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Novel biomarkers and the development of a point-of-care test for fatty liver disease

Ahead of The 3rd Global NASH Congress, Dr Bevin Gangadharan explains how he helped to discover novel NAFLD biomarkers and their role in the development of a point-of-care test for NAFLD.

Professors Nicole Zitzmann and Raymond Dwek at the University of Oxford have used proteomics techniques to identify potential novel protein biomarkers for the most common liver disorder in the Western world and are currently developing a point-of-care test (POCT) for the disease which affects more than a billion people worldwide.

Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of progressive liver disease ranging from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH). Approximately 1 in 3 people in the UK and 25% of adults worldwide are expected to have some degree of NAFLD and most affected individuals are unaware they have the disease. Currently, the reference standard for assessing NAFLD is the liver biopsy: an invasive, painful procedure that can be unreliable and costly. Consequently, there is a need for improved, minimally-invasive methods of determining stages of NAFLD in patients.

Two postdocs in the Zitzmann lab – myself (Bevin Gangadharan) and Abhinav Kumar – have used 2D gels and mass spectrometry to separate proteins in serum from patients with NAFLD at various stages of the disease and have identified several potential biomarkers. Most importantly including ones which can differentiate NAFL from NASH.1 The proteomics techniques were used to separate proteins in serum from healthy individuals and patients with NAFLD (including NAFL and NASH with various fibrosis stages). 

One of their most promising NAFLD biomarkers, apolipoprotein F (ApoF), was first identified by the Zitzmann lab as a biomarker for liver fibrosis in hepatitis C patients and they identified several other biomarkers which also tracked fibrosis severity.2 They have shown that their fibrosis biomarkers are promising compared to the biomarkers used in current tests such as FibroTest, ELF test, Hepascore, and FIBROSpect.3 

For NAFLD, the biomarkers identified at Oxford by 2D gels included ApoF which successfully tracked disease severity. Using mass spectrometry, they identified 46 potential NAFLD biomarkers including ApoF and other biomarkers capable of diagnosing the early stages where the disease is more easily reversible. The biomarkers identified by these two proteomics methods were evaluated using parallel reaction monitoring mass spectrometry. Their biomarkers were found to be promising compared to cleaved cytokeratin-18 and the protein biomarkers in NashTest. 

Since NAFLD affects 1 in 4 people worldwide, there is an urgent need for a rapid and inexpensive test to screen at-risk individuals such as those who are clinically obese, who have a 90% chance of having the disease. The Zitzmann lab is therefore currently looking into ways of developing a rapid POCT which can determine biomarker levels in less than 5 minutes from a single drop of finger-pricked capillary blood. They have also successfully developed the first antibody-free serum protein biomarker assay for NAFLD and liver fibrosis where a number of biomarkers can be measured with only a few microliters of serum or plasma, i.e. less than a drop of blood.4

The novel Oxford biomarkers would allow clinicians to identify NAFLD patients so they could be advised about lifestyle changes, such as diet and exercise, which could help to reverse the disease and thus save money for health providers. Once the markers are validated, the technology could potentially be used to diagnose, prognose or monitor the progression of NAFLD in an individual thereby reducing the need for a liver biopsy. These biomarkers could also help to reduce disease progression since patients could be stratified according to the stage of NAFLD ensuring appropriate measurements can be taken.

Dr. Bevin Gangadharan is a research scientist in Professor Nicole Zitzmann’s lab in the Department of Biochemistry at the University of Oxford. He will be presenting at The 3rd Global NASH Congress on ‘Novel serum biomarkers for NAFLD Identified using proteomics’.

To view resources from the Global NASH Congress series click here.

References

1. Bevin Gangadharan, Abhinav Kumar, Raymond Dwek, Nicole Zitzmann, Mark Thursz, Jeremy Cobbold (2018) Clinical diagnosis of non-alcoholic fatty liver disease using a panel of human blood protein biomarkers, Patent Publication Number: WO/2018/037229.

2. Bevin Gangadharan, Nicole Zitzmann, Raymond Dwek (2010) Clinical diagnosis of hepatic fibrosis using a novel panel of low abundant human plasma protein biomarkers. Patent Publication Number: US 2010/0291602 A1.

3. Bevin Gangadharan, Manisha Bapat, Jan Rossa, Robin Antrobus, David Chittenden, Bettina Kampa, Eleanor Barnes, Paul Klenerman, Raymond Dwek, Nicole Zitzmann (2012) Discovery of novel biomarker candidates for liver fibrosis in hepatitis C patients: a preliminary study, PLoS One, 7, e39603.

4. Abhinav Kumar, Bevin Gangadharan, Jeremy Cobbold, Mark Thursz, Nicole Zitzmann (2017) Absolute quantitation of disease protein biomarkers in a single LC-MS acquisition using apolipoprotein F as an example, Scientific Reports, 7, 12072.

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