Neuropsychological and psychophysical tests are recommended for the risk assessment for overt hepatic encephalopathy (OHE), but their accuracy is limited. Hyperammonaemia is central in the pathogenesis of OHE, but its predictive utility is unknown. This study aimed to determine the role of neuropsychological or psychophysical tests and ammonia and to develop the AMMON-OHE model for risk stratification regarding subsequent OHE development in outpatients with cirrhosis. This observational, prospective study included 426 outpatients without previous OHE from 3 liver units followed for a median of 2.5 years. Psychometric hepatic encephalopathy score (PHES) <-4 or critical flicker frequency (CFF) <39 was considered abnormal. Ammonia was normalized to upper limit of normal (AMM-ULN) at the respective reference laboratory. Multivariable frailty competing risk analyses and random survival forest were performed to predict future OHE and to develop the AMMON-OHE model. External validation was carried out using 267 and 381 patients from two independent units. Significant differences were found in time-to-OHE (log-rank p<0.001) according to PHES or CFF and ammonia, with the highest risk in patients with abnormal PHES plus high AMM-ULN (HR: 4.4; 95% CI: 2.4-8.1, p<0.001 compared with normal PHES and AMM-ULN). On multivariable analysis, AMM-ULN but not PHES or CFF was an independent predictor for development of OHE (HR: 1.4; 95%CI: 1.1-1.9; p=0.015). The AMMON-OHE model included sex, diabetes, albumin, creatinine and AMM-ULN, and showed a C-index of 0.844 and 0.728 in the two external validation cohorts. This study developed and validated the AMMON-OHE model, comprising readily available clinical and biochemical variables that can be used to identify outpatients at highest risk for developing a first episode of OHE.

Neurofilament light chain protein (NfL) levels reflect neuronal damage in several neurological diseases and have been proposed as a possible biomarker. Plasma extracellular vesicles (EVs) could play an important role as mediators of the inflammatory changes associated with inducing minimal hepatic encephalopathy (MHE) in cirrhotic patients. This study investigated the association of NfL levels in plasma and EVs with the presence of MHE in cirrhotic patients, and with responses to rifaximin treatment. The NfL levels in plasma and EVs were assessed in 71 patients with liver cirrhosis (40 with MHE and 31 without MHE) and 26 controls. A total of 31 patients with MHE received rifaximin treatment. We examined changes in NfL levels in plasma and EVs before and after 6 months of rifaximin treatment. The NfL measures were correlated with cognitive alterations and plasma inflammatory cytokines. MHE patients showed increased plasma levels of NfL, which were reverted after rifaximin treatment in patients who responded to treatment. The NfL content in EVs also showed a reversal pattern in MHE patients treated with rifaximin. In multivariable analyses, NfL levels were independently associated with the presence of MHE. We also showed that patients with high levels of both ammonia and fractalkine had significantly higher NfL levels than patients with low levels of least one of these parameters. Rifaximin treatment in MHE patients showed promising results in improving axonal damage, suggesting that rifaximin may have therapeutic benefits against disease progression in MHE.

Ammonia is a product of amino acid metabolism that accumulates in the blood of patients with liver cirrhosis, leading to neurotoxic effects and hepatic encephalopathy (HE). HE manifestations can range from mild, subclinical disturbances in cognition, or minimal HE (mHE) to gross disorientation and coma, a condition referred to as overt HE. Many blood-based biomarkers reflecting these neurotoxic effects of ammonia and liver disease can be measured in the blood allowing the development of new biomarkers to diagnose cirrhosis patients at risk of developing HE. The effect of ammonia on the brain is modulated by severity of systemic inflammation, and both hyperammonemia and inflammation can induce oxidative stress, which may mediate the neurological alterations associated to HE. This review aims to provide the latest evidence on biomarkers of HE beyond ammonia. We present different approaches to predict overt HE based on the combination of blood ammonia with some analytical and clinical parameters. Magnetic resonance analysis of brain images could also provide sensitive diagnostic biomarkers based on neuroimaging parameters. Some reports suggest that markers of systemic inflammation, oxidative stress, and central nervous system-derived components, may serve as additional biomarkers of HE. The involvement of extracellular vesicles and microbiota in the pathophysiology of mHE and HE has recently acquired importance and it would be interesting to explore their usefulness as early biomarkers of the disease. It is important to have a biomarker or a combination of them for early diagnosis of mHE to improve its treatment and prevent progression to overt HE.

Patients with nonalcoholic fatty liver disease (NAFLD) may show mild cognitive impairment (MCI). The mechanisms involved remain unclear. The plasma concentrations of several cytokines and chemokines were measured in 71 NAFLD patients (20 with and 51 without MCI) and 61 controls. Characterization and activation of leukocyte populations and CD4⁺ sub-populations were carried out and analyzed by flow cytometry. We analyzed the cytokines released from CD4⁺ cell cultures and the mRNA expression of transcription factors and receptors in peripheral blood mononuclear cells. The appearance of MCI in NAFLD patients was associated with increased activation of CD4⁺ T lymphocytes, mainly of the Th17 subtype, increased plasma levels of pro-inflammatory and anti-inflammatory cytokines such as IL-17A, IL-23, IL-21, IL-22, IL-6, INF-γ, and IL-13, and higher expression of the CCR2 receptor. Constitutive expression of IL-17 was found in cultures of CD4⁺ cells from MCI patients, reflecting Th17 activation. High IL-13 plasma levels were predictive of MCI and could reflect a compensatory anti-inflammatory response to the increased expression of pro-inflammatory cytokines. This study identified some specific alterations of the immune system associated with the appearance of neurological alterations in MCI patients with NAFLD that could be the basis to improve and restore cognitive functions and quality of life in these patients.