Most physiological and behavioral human variables, including sleep and wakefulness, endocrine function, thermoregulation and metabolism exhibit circadian patterns. Circadian rhythms are controlled by central neural pacemakers, of which the Suprachiasmatic Hypothalamic Nucleus (SCN) is the best characterized 12345 Hepatic encephalopathy, a major cirrhosis complication, is a clinical syndrome characterized by mental status abnormalities in patients with severe hepatic failure. In contrast, the term "minimal (subclinical) hepatic encephalopathy" describes milder disturbances of biological parameters such as sleep, and abnormalities in every day life activities, in the absence of clinical encephalopathy. 6789101112 Liver diseases are often associated with hormonal disorders, and metabolic changes in cirrhosis can result in circadian rhythm abnormalities. Previous studies have shown that disruption of the diurnal rhythm of melatonin reflects circadian function alterations that contribute to the disturbances of the sleep-wake cycle frequently seen in patients with cirrhosis. 131415. This study was designed to evaluate the melatonin profile of cirrhotic patients and correlate melatonin abnormalities to cirrhosis severity in the absence of clinical hepatic encephalopathy.

Abnormal circadian rhythms have been described for several biological parameters in cirrhosis. The existence of a "biologic clock" in the SCN allows the body to anticipate external environment modifications during the day-night cycle. Current views propose two explanations for circadian alterations seen in chronic liver disease. According to the first hypothesis, circadian rhythm abnormalities arise from the effect of neurotoxins on the SCN and/or its afferent/efferent connections. According to the second hypothesis, impaired hepatic melatonin clearance, probably due to decreased liver blood flow, lower 6-beta-hydroxylase activity, and competition with bilirubin in the intrahepatic transport system 14202122, results in elevated morning melatonin levels, thereby causing a circadian clock phase-shift. It is possible that both proposed mechanisms, by combining the effects of hepatocellular dysfunction and portal systemic shunting, are responsible for circadian abnormalities in liver disease 1232425.

Melatonin has been proposed to act as "internal synchronizer" for circadian rhythms generated at different levels of the human body. Melatonin alterations have been described in many biological rhythm disorders, including sleep disorders related to jet lag, shift work, blindness and aging 13152627282930. Cirrhosis patients have markedly elevated daytime melatonin levels, significantly delayed melatonin increase onset and consistently delayed melatonin peak 14202122. High daytime melatonin levels cause an endogenous biologic clock phase-shift; this diurnal melatonin disruption probably has clinical implications and may be related to the high prevalence of sleep disturbances 731 and pituitary hormone abnormalities 32 in cirrhosis. Hyperammonemia, cerebral accumulation of false neurotransmitters, abnormal dopaminergic activity, GABA-ergic neurotransmission disturbances, and stress are additional factors possibly affecting the biologic clock in cirrhosis.

Sleep disturbances, such as delayed sleep onset and short night sleep duration, which are well documented in cirrhosis, were also observed in our minimal encephalopathy cirrhosis population. Cirrhosis patients generally exhibit sleep-wake cycle abnormalities, manifesting as chronotypology other than Morningness Type. However, in striking disagreement with previous findings 131424, 23 of 26 (88.4%) of our cirrhosis patients exhibited Morningness (DM or MM) Chronotypology. This unexpected finding may in part be explained by the opposing effects of melatonin and bright morning light in our Mediterranean population. Although other factors may also affect chronotypology, we believe that the true importance of melatonin deserves further investigation.

Limitations of this study include study design (observational study, no intervention, no randomization, no power analysis), the small number of patients both in the cirrhosis and in the control group, and the fact that we have tried to make inferences about melatonin secretion from only three measurements per day.

The most important finding of our study is the presence of abnormal plasma melatonin levels in cirrhosis patients before the onset of clinical hepatic encephalopathy. This is not an entirely new finding, as it has also been reported by Steindl et al 14. However, the study by Steindl, which only included 7 cirrhosis patients, compared cirrhosis patients with subclinical encephalopathy vs. healthy controls. In contrast, our study included 26 cirrhosis patients and 13 controls, and we used patients with other diseases, rather than healthy people, as controls. Compared to the Steindl study 14 our study shows two additional important findings: i) the observation that evening melatonin levels are lower in patients with more severe cirrhosis (Figure 1) and ii) the observed morning type chronotypology in our cirrhosis population.

These findings are important because they are highly abnormal and markedly different compared to values reported in the literature for normal subjects. The finding that patients with more severe cirrhosis have lower evening melatonin levels has not, to our knowledge, been described before. In addition, the observed chronotypology raises questions regarding the influence of melatonin on sleep patterns in cirrhosis.

Further studies are needed to accurately describe melatonin secretion and clarify the possible association between melatonin levels and chronotypology in cirrhosis.

Melatonin abnormalities occur in patients with liver cirrhosis in the absence of clinical encephalopathy and are related to severity of cirrhosis. Morning type chronotypology was identified in our cirrhosis patients. The influence of these melatonin abnormalities on chronotypology patterns in cirrhosis patients with subclinical encephalopathy deserves further investigation.

HBV: Hepatitis B Virus; HCV: Hepatitis C Virus; DM: Definitely Morning Type; MM: Moderately Morning Type; NT: Neither Type; ME: Moderately Evening Type; DE: Definitely Evening Type; BNSQ: The Basic Nordic Sleep Questionnaire; NCT-A: Number Connecting Test A; DST: Digit Symbol Test; SIP: Sickness Impact Profile; SD: Standard Deviation; ANOVA: Analysis Of Variance.

The authors declare that they have no competing interests.

DV is the principal investigator, participated in the design of the study and performed research. VK helped with data collection, paper editing and submission. PP did all neurologic and chronotypology evaluations. MK performed the statistical analysis, interpreted results and wrote the paper. All authors read and approved the final manuscript.