complement C3 increased in cirrhosis and was observed on the 2-DE gel at 38 kDa with an approximate isoelectric point of pI 4.9 (Figure 2B). The peptide sequences identified by mass spectrometry are shown in Figure 3 and span from amino acids 955 to 1201. The amino acids for complement C3dg span from 955 to 1303 and its theoretical molecular weight and isoelectric point (39 kDa and pI 5) are in line with the observed gel feature indicating that the fragment of complement C3 in the feature is complement C3dg. Western blots using anti-complement C3d antibodies showed one band decreasing with increasing fibrosis stage (Figures 4 and 5), which is possibly thioester cleaved Complement C3 containing C3d.


Discovery of novel biomarker candidates for liver fibrosis in hepatitis C patients: a preliminary study.

Gangadharan B, Bapat M, Rossa J, Antrobus R, Chittenden D, Kampa B, Barnes E, Klenerman P, Dwek RA, Zitzmann N - PLoS ONE (2012)

Western blot band densitometry.The five plots on the left show densitometry data for our five markers; from top to bottom: LTIP, complement C3d, apolipoprotein L1, apolipoprotein J and corticosteroid-binding globulin. The five plots on the right show densitometry data for all the markers that were blotted for in the ELF test (TIMP1 and PIIIP), FibroTest (apolipoprotein A1, alpha 2 macroglobulin and haptoglobin), Hepascore (alpha 2 macroglobulin) and FIBROSpect (TIMP-1, and alpha 2 macroglobulin). Each point represents the average band intensity for four patient samples. Error bars show +/− standard error.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3383672&req=5

pone-0039603-g005: Western blot band densitometry.The five plots on the left show densitometry data for our five markers; from top to bottom: LTIP, complement C3d, apolipoprotein L1, apolipoprotein J and corticosteroid-binding globulin. The five plots on the right show densitometry data for all the markers that were blotted for in the ELF test (TIMP1 and PIIIP), FibroTest (apolipoprotein A1, alpha 2 macroglobulin and haptoglobin), Hepascore (alpha 2 macroglobulin) and FIBROSpect (TIMP-1, and alpha 2 macroglobulin). Each point represents the average band intensity for four patient samples. Error bars show +/− standard error.
Mentions: Complement C3 has several cleavage products within its sequence and was chosen for further peptide sequence analysis to identify which cleavage product was higher in cirrhotic patient plasma. A fragment of complement C3 increased in cirrhosis and was observed on the 2-DE gel at 38 kDa with an approximate isoelectric point of pI 4.9 (Figure 2B). The peptide sequences identified by mass spectrometry are shown in Figure 3 and span from amino acids 955 to 1201. The amino acids for complement C3dg span from 955 to 1303 and its theoretical molecular weight and isoelectric point (39 kDa and pI 5) are in line with the observed gel feature indicating that the fragment of complement C3 in the feature is complement C3dg. Western blots using anti-complement C3d antibodies showed one band decreasing with increasing fibrosis stage (Figures 4 and 5), which is possibly thioester cleaved Complement C3 containing C3d.

Bottom Line: Identified markers were validated across all Ishak fibrosis stages and compared to the markers used in FibroTest, Enhanced Liver Fibrosis (ELF) test, Hepascore and FIBROSpect by Western blotting.These five novel fibrosis markers which are secreted in blood showed a promising consistent change with increasing fibrosis stage when compared to the markers used for the FibroTest, ELF test, Hepascore and FIBROSpect.This study identifies 20 novel fibrosis biomarker candidates.

View Article: PubMed Central - PubMed

Affiliation: Oxford Antiviral Drug Discovery Unit, Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, Oxford, United Kingdom. Bevin.Gangadharan@bioch.ox.ac.uk

ABSTRACT

Background: Liver biopsy is the reference standard for assessing liver fibrosis and no reliable non-invasive diagnostic approach is available to discriminate between the intermediate stages of fibrosis. Therefore suitable serological biomarkers of liver fibrosis are urgently needed. We used proteomics to identify novel fibrosis biomarkers in hepatitis C patients with different degrees of liver fibrosis.

Methodology/principal findings: Proteins in plasma samples from healthy control individuals and patients with hepatitis C virus (HCV) induced cirrhosis were analysed using a proteomics technique: two dimensional gel electrophoresis (2-DE). This technique separated the proteins in plasma samples of control and cirrhotic patients and by visualizing the separated proteins we were able to identify proteins which were increasing or decreasing in hepatic cirrhosis. Identified markers were validated across all Ishak fibrosis stages and compared to the markers used in FibroTest, Enhanced Liver Fibrosis (ELF) test, Hepascore and FIBROSpect by Western blotting. Forty four candidate biomarkers for hepatic fibrosis were identified of which 20 were novel biomarkers of liver fibrosis. Western blot validation of all candidate markers using plasma samples from patients across all Ishak fibrosis scores showed that the markers which changed with increasing fibrosis most consistently included lipid transfer inhibitor protein, complement C3d, corticosteroid-binding globulin, apolipoprotein J and apolipoprotein L1. These five novel fibrosis markers which are secreted in blood showed a promising consistent change with increasing fibrosis stage when compared to the markers used for the FibroTest, ELF test, Hepascore and FIBROSpect. These markers will be further validated using a large clinical cohort.

Conclusions/significance: This study identifies 20 novel fibrosis biomarker candidates. The proteins identified may help to assess hepatic fibrosis and eliminate the need for invasive liver biopsies.

Show MeSH
Related in: MedlinePlus