S100B protein as a marker of minimal hepatic encephalopathy

Introduction

Hepatic encephalopathy (HE) is a complex issue affecting patients with acute and chronic liver diseases. It is an interdisciplinary problem, addressed not only by gastroenterologists but also by internists, psychiatrists, neurologists, and family medicine doctors.

HE is a potentially reversible condition characterized by impaired central nervous system function. The impairments involve intellectual and motor functions, as well as activities characteristic of the central nervous system, including personality changes. Typical symptoms of HE include cognitive decline, ranging up to hepatic coma, disturbances in the sleep-wake cycle, muscle tremors, memory problems, and prolonged reaction times to various stimuli.

The symptoms of HE result from one of two mechanisms. In acute liver failure, due to a rapid decline in the number of normal hepatocytes, the liver’s detoxification capacity fails. In chronic liver failure, besides the aforementioned mechanism, there is also portal-systemic shunting, portal hypertension, and the formation of non-anatomical portal-systemic connections. As a result, blood from the portal system enters the systemic circulation bypassing the liver, thus not undergoing detoxification. In both cases, toxic substances and false neurotransmitters cross the damaged blood-brain barrier, leading to the inhibition of central nervous system (CNS) activity [1]. A similar effect occurs with the artificial creation of a portal-systemic shunt to reduce pressure in the portal system (TIPS). A potential unintended consequence of such a procedure is the occurrence or exacerbation of HE symptoms.

According to the recommendations of the European Association for the Study of the Liver (EASL) and the American Association for the Study of Liver Diseases (AASLD) [2], HE is classified based on its relation to the underlying disease, clinical manifestation, duration, and the presence or absence of precipitating factors. Minimal hepatic encephalopathy (MHE), along with grade I encephalopathy according to the West Haven Criteria (WHC), corresponds to covert encephalopathy according to the criteria of the International Society for Hepatic Encephalopathy and Nitrogen Metabolism (ISHEN) [3].

MHE is usually the first, subtle stage of CNS dysfunction and is therefore difficult to diagnose.

According to the current definition, MHE is a CNS dysfunction detected through targeted diagnostic tests or based on the presence of subtle clinical symptoms. It does not cause the typical disorientation and pathognomonic hand tremor (asterixis) seen in overt forms of HE. Available studies indicate that it may affect up to 80% of patients with liver cirrhosis [3]. The subclinical symptoms of MHE are extremely significant for the patient’s daily functioning. MHE impairs the ability to make quick reactions, such as when driving vehicles [4]. The quality of life for patients with this form of HE is reduced [5]. Moreover, the occurrence of MHE increases the risk of overt HE and worsens the overall prognosis [6].

Due to the lack of typical clinical symptoms, the detection of MHE relies on the application of psychometric tests, neurophysiological tests, and neuroimaging studies. Among the psychometric tests used are the Psychometric Hepatic Encephalopathy Score (PHES test), Animal Naming Test (ANT), Continuous Reaction Time (CRT) test, and Inhibitory Control Test (ICT). Recommended neurophysiological methods include electroencephalography (EEG), the Critical Flicker Frequency (CFF) test, and evoked potentials. The neuroimaging studies used include computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET).

AASLD/EASL guidelines recommend using at least two different tests in clinical studies. One of them should be the PHES test, and the other should be either another computer-assisted psychometric test or a neurophysiological study.

The aforementioned, somewhat unclear recommendations prompt the search for a simple serum biomarker that could aid in diagnosing MHE. Currently, the most commonly used indicator of HE is serum ammonia levels. However, elevated serum ammonia levels have been observed in patients with liver cirrhosis who do not exhibit signs of HE [7]. Additionally, factors such as active smoking and physical activity influence its serum level [8]. Moreover, its levels do not reflect the severity of HE.

In the described literature on astrocyte damage in HE [9, 10], attention is drawn to laboratory markers of central nervous system (CNS) injury. One such marker is SB100 protein. It is a small, acidic protein that binds calcium ions and is found in various organs. The highest concentration of this protein has been reported in brain tissue of many mammalian species. It is also present in lower concentrations in adipose tissue and skin. Elevated levels have been observed in patients with mechanical brain injury [11] and ischemic stroke [12].

Aim

The aim of the study was to evaluate the potential correlation between the serum levels of SB100P protein in patients with MHE symptoms and the levels of SB100P protein in patients with MHE.

Material and methods

The study enrolled patients with liver cirrhosis accompanied by MHE. MHE was diagnosed based on the PHES test using standardization for the Polish population [13]. According to Polish population standards, a cutoff score of -5 on the PHES test was used for diagnosis.

The PHES test was conducted on patients with liver cirrhosis of various etiologies who were hospitalized at the Department of Gastroenterology and Hepatology at the University Clinical Hospital in Wroclaw. Patients with overt HE and those with cognitive impairments suggestive of dementia were excluded from the study. The Mini-Mental State Examination (MMSE), a standardized set of 11 scored questions, was used for this purpose. Patients were eligible for the study if they had an MMSE score of 25 or higher, indicating no cognitive impairment.

For the quantitative determination of SB100 protein concentration in serum, the CanAg S100 EIA kit produced by Fujirebio Diagnostic Inc. was used. This is a two-step immunoassay based on two mouse monoclonal antibodies specific for two different epitopes expressed in the S100B protein. During the enzymatic reaction occurring in the test, the appearance of a blue color confirms the presence of S100B, and the color intensity increases proportionally to the amount of protein in the sample. The color intensity was measured spectrometrically at a wavelength of 620 nm. The results were read from the calibration curves included in the kit. The CanAg S100 EIA test is designed to measure S100B concentrations in the range of 10–3500 pg/ml.

For the study, venous blood was collected from fasting patients, and then the serum was separated. The serum samples were stored at –20°C, and the immunoenzymatic analysis was conducted after thawing the samples and bringing them to room temperature.

In addition to the concentration of S100B protein, serum aminotransferases activity, bilirubin, creatinine, total protein, sodium, and prothrombin time were measured in all patients, along with total protein and albumin levels. All patients included in the study were assessed using the MELD-Na scale, with scores ranging from 7 to 40 points (average of 20.6 points).

Results

The study included 36 patients diagnosed with MHE based on the PHES test. Among them were 14 (35%) women and 22 (65%) men. In half of the patients (55%), chronic liver damage was caused by the toxic effects of alcohol. The remaining 45% had an autoimmune background. The average age of the patients was 51.4 years. The results of the PHES test, used to diagnose HE, ranged from –5 to –11 points. The serum S100B protein level, measured using the CanAg S100 EIA test according to the manufacturer’s recommendations, ranged from 21 to 194.2 ng/l (mean: 84.9, median: 85.9) (Table I).

Based on the performed statistical analysis, a statistically significant correlation was found between serum S100B protein levels and PHES test results (rho = –0.35, p = 0.036). No statistically significant correlation was found between S100B protein levels and liver function as expressed by the MELD-Na score (rho = 0.27, p = 0.1). Analyzing the individual components of the MELD-Na score, a statistically significant correlation was found between serum S100B protein levels and sodium levels (rho = 0.42, p = 0.01) and alanine aminotransferase activity (rho = 0.33, p = 0.047). The other components of the MELD-Na score did not show a correlation with S100B protein levels. Additionally, no statistically significant correlation was found between serum S100B protein levels and ammonia levels.

The level of S100B protein in patients with MHE was higher than the average for the healthy population provided by the test manufacturer (15.6 ng/l) in 30 (83%) patients. The S100B protein level also showed a statistically significant correlation with the age of the patients (rho = 0.38, p = 0.022).

Discussion

Published studies on the role of serum S100B protein levels in patients with HE have been inconclusive. A 1999 study (Witlfang) measured this protein in patients with HE caused by portosystemic shunting. The results indicated a correlation between S100B protein levels and HE, suggesting that S100B may have greater diagnostic value for MHE than ammonia. Similarly, a 2007 study [14] demonstrated statistically significantly higher S100B protein levels in patients with overt HE (grade I and II according to the West Haven Criteria) compared to a healthy control group.

In a study by Duarte Rojo et al. [15], significantly elevated S100B protein levels were found in patients with liver cirrhosis, with the highest levels observed in patients with HE. A more recent study by Strobel et al. [16] also showed an association between elevated S100B protein levels and both overt and covert HE. However, this study did not establish a correlation between serum S100B levels and the severity of HE.

Conversely, some studies have presented different conclusions. In a 2003 study by Vaquero (2003) [17], no statistically significant correlation was found between S100B levels and the severity of HE or liver function. One pediatric study [18] conducted in patients with acute liver injury of various etiologies demonstrated a link between S100B protein levels and the presence of HE (p = 0.04), but did not find a correlation between S100B levels and the severity of HE.

The number of studies published to date and their differing results do not justify considering S100B protein as a marker of HE. All cited studies were conducted on small, often heterogeneous groups of patients. Additionally, they primarily focused on patients with overt HE, where the search for a biomarker appears to be less critical compared to MHE.

The results of the present study showed a higher level of S100B protein in 83% of patients with MHE compared to the healthy population. A significant correlation (p = 0.036) was found between the level of S100B protein and the PHES test results (expressed in negative values), indicating a possible role of S100B protein as a useful biomarker in MHE diagnosis. There was no significant correlation between the level of the examined protein and liver function expressed by the commonly used MELD-Na index. Therefore, it seems that it should not be considered as a marker of liver damage severity.

An important limitation of the study is the number of patients included in the study, which is associated with narrowing the study group to patients with MHE, without other cognitive impairments. Further research on a significantly larger group of patients is necessary to definitively verify S100B protein as a potential biomarker of HE. Considering the significant correlation between the level of S100B protein and the age of the studied patients (p = 0.22), this parameter will be extremely important in future studies.

Conclusions

The results of the conducted study suggest a potential role of serum S100B protein levels in patients with MHE. A significant correlation was found between the score obtained in the PHES test and the level of S100B protein. However, a limitation of the study is the small sample size, which does not allow for the definitive recognition of S100B protein as a significant biomarker for MHE. Therefore, it was not possible to establish a cutoff point confirming or excluding the diagnosis. Nevertheless, these results are encouraging for further research into the role of S100B protein in the diagnosis of MHE.