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Elevated serum levels of glial fibrillary acidic protein are associated with covert hepatic encephalopathy in patients with cirrhosis

  • Simon Johannes Gairing
    Affiliations
    Department of Internal Medicine I, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

    Cirrhosis Center Mainz (CCM), University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
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  • Sven Danneberg
    Affiliations
    Department of Medicine I, University Hospital Schleswig-Holstein, Lübeck, Germany
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  • Leonard Kaps
    Affiliations
    Department of Internal Medicine I, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

    Cirrhosis Center Mainz (CCM), University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
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  • Michael Nagel
    Affiliations
    Department of Internal Medicine I, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

    Cirrhosis Center Mainz (CCM), University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
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  • Eva Maria Schleicher
    Affiliations
    Department of Internal Medicine I, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

    Cirrhosis Center Mainz (CCM), University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
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  • Charlotte Quack
    Affiliations
    Department of Internal Medicine I, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

    Cirrhosis Center Mainz (CCM), University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
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  • Sinah Engel
    Affiliations
    Department of Neurology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
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  • Stefan Bittner
    Affiliations
    Department of Neurology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
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  • Peter Robert Galle
    Affiliations
    Department of Internal Medicine I, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

    Cirrhosis Center Mainz (CCM), University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
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  • Jörn Markus Schattenberg
    Affiliations
    Department of Internal Medicine I, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

    Metabolic Liver Research Program, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
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  • Marcus-Alexander Wörns
    Affiliations
    Department of Internal Medicine I, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

    Cirrhosis Center Mainz (CCM), University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

    Department of Gastroenterology, Hematology, Oncology and Endocrinology, Klinikum Dortmund, Germany
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  • Author Footnotes
    # these authors share senior-authorship
    Felix Luessi
    Footnotes
    # these authors share senior-authorship
    Affiliations
    Department of Neurology, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
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  • Author Footnotes
    # these authors share senior-authorship
    Jens Uwe Marquardt
    Footnotes
    # these authors share senior-authorship
    Affiliations
    Department of Medicine I, University Hospital Schleswig-Holstein, Lübeck, Germany
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  • Author Footnotes
    # these authors share senior-authorship
    Christian Labenz
    Correspondence
    Corresponding author: Department of Internal Medicine I, University Medical Center of the Johannes Gutenberg-University, Langenbeckstrasse 1, 55131 Mainz, Germany, Telephone: +49 (0) 6131 17 2380, Telefax: +49 (0) 6131 17 477282,
    Footnotes
    # these authors share senior-authorship
    Affiliations
    Department of Internal Medicine I, University Medical Center of the Johannes Gutenberg-University, Mainz, Germany

    Cirrhosis Center Mainz (CCM), University Medical Center of the Johannes Gutenberg-University, Mainz, Germany
    Search for articles by this author
  • Author Footnotes
    # these authors share senior-authorship
Open AccessPublished:January 17, 2023DOI:https://doi.org/10.1016/j.jhepr.2023.100671

      Highlights

      • Blood biomarkers for covert hepatic encephalopathy (CHE) in patients with cirrhosis are lacking
      • Patients with CHE had significantly higher serum GFAP (sGFAP) levels than patients without CHE or healthy controls
      • sGFAP correlated with ammonia and interleukin-6 serum levels
      • sGFAP levels did not differ between patients with alcoholic vs non-alcoholic cirrhosis or between ongoing vs discontinued alcohol use

      Abstract

      Background & Aims

      Blood biomarkers facilitating the diagnosis of covert hepatic encephalopathy (CHE) in patients with cirrhosis are lacking. Astrocyte swelling is a major component of HE. Thus, we hypothesized that glial fibrillary acidic protein (GFAP), the major intermediate filament of astrocytes, might facilitate early diagnosis and management. This study aimed to investigate the utility of GFAP serum (sGFAP) levels as a biomarker of CHE.

      Methods

      In this bicentric study, 135 patients with cirrhosis, 21 patients with ongoing harmful alcohol use and cirrhosis, and 15 healthy controls were recruited. CHE was diagnosed using psychometric hepatic encephalopathy score (PHES). sGFAP levels were measured by highly sensitive single molecule array (SiMoA) immunoassay.

      Results

      In total, 50 (37%) patients presented with CHE at study inclusion. Patients with CHE displayed significantly higher sGFAP levels than patients without CHE (median sGFAP (163 pg/ml (interquartile range (IQR) 136; 268) vs 106 pg/ml (IQR 75; 153, p < 0.001) or healthy controls (p < 0.001). sGFAP correlated with results in PHES (Spearman's r = -0.326, p < 0.001), MELD score (Spearman's rho = 0.253, p = 0.003), ammonia (Spearman’s rho = 0.453, p = 0.002) and interleukin-6 (IL-6) serum levels (Spearman's rho = 0.323, p = 0.006). Additionally, sGFAP levels were independently associated with the presence of CHE in multivariable logistic regression analysis (odds ratio (OR): 1.009 (95% confidence interval (CI) 1.004 – 1.015; p < 0.001). sGFAP levels did not differ between patients with alcoholic vs non-alcoholic cirrhosis or between ongoing vs discontinued alcohol use.

      Conclusions

      sGFAP levels are associated with CHE in patients with cirrhosis. These results suggest that astrocyte injury may already occur in patients with cirrhosis and subclinical cognitive deficits and could be explored as a novel biomarker.

      Lay summary

      Blood biomarkers facilitating the diagnosis of covert hepatic encephalopathy (CHE) in patients with cirrhosis are lacking. In this study, we were able to demonstrate that sGFAP levels are associated with CHE in patients with cirrhosis. These results suggest that astrocyte injury may already occur in patients with cirrhosis and subclinical cognitive deficits and sGFAP could be explored as a novel biomarker.

      Graphical abstract

      Keywords

      Abbreviations:

      HE (hepatic encephalopathy), MHE (minimal hepatic encephalopathy), CHE (covert hepatic encephalopathy), OHE (overt hepatic encephalopathy), IL-6 (interleukin-6), GFAP (glial fibrillary acidic protein), sGFAP (glial fibrillary acidic protein serum levels), PSE (portosystemic encephalopathy), PHES (Psychometric Hepatic Encephalopathy Score), SiMoA (single molecule array), CLIA (Chemiluminescence immunoassay), ULN (upper limit of normal), AUC (area under the curve), ROC (receiver operating characteristic), CI (confidence interval), IQR (interquartile range), MELD (Model of end-stage liver disease), OR (odds ratio), AUROC (area under the receiver operating characteristics), WBC (white blood cell)

      Introduction

      Hepatic encephalopathy (HE) is a severe complication of cirrhosis
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      Hepatic encephalopathy: Novel insights into classification, pathophysiology and therapy.
      . The two lowest HE grades - minimal HE (MHE) and HE grade I - are collectively depicted as covert HE (CHE)
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      • et al.
      Hepatic encephalopathy in chronic liver disease: 2014 Practice Guideline by the American Association for the Study of Liver Diseases and the European Association for the Study of the Liver.
      . Although signs and symptoms of CHE are commonly subclinical and only detectable with specialized tests, CHE impairs health-related quality of life and is associated with an increased risk for the development of overt HE (OHE), hospitalization and death
      • Patidar K.R.
      • Bajaj J.S.
      Covert and Overt Hepatic Encephalopathy: Diagnosis and Management.
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      • et al.
      Covert hepatic encephalopathy is independently associated with poor survival and increased risk of hospitalization.
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      • et al.
      Prospective evaluation of the impact of covert hepatic encephalopathy on quality of life and sleep in cirrhotic patients.
      . In consequence, CHE often remains undiagnosed in routine clinical practice which prevents the initiation of medical therapy
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      Management der hepatischen Enzephalopathie in Deutschland: eine Umfrage unter Gastroenterologen und Allgemeinmedizinern.
      . Consequently, easy-to-use and reliable testing strategies would be beneficial to support the diagnosis. In this context, diagnostic serum biomarkers facilitating the diagnosis of CHE are of particular relevance. Moreover, they may contribute to a deeper understanding of the underlying pathophysiological processes. Recent studies indicate the diagnostic value of Interleukin-6 (IL-6) for the diagnosis of CHE as well as risk prediction of OHE development in patients with liver cirrhosis
      • Gairing S.J.
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      • et al.
      Evaluation of IL-6 for Stepwise Diagnosis of Minimal Hepatic Encephalopathy in Patients With Liver Cirrhosis.
      . Additionally, a recent study indicated the prognostic impact of serum ammonia on OHE development and mortality
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      • Alexandrino G.
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      • et al.
      Plasma ammonia levels predict hospitalisation with liver-related complications and mortality in clinically stable outpatients with cirrhosis.
      . However, these markers have the disadvantage of responding unspecifically to inflammatory processes (IL-6) or have susceptible pre-analytics (ammonia).
      From a pathophysiological perspective, hyperammonemia is a hallmark of HE. High ammonia levels induce astrocyte swelling, cerebral edema and neuronal cell death
      • Sepehrinezhad A.
      • Zarifkar A.
      • Namvar G.
      • Shahbazi A.
      • Williams R.
      Astrocyte swelling in hepatic encephalopathy: molecular perspective of cytotoxic edema.
      ,
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      • Kerbert A.J.C.
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      • Hall A.
      • Abramov A.Y.
      • Jalan R.
      Hyperammonaemia induces mitochondrial dysfunction and neuronal cell death.
      . Thus, markers of neuronal or astrocyte cell injury may be valuable for the diagnosis of HE. In this context, glial fibrillary acidic protein (GFAP), the most abundant intermediate filament protein in the cytoskeleton of astrocytes
      • Abdelhak A.
      • Foschi M.
      • Abu-Rumeileh S.
      • Yue J.K.
      • D'Anna L.
      • Huss A.
      • et al.
      Blood GFAP as an emerging biomarker in brain and spinal cord disorders.
      , might be of particular relevance. In general, GFAP is a biomarker indicating astrocyte injury or activation
      • Abdelhak A.
      • Foschi M.
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      • Yue J.K.
      • D'Anna L.
      • Huss A.
      • et al.
      Blood GFAP as an emerging biomarker in brain and spinal cord disorders.
      . GFAP is a critical cell structural protein and plays a role in various physiological processes such as the maintenance of the blood-brain barrier, astrocyte migration and proliferation
      • Middeldorp J.
      • Hol E.M.
      GFAP in health and disease.
      ,
      • Butterworth R.F.
      Pathogenesis of hepatic encephalopathy and brain edema in acute liver failure.
      . In addition, various diseases such as traumatic brain injuries or neurodegeneration result in reactive (astro)gliosis and an increased expression of intermediate filament proteins such as GFAP
      • Middeldorp J.
      • Hol E.M.
      GFAP in health and disease.
      . Today, even very low GFAP levels in the blood can be detected using high-sensitivity assays
      • Abdelhak A.
      • Huss A.
      • Kassubek J.
      • Tumani H.
      • Otto M.
      Serum GFAP as a biomarker for disease severity in multiple sclerosis.
      .
      In this proof-of-concept study, we hypothesized that ammonia-driven astrocyte swelling and mild neuronal cell death with consecutive reactive gliosis in patients with CHE may lead to elevated GFAP serum (sGFAP) levels, which could be used as a novel biomarker for CHE in patients with cirrhosis.

      Patients and methods

      Study cohort

      In this proof-of-concept study, the data of prospectively recruited patients (between 07/2019 and 07/2021) from two German tertiary care centers (University of Mainz, University of Lübeck) were retrospectively analyzed. Patients from Mainz were recruited during outpatient visits or during elective measurement of the hepatic venous pressure gradient. Patients from Lübeck were recruited during outpatient visits or during hospital admission due to reasons other than HE. Diagnosis of liver cirrhosis was based on histology, conclusive appearance in ultrasound or radiological imaging, endoscopic features of portal hypertension, and medical history. Patients with liver cirrhosis with previous episode of OHE during the last six weeks, pre-terminal comorbidities, severe neurological comorbidities, a history of or ongoing chemotherapy with paclitaxel and carboplatin, or ongoing chronic harmful alcohol consumption were not approached for this study. In addition to the main cohort, a second cohort including 21 patients with ongoing chronic harmful alcohol use was recruited in Lübeck. Additionally, we obtained blood from 10 patients with HE grade 2 (HE2) according to the West-Haven-Criteria (Lübeck n = 8, Mainz n = 2). Furthermore, 15 healthy individuals were included, who did not meet criteria for neurological or psychiatric diseases.
      Blood was collected from all patients for biochemical analysis immediately after study inclusion (between 9 a.m. and 2 p.m.). Specifically, blood samples for the determination of venous ammonia levels were immediately cooled on ice and analysed without delay.

      Diagnosis of CHE

      First, all patients were screened for clinical signs of OHE according to the West-Haven criteria
      • Vilstrup H.
      • Amodio P.
      • Bajaj J.
      • Cordoba J.
      • Ferenci P.
      • Mullen K.D.
      • et al.
      Hepatic encephalopathy in chronic liver disease: 2014 Practice Guideline by the American Association for the Study of Liver Diseases and the European Association for the Study of the Liver.
      . Next, detailed medical history, physical examination and, if indicated, additional examinations were performed to rule out active infections. If OHE and active infections were excluded, the portosystemic encephalopathy (PSE) syndrome test (gold standard) was used to screen for CHE. Due to the bicentric character of the study and uncertainty of diagnosis of grade 1 HE between different centers, we decided to use the term CHE and not to distinguish between grade 1 HE and MHE
      • Reuter B.
      • Walter K.
      • Bissonnette J.
      • Leise M.D.
      • Lai J.
      • Tandon P.
      • et al.
      Assessment of the spectrum of hepatic encephalopathy: A multicenter study.
      . CHE was diagnosed in patients with a psychometric hepatic encephalopathy score (PHES) below -4 according to the age-adjusted German norms published by Weissenborn et al. (Hannover, Germany)
      • Patidar K.R.
      • Bajaj J.S.
      Covert and Overt Hepatic Encephalopathy: Diagnosis and Management.
      . Both centres used the same test manual, which is commercially available with German norms, from Weissenborn et al. The PHES consists of the number connection test-A, the number connection test-B, the digit symbol test, the serial dotting test, and the line tracing test. To minimize distraction and other confounding factors, patients performed the test between 9 a.m. and 2 p.m. in a quiet, separate room.

      Determination of sGFAP levels

      Blood samples were spun at 2000g at room temperature for 10 minutes within 60 minutes after withdrawal at the day of study inclusion and stored in polypropylene tubes at -80°C until batched sGFAP analysis. sGFAP levels were determined using the highly sensitive SiMoA technology. Samples were measured in duplicates in several rounds by SiMoA HD-1 (Quanterix, USA) using the GFAP Discovery kits according to manufacturer’s instructions. Resorufin-β-D-galactopyranoside (RGP) was incubated at 33°C for 60 minutes prior to running the assay. The coefficient of variation (CV, as a percentage) of the two replicates was obtained by dividing the standard deviation of both replicates by the mean of both replicates multiplied by 100. CVs above 20% (or missing replicate result) were measured twice. Measurements were performed in a blinded fashion without information about clinical data.

      Determination of IL-6 and ammonia serum levels

      IL-6 serum levels were determined in a subset of patients at the day of study inclusion immediately after testing with PHES using a commercially available Chemiluminescence immunoassay (CLIA) (Cobas e 411 Analyzer, F. Hoffmann-La Roche AG, Basel, Swiss)
      • Labenz C.
      • Nagel M.
      • Kämper P.
      • Engel S.
      • Bittner S.
      • Kaps L.
      • et al.
      Association Between Serum Levels of Neurofilament Light Chains and Minimal Hepatic Encephalopathy in Patients With Liver Cirrhosis.
      .
      Venous ammonia levels were measured according to a standard operating procedure that involved rapid sample transport on ice to the central laboratory within five minutes and the upper limit of normal (ULN) was 72 μmol/l.

      Ethics

      The study was conducted in accordance with the ethical guidelines of the 1975 Declaration of Helsinki (6th revision, 2008). The studies for both cohorts (Mainz and Lübeck) were approved by the ethics committee of the Landesärztekammer Rheinland-Pfalz and of the University of Lübeck. Written informed consent was obtained from all participants.

      Statistical analysis

      Categorical data are expressed as frequencies and percentages, metric data as medians with interquartile ranges (IQR). Differences between two independent groups (e.g. CHE vs no CHE) with metric data (e.g. sGFAP) were evaluated using an unpaired t-test or a Mann-Whitney U Test depending on data distribution. Differences between three or more groups with metric data were evaluated using an ordinary one-way ANOVA with Tukey’s multiple comparisons test. For comparison of two or more patient groups with categorical variables a chi-square test was applied. For correlation analyses, two-tailed spearman’s rank correlation coefficient (Spearman’s ρ) was used. To identify predictors for the presence of CHE, multivariable logistic regression models were used. Multivariable linear regression models were performed to determine associations of variables with continuous dependent variables (PHES or sGFAP). The discriminate ability of sGFAP for the identification of patients with CHE was analysed with help of the area under the curve (AUC) of receiver operating characteristic (ROC) curves and its respective 95% confidence interval (95% CI). P values below 0.05 were considered significant. Statistical analyses were performed with IBM SPSS Statistics (version 27, Armonk, NY: IBM Corp) and GraphPad Prism (version 9.4, GraphPad Software, San Diego, California, USA).

      Results

      Demographics and baseline characteristics

      In total, 135 patients were included into this study (Mainz: 85, Lübeck: 50). Baseline characteristics of the study cohort are displayed in Table 1. At study inclusion, 50 patients (37%) were diagnosed with CHE by PHES. The characteristics of the patients stratified by study sites (Mainz and Lübeck) are displayed in Supplementary Table 1.
      Table 1Baseline characteristics of the study cohort.
      VariableAll patients n = 135Patients with CHE n = 50Patients without CHE n = 85p-value
      Age, y (IQR)60 (53; 66)61 (54; 68)60 (53; 66)0.356
      Male gender, n (%)67 (50)28 (56)39 (46)0.256
      AetiologyAlcohol, n (%)80 (59)37 (74)43 (51)0.008
      Other, n (%)55 (41)13 (26)42 (49)
      Median MELD score (IQR)11 (9; 16)14 (11; 18)10 (8; 15)<0.001
      Child-Pugh A/B/C, n (%)64/60/11 (47/44/8)11/32/7 (22/64/14)53/28/4 (62/33/5)<0.001
      History of OHE, n (%)17 (13)12 (24)5 (6)0.002
      History of ascites, n (%)84 (62)40 (80)44 (52)0.001
      Sodium, mmol/l (IQR)138 (135; 140)137 (133; 140)138 (137; 140)0.016
      Albumin, g/l (IQR)32 (27; 39)28 (24; 35)35 (29; 43)<0.001
      WBC, /nl (IQR)5.6 (3.8; 7.6)5.3 (3.8; 7.5)5.8 (3.9; 8.0)0.693
      IL-6, pg/ml (IQR)
      measured in 71 patients.
      19 (9; 40)33 (16; 66)13 (5; 30)0.001
      Ammonia, μmol/l (IQR)
      measured in 43 patients.
      44 (35; 58)55 (44; 65)38 (31; 47)<0.001
      GFAP, pg/ml (IQR)138 (87; 196)163 (136; 268)106 (75; 153)<0.001
      CHE, n (%)50 (37)50 (100)0 (0)
      Median PHES (IQR)-3 (-8; 0)-10 (-12; -7)-1 (-3; 1)<0.001
      Data are expressed as medians and interquartile ranges (IQR) or as frequencies and percentages. Differences between patients with CHE vs without CHE with metric data were evaluated using an unpaired t-test or a Mann-Whitney U Test depending on data distribution. Regarding categorical variables, a chi-square test was applied. MELD, model for end-stage liver disease; WBC, white blood cell count; IL-6, Interleukin-6; OHE, overt hepatic encephalopathy; CHE, covert hepatic encephalopathy; PHES, psychometric hepatic encephalopathy score.
      # measured in 71 patients.
      measured in 43 patients.

      Correlation of sGFAP with MELD, PHES, IL-6 and ammonia levels

      sGFAP levels were inversely correlated with PHES (Spearman’s rho = -0.326, p < 0.001, n = 135). In addition, sGFAP levels were positively correlated with the MELD score (Spearman’s rho = 0.253, p = 0.003, n = 135), IL-6 serum levels (Spearman’s rho = 0.323, p = 0.006, n = 70) and ammonia levels (Spearman’s rho = 0.453, p = 0.002, n = 43) (Figure 1).
      Figure thumbnail gr1
      Figure 1Correlation between glial fibrillary acidic protein (GFAP) and PHES, MELD, Interleukin-6 or ammonia. A displays the correlation between serum levels of GFAP and PHES. B displays the correlation between serum levels of GFAP and MELD. C displays the correlation between serum levels of GFAP and Interleukin-6 (IL-6). D displays the correlation between serum levels of GFAP and ammonia. A two-tailed spearman’s rank correlation coefficient (Spearman’s ρ) was used, respectively. r: spearman's ρ.

      Association of sGFAP with CHE

      Patients diagnosed with CHE according to the PHES had significantly higher sGFAP levels than patients without CHE (163 pg/ml (IQR 136; 268) vs 106 pg/ml (IQR 75; 153, p < 0.001, Figure 2A). This finding was confirmed in sensitivity analyses for both centers (Figure 2B/C). In addition, patients without CHE had higher sGFAP levels compared to healthy controls (median sGFAP levels 106 pg/ml vs 60 pg/ml, p < 0.05, Figure 2A). sGFAP levels were also significantly elevated in patients with CHE compared to patients without CHE, when all patients with a history of OHE were excluded (median sGFAP levels 169 vs 109, p < 0.001, Figure 2D). Patients with HE2 had numerically higher sGFAP levels compared to patients with CHE (222 pg/ml (IQR 176; 280) vs 163 pg/ml (IQR 136; 268), p = 0.744, Figure 2E)
      Figure thumbnail gr2
      Figure 2Serum levels of glial fibrillary acidic protein (GFAP) in different patient cohorts. A displays GFAP serum levels in patients with cirrhosis with (n=50) or without CHE (n=85) and healthy individuals (n=15). B displays GFAP serum levels in patients from the Mainz cohort with cirrhosis with or without CHE. C displays GFAP serum levels in patients from the Lübeck cohort with cirrhosis with or without CHE. D displays GFAP serum levels in patients without a history of OHE with or without CHE. E displays GFAP serum levels in patients with HE2, CHE and without HE. Data are presented as boxplots with median, IQR and range. Differences between two independent groups (e.g., CHE vs no CHE) with metric data were evaluated using a Mann-Whitney U test. Differences between three or more groups with metric data were evaluated using an ordinary one-way ANOVA with Tukey’s multiple comparisons test. ns: not significant (E: HE2 vs CHE, p = 0.744), *: p < 0.05, **: p < 0.01, ***: p < 0.001. ****: p < 0.0001.
      To investigate whether sGFAP is independently associated with the presence of CHE in patients with cirrhosis, a multivariable logistic regression model was built. Besides sGFAP, variables that are known to be potentially associated with CHE were included into the regression model (MELD, age, albumin serum levels, history of OHE). In this model, sGFAP levels remained significantly associated with the presence of CHE with an odds ratio (OR) of 1.009 (95% CI 1.004 – 1.015; p < 0.001) (Table 2). Moreover, we repeated this analysis after exclusion of all patients with a history of OHE (n = 118 patients included in the model). Again, sGFAP levels remained significantly associated with the presence of CHE (OR 1.009, 95% CI 1.004 – 1.014; p < 0.001) (Table 2).
      Table 2Multivariable logistic regression models to analyze the association of variables with covert hepatic encephalopathy (CHE).
      VariablesOR (95% CI)p-value
      Total cohort (n = 135)
      GFAP1.009 (1.004 – 1.015)<0.001
      MELD1.038 (0.945 – 1.140)0.433
      Male gender1.482 (0.642 – 3.422)0.357
      Albumin0.933 (0.877 – 0.992)0.028
      History of OHE4.662 (1.297 – 16.755)0.018
      Only patients without a history of OHE (n = 118)
      GFAP1.009 (1.004 – 1.014)<0.001
      MELD1.035 (0.939 – 1.140)0.489
      Male gender1.385 (0.573 – 3.346)0.470
      Albumin0.934 (0.876 – 0.995)0.035
      OR, odds ratio; 95% CI, 95% confidence interval; GFAP, Glial fibrillary acidic protein; MELD, model for end-stage liver disease; OHE, overt hepatic encephalopathy.
      Next, we investigated whether sGFAP is independently associated with poorer results in PHES in patients with cirrhosis. Here, we conducted a linear regression analysis including the same variables as mentioned above. In this regression analysis, higher sGFAP levels remained significantly associated with poorer results in PHES (β -0.251, p < 0.001) (Table 3).
      Table 3Multivariable linear regression model to analyze the association of variables with PHES.
      VariablesRegression coefficient95 % CI of regression coefficientβp-value
      GFAP-0.013-0.021 – -0.006-0.251<0.001
      MELD-0.131-0.297 – 0.034-0.1340.118
      Gender-0.782-2.202 – 0.639-0.0780.278
      Albumin0.1650.059 – 0.2710.2660.002
      History of OHE4.0901.921 – 6.2580.270<0.001
      95% CI. 95% confidence interval; GFAP, Glial fibrillary acidic protein; MELD, model for end-stage liver disease; OHE, overt hepatic encephalopathy.
      R2: 0.357
      Finally, we sought to detect variables independently associated with higher sGFAP levels by using linear regression analysis. The only factors associated with higher sGFAP levels were higher age (β 0.281, p < 0.001) and the presence of CHE (β 0.336, p < 0.001) (Table 4).
      Table 4Multiple linear regression model to analyze the association of variables with sGFAP levels.
      VariablesRegression coefficient95 % CI of regression coefficientβp-value
      CHE67.02833.041 – 101.0140.336<0.001
      MELD1.126-2.364 – 4.6160.0600.524
      Gender-0.531-30.750 – 29.688-0.0350.972
      Albumin-1.000-3.313 – 1.313-0.0840.394
      History of OHE17.494-29.352 – 64.3400.0600.461
      Age2.6631.148 – 4.1770.281<0.001
      95% CI. 95% confidence interval; GFAP, Glial fibrillary acidic protein; MELD, model for end-stage liver disease; CHE, covert hepatic encephalopathy; OHE, overt hepatic encephalopathy.
      R2: 0.228
      To examine the diagnostic accuracy of sGFAP in detecting CHE, ROC curve analyses were performed. The area under the ROC (AUROC) curve for sGFAP was 0.736 (95% CI 0.649 – 0.823, p < 0.001) in the total cohort.

      Association of harmful alcohol use with sGFAP levels

      To investigate the association of a history of harmful alcohol use with sGFAP levels, we conducted a sensitivity analysis between patients with and without an alcoholic aetiology of cirrhosis. As displayed in Figure 3A, patients with an alcoholic aetiology and CHE did not differ in terms of sGFAP levels compared to patients with a non-alcoholic aetiology and CHE. The same was true for patients without CHE and an alcoholic aetiology compared to non-alcoholic patients (Figure 3A).
      Figure thumbnail gr3
      Figure 3Serum levels of glial fibrillary acidic protein (GFAP) in patients with and without alcoholic liver cirrhosis. A displays GFAP serum levels in patients with cirrhosis with or without CHE stratified by alcoholic aetiology or non-alcoholic aetiology of cirrhosis. B displays GFAP serum levels in patients with active or discontinued harmful alcohol use and PHES ≥ - 4. Data are presented as boxplots with median, IQR and range. Differences between two independent groups with metric data were evaluated using a Mann-Whitney U test. Differences between three or more groups with metric data were evaluated using an ordinary one-way ANOVA with Tukey’s multiple comparisons test. p-values are displayed in the respective figure.
      Additionally, we investigated the impact of ongoing harmful alcohol use on sGFAP levels in patients with and without CHE. For this purpose, we compared the data of the main cohort with 21 additionally recruited patients with ongoing harmful alcohol use. Of these, 12 (57%) were classified as having “CHE” according to PHES. Although those patients were not tested in a state of acute intoxication, it has to be acknowledged in these cases that CHE cannot be sufficiently diagnosed due to the confounding effect of alcohol. As displayed in Figure 3B, sGFAP levels did not differ between patients with an active and a discontinued harmful alcohol use in the subcohorts of patients with a PHES ≥ -4. This finding was also validated in a comparison of sGFAP levels between patients with an ongoing and a discontinued harmful alcohol use and a PHES < - 4 (p = 0.072).

      Discussion

      Identification of novel blood biomarkers to facilitate CHE diagnosis and to elucidate underlying pathophysiological processes accompanying CHE development are urgently required. In this bicentric study, we demonstrate that sGFAP levels are significantly increased in patients with cirrhosis and CHE compared to patients without CHE. In addition, sGFAP levels correlate with the PHES, ammonia and systemic inflammation, as reflected by IL-6 serum levels. Our data may therefore provide indirect evidence of a potential astrocyte injury and/or activation with subsequent release of GFAP into the serum in patients with liver cirrhosis and CHE.
      As the major intermediate filament protein in astrocytes, GFAP is a biomarker of astrocyte injury and/or activation
      • Abdelhak A.
      • Foschi M.
      • Abu-Rumeileh S.
      • Yue J.K.
      • D'Anna L.
      • Huss A.
      • et al.
      Blood GFAP as an emerging biomarker in brain and spinal cord disorders.
      . High-sensitivity assays allow the detection of even low GFAP concentrations in the blood
      • Abdelhak A.
      • Huss A.
      • Kassubek J.
      • Tumani H.
      • Otto M.
      Serum GFAP as a biomarker for disease severity in multiple sclerosis.
      . In addition, point-of-care tests are currently under investigation, which could provide sGFAP levels within no more than 15 minutes
      • Abdelhak A.
      • Foschi M.
      • Abu-Rumeileh S.
      • Yue J.K.
      • D'Anna L.
      • Huss A.
      • et al.
      Blood GFAP as an emerging biomarker in brain and spinal cord disorders.
      ,
      • Yue J.K.
      • Yuh E.L.
      • Korley F.K.
      • Winkler E.A.
      • Sun X.
      • Puffer R.C.
      • et al.
      Association between plasma GFAP concentrations and MRI abnormalities in patients with CT-negative traumatic brain injury in the TRACK-TBI cohort: a prospective multicentre study.
      . However, it must be acknowledged that the determination of GFAP is currently very costly. Recently, sGFAP levels were found to be associated with disease severity in patients with chronic inflammatory central nervous system diseases such as progressive multiple sclerosis
      • Abdelhak A.
      • Huss A.
      • Kassubek J.
      • Tumani H.
      • Otto M.
      Serum GFAP as a biomarker for disease severity in multiple sclerosis.
      . In addition, normal sGFAP levels after out-of-hospital cardiac arrest were associated with favorable neurological outcomes after six months
      • Moseby-Knappe M.
      • Mattsson-Carlgren N.
      • Stammet P.
      • Backman S.
      • Blennow K.
      • Dankiewicz J.
      • et al.
      Serum markers of brain injury can predict good neurological outcome after out-of-hospital cardiac arrest.
      . Importantly, sGFAP levels are more stable in the context of preanalytics and showed a higher association with the pathology of cerebral amyloid beta compared to GFAP levels in the cerebrospinal fluid
      • Simrén J.
      • Weninger H.
      • Brum W.S.
      • Khalil S.
      • Benedet A.L.
      • Blennow K.
      • et al.
      Differences between blood and cerebrospinal fluid glial fibrillary Acidic protein levels: The effect of sample stability.
      . Thus, sGFAP is a fast, readily accessible and reliable biomarker. Our current study expands this growing body of evidence on the potential clinical usefulness of measuring sGFAP beyond neuro-degenerative applications by demonstrating higher sGFAP levels in patients with liver cirrhosis and CHE than in patients with cirrhosis and no CHE or healthy controls. Nevertheless, the aforementioned evidence clearly suggests that sGFAP levels are not specific for HE in patients with cirrhosis. Consequently, sGFAP levels must be interpreted under careful consideration of potential comorbidities. One of the most important confounders for interpreting sGFAP levels in patients with cirrhosis, especially in the Western world, may be harmful alcohol use
      • Dalçik H.
      • Yardimoglu M.
      • Filiz S.
      • Gonca S.
      • Dalçik C.
      • Erden B.F.
      Chronic ethanol-induced glial fibrillary acidic protein (GFAP) immunoreactivity: an immunocytochemical observation in various regions of adult rat brain.
      . Of note, GFAP is also expressed in Schwann cells and alcohol-related peripheral neuropathy may also lead to increased sGFAP levels
      • Notturno F.
      • Capasso M.
      • DeLauretis A.
      • Carpo M.
      • Uncini A.
      Glial fibrillary acidic protein as a marker of axonal damage in chronic neuropathies.
      . Surprisingly, despite the potential impact on astrocyte integrity and peripheral nerves, we did not find a significant difference in sGFAP levels neither between patients with alcoholic cirrhosis and non-alcoholic cirrhosis, nor between patients with ongoing or discontinued harmful alcohol use. Importantly, our results indicates that alcohol may not confound the interpretation of sGFAP levels and, thus, could be used in patients with cirrhosis in the context of HE, irrespective of the most common etiologies. Consequently, elevated sGFAP levels may, thus, be related to HE and not to alcohol-associated cerebral injury in these patients.
      Several HE-related processes have the potential to contribute to the observed association between HE and elevated sGFAP levels. First and foremost, astrocyte injury caused by cell swelling and neuronal cell death with reactive gliosis and astrocyte activation are two mechanisms which may lead to increased sGFAP levels. In line, Angelova et al. recently found a significant neuronal cell death with a consecutive rise in GFAP-labelled astrocytes in bile duct-ligated rats, which represents an established animal model of MHE
      • Angelova P.R.
      • Kerbert A.J.C.
      • Habtesion A.
      • Hall A.
      • Abramov A.Y.
      • Jalan R.
      Hyperammonaemia induces mitochondrial dysfunction and neuronal cell death.
      . Moreover, it is well known that leakage through the blood brain barrier, allowing GFAP to pass into the blood stream, is a prerequisite for detectable levels in the peripheral blood
      • Brunkhorst R.
      • Pfeilschifter W.
      • Foerch C.
      Astroglial proteins as diagnostic markers of acute intracerebral hemorrhage-pathophysiological background and clinical findings.
      . However, the detailed mechanism on how astrocyte activation leads to elevated sGFAP levels is currently not fully understood. Interestingly, GFAP is not exclusively expressed in astrocytes but also in peripheral cells such as enteric glia or hepatic stellate cells
      • Abdelhak A.
      • Foschi M.
      • Abu-Rumeileh S.
      • Yue J.K.
      • D'Anna L.
      • Huss A.
      • et al.
      Blood GFAP as an emerging biomarker in brain and spinal cord disorders.
      , and the proportion of GFAP-positive glia cells in the gut increases when treated with cytokines
      • Boyen GBT von
      • Steinkamp M.
      • Reinshagen M.
      • Schäfer K.-H.
      • Adler G.
      • Kirsch J.
      Proinflammatory cytokines increase glial fibrillary acidic protein expression in enteric glia.
      . Since low-grade systemic inflammation is associated with the development of HE, and blood levels of the proinflammatory cytokine IL-6 are associated with the presence of MHE
      • Gairing S.J.
      • Anders J.
      • Kaps L.
      • Nagel M.
      • Michel M.
      • Kremer W.M.
      • et al.
      Evaluation of IL-6 for Stepwise Diagnosis of Minimal Hepatic Encephalopathy in Patients With Liver Cirrhosis.
      , the elevation of sGFAP levels in patients with CHE might partly be cytokine/inflammation-mediated. However, the design of the current study does not allow to assess to which extent each of the potential GFAP sources actually contributes to the observed elevation.
      It is an interesting finding that even patients without CHE according to PHES had significantly higher sGFAP levels compared to healthy controls. On the one hand, this could be explained by the fact that HE is not a dichotomous disease, as possibly indicated by cut-offs of tests such as the PHES, but a disorder that encompasses a continuum of cognitive deficits. Therefore, even patients with cirrhosis but without CHE might already suffer from mild cerebral injury, as reflected by higher sGFAP levels, caused by the complex interplay of ammonia and systemic inflammation. On the other hand, it has to be acknowledged that sGFAP levels are age-dependent and this may have contributed to lower sGFAP levels in the control groups
      • Abdelhak A.
      • Foschi M.
      • Abu-Rumeileh S.
      • Yue J.K.
      • D'Anna L.
      • Huss A.
      • et al.
      Blood GFAP as an emerging biomarker in brain and spinal cord disorders.
      ,
      • Nichols N.R.
      • Day J.R.
      • Laping N.J.
      • Johnson S.A.
      • Finch C.E.
      GFAP mRNA increases with age in rat and human brain.
      .
      One strength of this study is its bicentric design including a total of 181 patients with cirrhosis or healthy controls. Yet, several limitations have to be acknowledged. First, the sample size of some subgroups leading to a potential lack of power, as well as the fact that sGFAP levels appear to be age-dependent prevents us from establishing potential cut-offs for sGFAP for distinguishing between patients with and without CHE in the here presented study. However, our result clearly indicate that larger and prospective multicenter studies are warranted. Second, due to our cross-sectional study design, we are only able to reveal associations and are unable to prove causality. Therefore, future studies should focus on longitudinal assessments of CHE and associated changes in sGFAP levels. Third, it remains unknown when the ideal time point for sGFAP measurement is. This question should be addressed in a future longitudinal study to elucidate the dynamics of sGFAP levels in patients with cirrhosis. Fourth, sGFAP assays are currently not available in standard hospital laboratories and are still expensive.
      In conclusion, sGFAP levels are independently associated with the presence of CHE in patients with cirrhosis. This finding may add valuable insights into pathophysiological processes of HE, even at early stages. In addition, sGFAP might be a future candidate biomarker for CHE. Our results clearly indicate that large prospective trials are warranted to elucidate the prognostic value of sGFAP for predicting higher grades of HE and the longitudinal changes under therapy. In addition, larger studies may develop prediction models including sGFAP for diagnosing CHE.

      Conflict of interest

      The authors disclose no potential financial or non-financial conflict of interests regarding this study.

      Financial support statement

      This work was not supported by any grant or funding source.

      Authors’ contributions

      Performed research: S.J.G., C.Q., F.L., J.U.M., C.L.
      Contributed to acquisition of data: S.J.G., S.D., L.K., M.N., E.M.S., C.Q., S.E., S.B., J.M.S., M.-A. W., F.L., J.U.M., C.L.
      Designed the experiments and analyzed the data: S.J.G., C.L.
      Contributed reagents/materials/analysis tools: S.J.G., S.B., P.R.G., J.M.S., F.L., S.E., S.B., J.U.M., C.L.
      Wrote the paper: S.J.G., C.L.
      Statistical analysis: S.J.G., C.L.
      All authors approved the final version of the manuscript and the authorship list.
      Guarantor of the article: C.L.

      Data availability statement

      The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.

      Acknowledgments

      We thank Paula Kämper for excellent technical assistance. This study contains parts of the medical thesis of Charlotte Quack and Sven Danneberg. S.J.G. and E.M.S. are supported by the Clinician Scientist Fellowship “Else Kröner Research College: 2018_Kolleg.05”.

      Appendix A. Supplementary data

      The following is/are the supplementary data to this article:

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