Clinical Chemistry in NAFLD Part 2 – Biomarkers, Hepatic Steatosis and NASH

INTRODUCTION ABOUT DR. GUILLAUME GRZYCH

In today’s critical discussion on the role of Clinical Chemistry in the landscape of Non-Alcoholic Fatty Liver Disease (NAFLD), we are pleased to have Dr. Guillaume Grzych as our esteemed guide. He serves as an Associate Professor and Hospital Practitioner at faculté de Pharmacie de Lille, where he specializes in medical biology with a focus on biochemistry.

He comes equipped with a wealth of knowledge, not only in the medical biological sciences but also in advanced computational techniques like next-generation sequencing, bioinformatics, and biostatistics. Dr. Grzych’s cross-disciplinary expertise places him at the forefront of modern medical research, and he is uniquely positioned to discuss the value and utility of biomarkers in NAFLD.

In this enlightening series, Dr. Grzych aims to shed light on the specific interests of clinical chemists in the NAFLD realm. The series is segmented into three parts, and in this second installment, Dr Grzych will delve into specific Biomarkers and Hepatic Steatosis and NASH.

OVERVIEW OF CLINICAL CHEMISTRY IN NAFLD PART 2 – Biomarkers and Hepatic Steatosis and NASH

Biomarkers in Diagnosing and Managing NAFLD Stages

In the first video of Dr. Guillaume Grzych’s enlightening video, he draws attention to the significant role that biomarkers can play in both diagnosing and monitoring the various stages of Non-Alcoholic Fatty Liver Disease (NAFLD). NAFLD manifests in multiple forms, ranging from simple steatosis to Non-Alcoholic Steatohepatitis (NASH) and liver fibrosis. Dr. Grzych focus in on the most used combinations of biomarkers to tackle these stages effectively.

For Simple Steatosis Diagnosis and in NASH: Dr. Grzych particularly emphasizes the importance of the Fatty Liver Index, Hepatic Steatosis Index, and SteatoTest. Utilizing these indices is vital for screening individuals who may be at higher risk of progressing from simple steatosis to more advanced conditions like NASH.

Increasing Prevalence of NAFL: Dr. Grzych further notes that the prevalence of Non-Alcoholic Fatty Liver (NAFL) is rising globally. Astonishingly, simple steatosis affects approximately 30-40% of the global population. Given this staggering number, screening through biological and clinical means is not just recommended—it’s imperative.

This tailored focus on varying biomarkers for different stages of NAFLD offers healthcare providers a more nuanced approach to diagnosis and treatment.

Understanding the Pathophysiology of NAFLD for Effective Biomarker Selection

To grasp the value of biomarkers in NAFLD, Dr. Guillaume Grzych underscores the need to first understand the disease’s pathophysiology. A sound comprehension of these underlying mechanisms allows for the selection of more accurate biomarkers.

What is Hepatic Steatosis?

Hepatic steatosis refers to the accumulation of fatty acids, particularly triglycerides, in the liver. These triglycerides can originate from various sources like:

• the liver from fatty acid
• from the gut
• from nutrition alimentation
• when you put a lot of fatty acid, you could have a lot of triglycerides in your liver. 
• Adipose tissue in the form of adipocytes

An increase in adipocytes triggers lipolysis, releasing fatty acids that are chemically converted into triglycerides in the liver. This leads to a buildup of triglycerides, which is further exacerbated by obesity.

Role of Obesity and Insulin Resistance

Obesity is a key player, as it significantly increases the risk of triglyceride accumulation due to higher levels of adipocytes and lipolysis. Likewise, insulin resistance and diabetes add another layer of complexity:

• Increased of adipose cells lead to higher lipolysis rates and accumulation of triglycerides
• There is enhanced hepatic lipogenesis, producing more fatty acids in the liver
• Decrease of beta-oxidation, the process that typically converts fatty acids into energy

In the context of insulin resistance, fatty acids are less frequently used for energy, leading to an even greater conversion to triglycerides and causing further insulin resistance. This sets up a vicious cycle of lipid accumulation and metabolic dysfunction.

Important Biomarkers for Hepatic Steatosis

Given these intricate pathways, it’s crucial to consider specific metabolic parameters when selecting biomarkers for hepatic steatosis:

• Triglyceride levels
• Obesity metrics, often represented by Body Mass Index (BMI)
• Insulin resistance indicators, such as glycemia or Oral Glucose Tolerance Test (OGTT)

By focusing on combinations of these metabolic parameters, healthcare providers can achieve a more comprehensive and effective approach to diagnosing and managing NAFLD.

A Deep Dive into SteatoTest by BioPredictive

Dr. Guillaume Grzych elaborates on the SteatoTest, a diagnostic tool developed by BioPredictive. This test offers a paid service that combines various analytical measurements to assess liver health.

How SteatoTest Works

The SteatoTest process involves several steps:

1. First, perform the analytical measurements of the following markers:

• Hepatic Markers
• Gamma-GT
• Total Bilirubin
• Alpha-2-macroglobulin
• Apolipoprotein A1
• Haptoglobin
• Glucose
• Triglycerides (TG)
• Cholesterol

2. Enter the patient’s age and sex, as these are also factored into BioPredictive’s calculations.
3. Feed these results into BioPredictive’s software to obtain the SteatoTest score.

Note: The precise calculations behind the scores are proprietary information, and a fee is charged for the score computation

Scoring Variability and Tests

While SteatoTest is the focus here, it’s important to mention that the same parameters used for this test can also yield scores for NASH and Fibrotest. BioPredictive’s algorithms can compute multiple scores based on the same input data.

The Importance of External Validation

It’s crucial to note that while these tests are backed by the manufacturing company, unbiased, third-party validation is necessary.

Dr Grzych refers to a study by Lassailly, published in the European Journal of Gastroenterology and Hepatology, which indicates that SteatoTest shows promise in diagnosing steatosis.

The score demonstrated an area under the curve around 0.7 when compared to biopsy, marking good performance, although there is room for improvement.

By understanding the mechanics and validation of the SteatoTest, HCPs can make more informed decisions in the screening and management of liver diseases such as NAFLD.

A Comparative Overview of SteatoTest 2 and Other Liver Health Indexes

Dr. Guillaume Grzych explores various diagnostic tools for liver health, including the new SteatoTest-2 by BioPredictive, the Fatty Liver Index, and the Hepatic Steatosis Index.

SteatoTest-2: An Adaptation for Specific Conditions

BioPredictive offers a modified version of the original SteatoTest, known as SteatoTest-2. This version omits BMI and Total Bilirubin from its calculations for specific reasons:

• BMI: Removed due to variability, especially with ageing, making it complicated to interpret.
• Total Bilirubin: Excluded due to its dependence on factors like hemolysis, with some samples difficult to collect on other pathology. Also pathology have a consequence on Total Bilirubin, such as the disease of Gilbert on other hepatic disease, called impact Total Bilirubin measurement. 

An evaluation comparing SteatoTest-2 with the original version showed no decrease in performance, making it a reliable alternative.

Fatty Liver Index: A Free and Robust Score

The Fatty Liver Index is another commonly used tool in the literature. Dr. Guillaume Grzych highlights it as a valuable resource, especially for diagnosing simple steatosis in NAFL cases.

This index is a freely accessible score based on measurements of triglycerides, Gamma-GT, and waist circumference, which can easily be entered into online algorithms to generate diagnostic scores.

In contrast to the SteatoTest, which scored around 0.7 in external validation, the Fatty Liver Index has proven its robustness with an impressive area under the curve of about 0.8. Unlike other metrics, this index is exclusively designed for identifying simple steatosis, making it unsuitable for assessing NASH stages.

By focusing solely on NAFL, this tool gives healthcare professionals a targeted approach for diagnosing simple steatosis.

Hepatic Steatosis Index: Another Free and Effective Option

The Hepatic Steatosis Index is another score used in the medical community. It combines the following factors:

• Aminotransferases measurements
• Body Mass Index (BMI)
• Type 2 diabetes status
• Patient’s sex

Based on Non-Alcoholic Fatty Liver (NAFL) pathophysiology, having diabetes or being female can increase the score, indicating a greater risk of steatosis. 

In terms of reliability, like the Fatty Liver Index, the Hepatic Steatosis Index also demonstrated an area under the curve around 0.8 in external validation studies, confirming its effectiveness in evaluating liver steatosis.

Because the algorithm is free and easily accessible, healthcare professionals can confidently use this index as a diagnostic tool. The higher the score, the greater the grade of liver steatosis, making it an essential tool for those managing NAFL.

Evaluating NAFLD Steatosis & NASH Progression

The most commonly used algorithms for diagnosing liver steatosis include the Fatty Liver Index (FLI), SteatoTest, and Hepatic Steatosis Index (HSI). Both FLI and HSI have shown high performance in external validation studies, making them highly reliable tools for steatosis evaluation. Therefore, these two tests are particularly recommended for assessing liver steatosis.

After steatosis, the next stage in NAFLD progression is Non-Alcoholic Steatohepatitis (NASH), which involves liver inflammation and steatohepatitis. Further diagnostic measures are needed to assess this stage.

NASH Pathophysiology & Challenges in Biomarker Identification

Inflammation’s Role

For effective diagnosis and management of steatosis in non-alcoholic fatty liver disease (NAFLD), understanding its pathophysiology is crucial. to understand what could be a good biomarker for this stage of NAFLD. 

The transition from simple fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH) is complex and not fully understood. So this is some leads, it’s very incomplete, it is just a summary here. 

The process appears to involve activation of hepatic cells, leading to inflammation in the liver. Both hepatic and peripheral blood cells, like neutrophils and lymphocytes, are recruited, resulting in significant inflammation within the liver. While research is ongoing to understand the exact mechanisms, it’s important to note that inflammation plays a critical role in this transition.

Given the centrality of inflammation, biomarkers targeting liver inflammation are under investigation. However, these markers often capture generic signs of inflammation and cellular activation, lacking the specificity needed for accurate diagnosis or monitoring of NASH.

Biomarker Limitations

Currently, there is no ideal biomarker for diagnosing or monitoring NASH. Various markers related to inflammation and cell activation have been proposed, but they lack specificity. Given the complexity of NASH pathophysiology, relying on a single biomarker is not feasible. Therefore, combinations of biomarkers are typically used, although these also have limitations.

NASH: Non-Validated Markers and Tests

There are several markers and tests for evaluating NASH, though they are not yet validated and are primarily used for research purposes rather than clinical diagnostics. Below are some of these exploratory tests and markers:

NASH Test by Biopredictive

Biopredictive offers a NASH test aimed at evaluating liver inflammation and assessing steatohepatitis. Interestingly, it employs the same markers as the SteatoTest but uses a different calculation algorithm. The markers include Gamma-GT, Total Bilirubin, Alpha-2-macroglobulin, Apolipoprotein A1, Haptoglobin, Glucose, Triglycerides, and Cholesterol.

In external validation studies, higher NASH test numbers corresponded to higher levels on histology scores. However, the area under the curve (AUC) was around 0.7, similar to the SteatoTest. This means that there’s a risk of missing some cases, making its performance inconsistent.

FAST Score: A Comprehensive Approach

The FAST Score introduces a more integrated approach by combining both biomarkers and blood tests with imaging techniques like FibroScan. Specifically, it incorporates FibroScan for liver stiffness and controlled attenuation parameter measurements. It boasts an AUC of 0.8, indicating potentially good performance.

Despite its effectiveness, the practical application of the FAST Score can be hindered by the lack of availability of required biomarkers and imaging equipment in some healthcare settings. While promising, its effectiveness can be constrained if either of these components is not readily accessible in a given hospital.

NIS4 Biomarker Combination

In research settings, the NIS4 score, developed by GENFIT, offers an intriguing biomarker combination with solid performance metrics, boasting an area under the curve (AUC) of 0.8 across two different cohorts.

In the field of research, Dr. Guillaume Grzych highlights an innovative biomarker combination score developed by a GENFIT. This novel score has garnered attention for its robust performance, showing an area under the curve (AUC) of 0.8 across different patient cohorts.

This score measures markers such as miRNA, alpha-2 macroglobulin, YKL-40, and glycated haemoglobin. This combination holds promise for providing a reliable diagnosis of non-alcoholic steatohepatitis (NASH).

However, Dr. Grzych cautions that the real-world applicability of this promising score is not without limitations. The availability of these specific markers is essential.

While certain markers like glycated hemoglobin may be commonly available in labs, others like alpha-2 macroglobulin and YKL-40 may not be. These less common markers are not only challenging but also costly to measure, potentially limiting their use to specialized laboratories, especially in general healthcare settings or for large-scale population screening. 

For healthcare providers interested in leveraging new research, Dr. Grzych advises consulting with clinical chemists to assess the availability of these specific biomarkers in their respective hospital laboratories.

Dr. Grzych emphasizes that the focus of this discussion has been primarily on simple steatosis and NASH. The next session will delve into the biomarkers associated with hepatic fibrosis.