The effect of induced hyperammonaemia on sleep and melanopsin-mediated pupillary light response in patients with liver cirrhosis: A single-blinded randomized crossover trial.

BACKGROUND & AIMS: Sleep disturbances are related to hepatic encephalopathy and hyperammonaemia in patients with cirrhosis. The circadian rhythm is regulated by light stimulation of the retina via melanopsin-containing ganglion cells. The study aimed to investigate whether induced hyperammonaemia affects the pupillary light response and sleep efficiency in patients with cirrhosis.
METHODS: The study was a single-blinded crossover trial including nine patients with cirrhosis. Sleep was evaluated by Pittsburgh Sleep Quality Index (PSQI) and monitored for twelve nights with wrist accelerometers and sleep diaries. On two experimental days, separated by one week, patients were randomized to ingest either an oral amino acid challenge (AAC) or an isocaloric glucose solution (GS). We measured pupillary light response, capillary ammonia, the Karolinska Sleepiness Scale (KSS), and two neuropsychological tests on both experimental days.
RESULTS: The patients had poor self-assessed sleep quality. The amino acid challenge led to a significant increase in capillary ammonia and KSS. The time spent in bed sleeping after AAC was longer and with a reduced movement index compared to baseline but not different from GS. We found no difference in the pupillary light response or neuropsychiatric tests when comparing the effect of AAC with GS.
CONCLUSIONS: Patients with cirrhosis had impaired sleep quality. Induced hyperammonaemia led to increased sleepiness but had no acute effect on pupillary light response or the neuropsychiatric tests. TRIAL REGISTRATION: Registration number: NCT04771104.

Introduction

About fifty percent of patients with cirrhosis experience sleep disturbances during life [1]. In previous studies, an association between hepatic encephalopathy (HE) and sleep disturbances has been proposed–e.g., inversion of the sleep-wake cycle and increased daytime sleepiness [2, 3]. The association is thought to be mediated through hyperammonaemia. For instance, Bersagliere et al. observed a parallel increase in subjective sleepiness and plasma ammonia levels [4], and Singh et al. found an improvement in sleep quality due to ammonia-lowering therapy [5]. Furthermore, it has previously been suggested that retinal impairment can be caused by hyperammonaemia and its detoxification by glial cells [6-8].

The circadian rhythm maintains a regular sleep pattern and is partly regulated by light stimulation of the retina via the melanopsin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs). Melanopsin is a photo pigment with maximal sensitivity to light with a wavelength of 480 nm corresponding to blue light. When retina is stimulated with blue light, the ipRGCs detect the light signals and transfer these to higher brain centers, including the suprachiasmatic nucleus (which affects circadian rhythm) and the olivary pretectal nucleus (which affects pupillary light response) [9]. Therefore, the function of ipRGCs can be evaluated by measuring the post-illumination pupillary response with chromatic pupillometry [10].

Based on the above, we proposed a new theory not previously investigated. We hypothesized that hyperammonaemia led to a dysfunction of the ipRGCs and thereby affected sleep regulation in patients with cirrhosis. We conducted a crossover trial to evaluate sleep and the pupillary light response with a pupillometry before and after an oral amino acid challenge. We hypothesized that an oral amino acid challenge induced hyperammonaemia, increased subjective sleepiness, worsened the psychometric tests for minimal encephalopathy, and impaired the pupillary response to blue light stimulation.

Materials and methods

Study design

The study was a single-blinded, randomized crossover study consisting of two experimental days separated by one week at the Department of Ophthalmology, Rigshospitalet, Denmark. The assessments on the two experimental days were identical and combined with a randomized intervention of either an oral amino acid challenge (AAC) or an isocaloric glucose solution (GS) followed by five hours of observation. Screening and inclusion of patients were done from 01-01-2017 until 01-03-2018.

Eligibility criteria

We included patients with Child-Pugh Class A or B cirrhosis. Patients were excluded if they were <20 years or >80 years of age, had alcohol misuse in the preceding six months, had episodes of hepatic decompensation leading to hospital admissions in the preceding month, or had prior overt HE, or received any HE treatment (lactulose, rifaximin, L-ornithine L-aspartate or branched-chain amino acid supplementation). Additional exclusion criteria were a history of significant head injury, severe sleep-wake disturbances, neurological/psychiatric comorbidity needing medical treatment, active use of neuroactive drugs or other medication known to affect sleep, traveling across more than two time zones in the preceding three months, shift work in the preceding five years, and diabetes (due to the high prevalence of retinopathy). On the first experimental day, all patients underwent an ophthalmological examination, including refraction, best-corrected visual acuity determination, slit-lamp examination, intraocular pressure measurement (Goldman tonometry), and spectral-domain optical coherence tomography of the macula (Spectralis; Heidelberg Engineering GmbH, Heidelberg, Germany). Inclusion required normal results in all ophthalmologic measurements except hypermetropia and myopia, which were allowed. For this reason, we decided only to assess non-diabetic patients for eligibility to avoid a high rate of screen failures. We also originally planned to include a control group of healthy subjects in the trial protocol, but due to time and resource constraints, we decided to limit the study population to cirrhotic patients only since the most significant treatment effect was expected here.

Intervention

With sealed envelopes, the patients were randomized to receive either AAC or GS on the first experimental day and the opposite on the second experimental day. We separated the experimental days by one week to eliminate or reduce the carry-over effect. The patients were blinded for randomization. The AAC consisted of 54 gram banana flavoured amino acid mixture added to 200–300 ml of water. The AAC simulated the composition of hemoglobin in approximately 400 ml of blood. The isocaloric glucose solution contained a similarly flavoured mixture of 72 g glucose added to 200–300 ml of water. Both solutions were ingested orally for 10 minutes [4, 11]. Random allocation sequence, enrolment of patients, and patient assignment to interventions were done by AEK.

The study assessments

The study assessments were performed at different time points during the inclusion period, as shown in Table 1. The experimental days ran from morning (8.00 am) until afternoon (3.00 pm) to reduce differences in the circadian rhythm between patients.

Table 1

Study assessments during the inclusion period.

Assessments	Before first experimental day	Day 1 (First experimental day)	Day 2	Day 8 (Second experimental day)	Day 9
PSQI	x
Sleep-wake evaluation	x		x		x
KSS		0h, 1h, 2h, 3h, 4h, 5h		0h, 1h, 2h, 3h, 4h, 5h
Capillary ammonia		0h, 1h, 2h, 3h, 4h, 5h		0h, 1h, 2h, 3h, 4h, 5h
PHES		0h, 4h		0h, 4h
CRT		0h, 2h, 4h		0h, 2h, 4h
Pupillometry		0h, 3h, 5h		0h, 3h, 5h

PSQI, Pittsburgh Sleep Quality Index; KSS, Karolinska Sleepiness Scale; PHES, psychometric hepatic encephalopathy score; CRT, continuous reaction time

Capillary ammonia

Capillary blood was obtained from the patient’s ear lobe at baseline and every hour in the subsequent five hours on each experimental day for ammonia measurements. The ammonia concentration was measured with a blood ammonia analyzer (Pocketchem Device BA, Arkray Factory, Kyoto, Japan). The method is a reliable alternative to arterial measurements. It is based on a micro-diffusion, where the ammonium ions are gasified and cause a colour development proportional to the ammonia concentration in the blood [12].

Sleep evaluation

Before the first experimental day, the patients completed the validated self-rated assessment tool Pittsburgh Sleep Quality Index (PSQI), which assesses sleep quality and disturbances within the preceding month. The questionnaire generates seven component scores: habitual sleep efficiency, daytime dysfunction, subjective sleep quality, sleep disturbances, sleep latency, sleep duration, and use of sleeping medication. A total score of over 5 indicates poor sleep quality [13].

At baseline and every subsequent hour during the experimental days, the patients reported their level of sleepiness by the Karolinska Sleepiness Scale (KSS). The scale is a 10-point scale going from 1 (extremely alert) to 10 (extremely sleepy, falls asleep all the time) [14, 15].

The patients were equipped with an accelerometer (GT3X+, ActiGraph, Florida, USA) from two days before the first experimental day to two days after the last experimental day. The accelerometer was worn on the wrist of the non-dominant arm during the day and night except when showering. The device records movements in three dimensions to provide data on physical activity and sleep efficiency, i.e., actigraphy. Throughout the study period, the patients completed a sleeping diary containing in-bed time, out-bed time, naps during the day, external factors that could disturb sleep, and un-wear times. The diary data was used for the subsequent data preparation and software analysis (ActiLife 6, Actigraph) [4, 16, 17]. We applied automatic sleep-wake detection of 1-minute epochs using the Cole-Kripke algorithm implemented in the software [17]. The following measures were calculated for each day in the sleep evaluation:

We defined the baseline sleep measures as an average of two nights preceding both experimental days, i.e., an average of four nights. We compared the baseline sleep to the sleep assessment of the night following each experimental day.

Neuropsychiatric assessment

The psychometric hepatic encephalopathy score (PHES) was obtained at baseline and after four hours on each experimental day. PHES is a psychometric paper-pencil test used to diagnose and quantify hepatic encephalopathy [18]. The test is divided into five subtests measuring different skills, e.g., number recognition and motor skill. The skills were scored according to age. The sum of the subtests yields a score from -18 to 6. A score under -4 indicates minimal hepatic encephalopathy.

Continuous Reaction Time (CRT) (EKHO, Aarhus, Denmark) was obtained at baseline and after two hours and four hours on each experimental day. It is an objective measure for hepatic encephalopathy. It consists of a headset and a button connected to a computer. The software generates 150 sound stimuli within different time intervals. The patient must press the button every time the software beeps. The CRT result is an index of all recorded reaction times, indicating steadiness of the reaction time: . A result below 1.9 indicates minimal hepatic encephalopathy [19].

Pupillometry

We measured the pupillary light response at baseline, after three hours, and after five hours on each experimental day from 8.00 am to 3.00 pm. We used a binocular chromatic pupillometer (DP-2000 Human Laboratory Pupillometer; NeurOptics, Inc., Irvine, CA, USA). In all patients, the right eye was the stimulus eye, while we recorded the pupillary responses of both eyes. Each assessment started with dark adaptation for five minutes, followed by stimulation with red light (633 nm), then five minutes of dark adaption followed by blue light (463 nm). The duration of light stimulation was 20 seconds, and the illuminance level of both light colors was 100 lux. The pupillary size was recorded continuously for 10 seconds before, 20 seconds during, and 60 seconds after light stimulation (Fig 1). The following pupillometric outcomes were calculated [10, 20, 21]:

Fig 1

A schematic example of the pupillometry measurement.

The figure shows a normal pupillometry. The mean pupillary diameter was normalized against the mean baseline pupil diameter. The pupils were video recorded for 10 seconds before light stimulation, 20 seconds during light stimulation called “Light ON” (blue arrow), and 40 seconds after light stimulation called “Light OFF” (black arrow). Early PIPR (light grey area) is the early post-illumination pupillary response recorded 0–10 seconds after the light turns off. Late PIPR (dark grey area) is the late post-illumination pupillary response (late PIPR) recorded 10–30 seconds after the light turns off [21].

The late post-illumination pupillary response (late PIPR) equals the mean pupil constriction from 10 to 30 seconds after light stimulation. The late PIPR outcome expresses the activation of the ipRGCs [21].

The early post-illumination pupillary response (early PIPR) equals the mean pupil constriction from start to 10 seconds after light stimulation. The early PIPR expresses the rod photoreceptors, cone photoreceptors, and ipRGCs’ response [10, 21].

Maximal contraction amplitude (CAmax) equals maximum pupil constriction 4 to 6 seconds after light stimulation. The CAmax expresses a combined response of rod photoreceptors, cone photoreceptors, and ipRGCs [10, 21].

Statistical analyses

We used a paired Wilcoxon signed-rank test to compare test results after either AAC or GS. The results were expressed as medians (25% quantile; 75% quantile). The length of the error bars on the plots is the median absolute deviation. P-values<0.05 were considered significant except for sleep evaluation, where we used a Bonferroni corrected p-value (P<0.0167) due to multiple comparisons.

Outcomes

The primary outcome was the difference in the late PIPR after AAC compared to GS. The secondary outcomes were differences in arterial ammonia, sleep quality, and measurements for hepatic encephalopathy (PHES and CRT) after AAC compared to GS.

Sample size

A power analysis was performed with the “pwr” package for R (v.3.1.0), suggesting a sample size of 15 for paired t-tests (effect size 0.8, significance level 0.05, power 0.8). Unfortunately, due to slow recruitment, we ended the study prematurely after the inclusion of nine patients.

Ethics

The study was approved by The Committee on Health Research Ethics of the Copenhagen Capital Region, Denmark (Project ID: H15000210). All patients provided written informed consent and were not offered financial compensation. The study was conducted under the Helsinki Declaration.

The study was registered on clinicaltrials.gov. We did not register the study before the initiation of patient enrolment, as it was not a requirement in The Committee on Health Research Ethics for this type of national study with no drug intervention. We have no ongoing or related trials.

Results

Fifteen patients were examined for eligibility (see Fig 2). Nine patients were included, and all finished the study according to the study protocol. Of the nine patients, 44% were men, and 67% were classified as a Child-Pugh A. Five patients received AAC on the first experimental day and four on the second experimental day. No critical harms or unintended effects were observed during the study period. The patient characteristics are presented in Table 2.

Fig 2

Flow diagram of enrolment in the study.

Table 2

The characteristics of the included patients with cirrhosis n = 9.

Characteristics	No. of patients (%) except when otherwise stated
Men	4 (44)
Age, MEDIAN(RANGE)	64 (53–73)
BMI, MEDIAN(RANGE)	26.2 (20.9–36.7)
Etiology of Liver Cirrhosis
HEPATITIS B	1 (11)
ALCOHOL	4 (44)
AUTOIMMUNE HEPATITIS	2 (22)
PRIMARY BILIARY CIRRHOSIS	1 (11)
PRIMARY SCLEROSING CHOLANGITIS	1 (11)
Child-Pugh Score, MEDIAN(RANGE)	6 (5–9)
Child-Pugh Classification
CLASS A	6 (67)
CLASS B	3 (33)
CLASS C	0 (0)
Randomization a
AMINO ACID CHALLENGE	5 (56)
GLUCOSE SOLUTION	4 (44)
Self-assessed sleep quality (PSQI)
NORMAL SLEEP QUALITY (SCORE ≤5)	3 (33)
POOR SLEEP QUALITY (SCORE >5)	6 (67)

BMI, Body Mass Index; PSQI, Pittsburgh Sleep Quality Index

a Intervention on the first experimental day

Pupillometry

We found no significant differences in the late PIPR on AAC day compared to GS day or over time during each day (Fig 3). Additionally, we found no differences in the maximum pupillary contraction or early PIPR on AAC day compared to GS day or over time (S1 and S2 Files).

Fig 3

The late post-illumination pupillary response during the two experimental days.

The late post-illumination pupillary response before, 2, and 4 hours after either amino acid challenge (AAC) or equicaloric glucose solution (GS).

Ammonia and KSS

The AAC intervention led to a significant increase in the median capillary ammonia with a maximum of 108 μM (72 μM;130 μM) three hours after intervention (Fig 4A). In addition, the AAC intervention was associated with a significant increase in the median self-rated sleepiness (KSS), with a maximum after 2 hours at a value of 7 (4;8) (Fig 4B). The increase in capillary ammonia and KSS were not seen on GS day.

Fig 4

A. Capillary ammonia concentration during the two experimental days. Capillary ammonia concentrations (μM) at baseline, 1, 2, 3, 4, and 5 hours after intervention with either an amino acid challenge (AAC) or equicaloric glucose solution (GS). *) Significantly different from GS (p = 0.0156, paired Wilcoxon signed-rank test). #) Significantly different from GS (p = 0.0223). B. Karolinska sleepiness score during the two experimental days. Karolinska sleepiness score (KSS) at baseline, 1, 2, 3, 4, and 5 hours after intervention with either an amino acid challenge (AAC) or equicaloric glucose solution (GS). *) Significantly different from GS (p = 0.0120, paired Wilcoxon signed-rank test).

Sleep evaluation

The median PSQI score was 6 (3;8). Six of nine patients (67%) had a PSQI score higher than five, indicating poor sleep quality. The sleep assessments by actigraphy showed increased sleep efficiency the night following both GS and AAC experimental day compared to the averaged baseline sleep efficiency (Table 3). Sleep efficiency the night after AAC did not differ from the night after GS. The increased sleep efficiency could mainly be explained by a decrease in the movement index.

Table 3

The average sleep evaluation at baseline compared to the night after amino acid challenge (AAC) and the night after glucose solution (GS) measured by Actigraph.

Median (25% quantile; 75% quantile) n = 9.

Sleep Parameters	Baseline sleepI	Sleep after GSII	Sleep after AACIII
Sleep Efficiency (%)	79 (77; 83)	84 (80; 87)*	87 (83; 90)**
Total Sleep Time (minutes)	354 (327; 371)	390 (381; 426)*	399 (396; 442)
Movement Index (%)	18 (16; 20)	15 (14; 2310)	14 (10; 18)*
Fragmentation Index (%)	15 (11; 16)	10 (0; 16)	9 (5; 13)
Sleep Fragmentation Index (%)	34 (27; 4114)	28 (23; 328)	21 (16; 32)
Wake After Sleep Onset (minutes)	79 (73; 98)	69 (63; 84)	59 (45; 71)***

AAC, amino acid challenge; GS, glucose solution

I Sleep measurements as an average of four nights; two nights before AAC day and two nights before GS.

II Sleep measurements the night after GS day

III Sleep measurements the night after AAC day

*Significantly different from baseline (p = 0.0117, paired Wilcoxon signed rank test)

**Significantly different from baseline (p = 0.0039, paired Wilcoxon signed rank test)

***Significantly different from baseline (p = 0.0128, paired Wilcoxon signed rank test)

Neuropsychiatric tests

Four of nine patients (44%) had a baseline CRT index lower than 1.9, indicating minimal hepatic encephalopathy. All patients had normal baseline PHES. We did not find significant differences in CRT index or PHES when comparing AAC vs. GS or over time within groups for each experimental day. However, a numerical reduction in the median CRT index following AAC was observed (Table 4).

Table 4

Continuous Reaction Time (CRT) Index and the Psychometric Hepatic Encephalopathy Score (PHES) according to randomization and time of examination from intervention.

Median (25% quantile; 75% quantile) n = 9.

Time from intervention	CRT index		PHES score
	AAC	GS	AAC	GS
0 hours (baseline)	1.973 (1365; 2000)	1.731 (1464; 2081)	0 (-1; 1)	-1 (-2; 1)
2 hours	1.840 (1535; 1965)	1.696 (1443; 2185)	-	-
4 hours	1.563 (1493; 2343)	1.714 (1440; 1985)	2 (-1; 3)	0 (-2; 2)

CRT, Continuous Reaction Rime; PHES, Psychometric Hepatic Encephalopathy Score, AAC, AAC, amino acid challenge; GS, glucose solution

Discussion

Our study included nine patients with cirrhosis. Two-thirds had impaired self-reported sleep quality. The AAC intervention was associated with an acute increase in capillary ammonia concentration and subjective sleepiness but had no immediate effect on the melanopsin-mediated pupillary light response or the psychometric tests. We observed more heavy sleep the night after both experimental days compared to baseline sleep but no independent effect of AAC on sleep.

Our hypothesis about impairment of the ipRGC function due to hyperammonemia was not confirmed because the late PIPR was not affected by AAC. Nonetheless, the median late PIPR ranged from 0.14 to 0.23 at the different time points, which is numerically higher than normal values seen in healthy subjects (0.12 (95% CI: 0.09–0.15) and lower than in patients with the eye disorder choroideremia (0.30 (95% CI: 0.18–0.41) [10]. These comparisons could indicate that patients with cirrhosis might have a slight degree of retinal ganglion cell dysfunction. However, our sample size and lack of a control group of healthy subjects do not allow us to make firm conclusions on baseline PIPR levels in patients with cirrhosis.

The increase in ammonia concentration and sleepiness after AAC was as we suspected and in line with previous studies [4, 11, 22]. We observed more coherent sleep with fewer movements and awakenings the night after AAC and GS, respectively, compared to baseline sleep. We would have expected that sleep after AAC decreased sleep efficiency because Bersagliere et al. [4] found superficial sleep after an equivalent AAC intervention in a similar population. However, differences in the study design could explain the divergence because Bersagliere et al. assessed sleep during a nap in the same afternoon with EEG analysis. Moreover, heavier sleep in our population could be due to exhaustion from a long experimental day rather than the intervention itself.

We found no significant effect of induced hyperammonemia on PHES and CRT, which is in line with previous studies, although slowing reaction time has been reported [4, 11]. Although we did not observe an association between hyperammonaemia and CRT, we found a tendency for a CRT index decline over time on AAC day, reaching values below the index threshold on 1.9. The lack of association between hyperammonemia and PHES could be caused by a potential learning effect of the short period between tests. However, we tried to prevent this by applying four unique PHES test sets per patient.

Plausible explanations of the limited responses we saw–apart from the null hypothesis–are addressed in the following. First, the patients may need a higher peak concentration of ammonia and a longer duration of hyperammonemia to affect the retinal ganglion cells and consequently impair sleep regulation. In addition, the daytime assessment may have affected the results, as ipRGCs are more sensitive to light stimulation in the evening [23]. Second, the composition of the included patients could have affected the results in different ways. For example, the patients’ different liver etiology could have contributed to the negative result. Further, the population might have been more susceptible if the patients had more severe liver disease, e.g., Child-Pugh class C, and a history of hepatic encephalopathy. By this, the effect of intervention might have been more pronounced. In the design phase of the study, we decided to use the current inclusion criteria due to a great need for practical cooperation during the time-consuming assessments, especially pupillometry.

A strength of our study was the crossover design with a biologically reasonable wash-out period and the use of non-parametric statistical methods, which gave acceptable protection against type 1 errors and still preserved enough power to detect clinically meaningful effects [24].

Conclusions

Our study showed that patients with cirrhosis had impaired sleep quality. An oral AAC resulted in an increase in ammonia concentration and associated subjective sleepiness within hours. We found no acute effect of the oral AAC on the melanopsin-mediated pupillary light response; therefore, we could not confirm our main hypothesis.

First part of original data.

The file contains all collected data except pupillometry data.

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Second part of original data.

The file contains all collected pupillometry data. Other data are provided in S1 File.

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The trial protocol.

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The consort checklist.

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13 Dec 2021

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Reviewer #1: The main points of concern in this manuscript are (a) the rather confused description of the known and hypothesized causal relationships and pathways and the light shed on these by this study and (b) the incomplete adherence to standard methods for analysis of cross-over studies.

(a) The introduction explains that liver cirrhosis can cause elevated ammonia level (hyperammonaemia) and hepatic encephalopathy (HE), which in turn can lead to sleep disturbances. The effect on sleep could hypothetically be mediated by damage to retinal cells leading to disturbance of the circadian rhythm. The causal connections and their direction, either known or suspected, between hyperammonaemia, HE and sleep are not clearly described. Ammonia levels were challenged and measures of ammonia level, HE-related variables, eye function and sleep were performed and pair-wise associations between these measures were investigated. It would be useful if the authors could summarize which associations are supported by the study results and what these results tell us about the possible causal pathways.

(b) Analysis of results from a cross-over design should look for a period effect by comparing the results from the first and second periods, since there may be systematic differences due to the subjects’ experience in period 1. It is clear that with an odd number of subjects, the intervention (AAC/GS) is unbalanced with respect to period, which might potentially bias the AAC/GS comparison. With a whole week between periods, we can presumably take the carry-over effect from the first to second period to be negligible – if so, please state this assumption. Further, where appropriate (e.g. PIPR, ammonia level, KSS) the measured values after intervention should be compared with the respective values before intervention (i.e. day 0 of the period), for instance by analyzing the changes (for each subject) during the period.

Minor points

1. Definition of movement index (p.6): why is denominator time spent in bed, rather than TST? As defined, longer TST could lead to increased movement index.

2. Definition of fragmentation index unclear (what is ‘Number of sleep epochs = 1 minute’?).

3. Please justify the assumed effect size (0.8 is unusually large).

4. The distribution of baseline values before AAC an GS respectively should be compared, since similar baselines are need to ensure a fair comparison.

5. In Fig. 3, are mean and standard deviation displayed? With only 9 subjects, it might be possible and more informative to plot the individual series.

6. Table 2: IQR should be given as (lower quartile, upper quartile).

7. Throughout Results, exact p-values should be quoted.

8. The graphic quality of Fig. 2 is very low, to the point of illegibility.

Reviewer #2: This is an interesting, innovative, well presented and honestly discussed piece of research.

The only suggestion I have is to replace commercial names with the actual compounds for the anti-HE drugs listed on page 4 (Hepa-Merz.....)

Reviewer #3: The authors present an interesting crossover trial of AAC vs GS in nine patients with cirrhosis where oral AAC was associated with increased sleepiness on Karolinska but not with hypothesized pupillary light response. I have a few comments regarding the methodology.

1. Did you consider potential learning effects of repeated PHES and CRT? Typically, PHES is not repeated in such a short interval (i.e. 1-day) and as with any neuropsych evaluation there can be a learning effect. Summarizing the details of PHES results (i.e. each component of the test and score), by #day (1st vs 2nd) and by AAC vs GS can be helpful in understanding how the subjects performed after intervention. The fact that CRT time was reduced after AAC also suggests that the learning effects may have been an important factor.

2. Please include more details of liver disease in the subjects (MELD score, complications of liver cirrhosis, etc.). The heterogeneity of cirrhosis etiology in a small sample could have contributed to the negative result.

3. The figure 2 (schematic representation of pupillary light response) is difficult to read, either due to rendering or due to the font. Please revise to help with readability.

4. PSQI questions ask sleep quality over the past 1 month. How was this handled? One-time intervention is unlikely to affect the scoring over 1-month interval over 2-day study period.

**********

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Reviewer #1: Yes: Jeremy Franklin

Reviewer #2: Yes: Sara Montagnese

Reviewer #3: No

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15 Mar 2022

PLEASE NOTE, THAT OUR RESPOND TO REVIEWERS IS ATTACHED AS A FILE UNDER "ATTACH FILES". THE FOLLOWING IS A COPY OF THIS DOCUMENT.

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Authors’ reply:

Thank you for this comment. We have now edited the style of our paper to meet the requirements.

2. Please ensure you have discussed any potential limitations of your study in the Discussion, including study design, sample size and/or potential confounders.

Authors’ reply:

Thank you for this comment. In the discussion, we have described the following with regard to sample size and study design: “However, our sample size and lack of a control group of healthy subjects do not allow us to make firm conclusions on baseline PIPR levels in patients with cirrhosis.”

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Authors’ reply:

Thank you very much for this comment. We apologize for the delay. We have now inserted the following in the method section to meet both comment 3a and 3b: “The study was registered in clinicaltrials.gov. We did not register the study before the initiation of patient enrolment, as it was not a requirement in The Committee on Health Research Ethics for this type of national study with no drug intervention. We have no ongoing or related trials.”

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(The project has been financially supported by a grant from the Novo Nordisk Foundation with grant no. NNF16OC0020490. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.)

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Authors’ reply:

Thank you for this comment. We have now re-written the paragraph:

“Anna Emilie Kann was supported by a pre-graduate scholarship from the Novo Nordisk Foundation (NNF16OC0020490). The funder had no role in study design, data collection, analysis, manuscript preparation, or decision to publish. No additional funders, either internally or externally.”

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Authors’ reply:

Thank you. We have now ensured that they are identical

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Authors’ reply:

Thank you for the comment. The ethics is written within the Method section:

“The study was approved by The Committee on Health Research Ethics of the Copenhagen Capital Region, Denmark (Project ID: H15000210). All patients provided written informed consent and were not offered financial compensation. The study was conducted under the Helsinki Declaration.”

Review Comments to the Author

Reviewer #1:

The main points of concern in this manuscript are:

(a) the rather confused description of the known and hypothesized causal relationships and pathways and the light shed on these by this study and

(b) the incomplete adherence to standard methods for analysis of cross-over studies.

(a) The introduction explains that liver cirrhosis can cause elevated ammonia level (hyperammonaemia) and hepatic encephalopathy (HE), which in turn can lead to sleep disturbances. The effect on sleep could hypothetically be mediated by damage to retinal cells leading to disturbance of the circadian rhythm. The causal connections and their direction, either known or suspected, between hyperammonaemia, HE and sleep are not clearly described.

Ammonia levels were challenged and measures of ammonia level, HE-related variables, eye function and sleep were performed and pair-wise associations between these measures were investigated. It would be useful if the authors could summarize which associations are supported by the study results and what these results tell us about the possible causal pathways.

(b) Analysis of results from a cross-over design should look for a period effect by comparing the results from the first and second periods, since there may be systematic differences due to the subjects’ experience in period 1. It is clear that with an odd number of subjects, the intervention (AAC/GS) is unbalanced with respect to period, which might potentially bias the AAC/GS comparison. With a whole week between periods, we can presumably take the carry-over effect from the first to second period to be negligible – if so, please state this assumption.

Further, where appropriate (e.g. PIPR, ammonia level, KSS) the measured values after intervention should be compared with the respective values before intervention (i.e. day 0 of the period), for instance by analyzing the changes (for each subject) during the period.

Authors’ reply to (a):

Thank you for your comment. We have now rephrased the introduction to make it more clear what is known and hypothesized.

We have now summarized the expected associations in the end of the introduction

“Based on the above, we proposed a new theory not previously investigated. We hypothesized that hyperammonaemia lead to a dysfunction of the ipRGCs and thereby affect the regulation of sleep in patients with cirrhosis. We conducted a crossover trial to evaluate sleep and pupillary light response with pupillomety before and after an oral amino acid challenge. We hypothesized that an oral amino acid challenge induced hyperammonaemia, increased subjective sleepiness, worsened the psychometric tests for minimal encephalopathy, and impaired the pupillary response to blue light stimulation.”

and the beginning of the discussion:

“Our study included nine patients with cirrhosis. Two-thirds had impaired self-reported sleep quality. The AAC intervention was associated with an acute increase in capillary ammonia concentration and subjective sleepiness but had no immediate effect on the melanopsin-mediated pupillary light response or the psychometric tests. We observed more heavy sleep the night after both experimental days compared to baseline sleep, but no independent effect of AAC on sleep.”

And in the conclusion:

“Our study showed that patients with cirrhosis had impaired sleep quality. An oral AAC resulted in an increase in the ammonia concentration and an associated subjective sleepiness within hours. We found no acute effect of the oral AAC on the melanopsin-mediated pupillary light response, and therefore we could not confirm our main hypothesis.”

Authors’ reply to (b):

Thank you for your comment. We agree. A cross over design was necessary, and by adding a one-week washout period, we presume that the effect of intervention is eliminated and would not affect the encephalopathy nor the sleeping. We have written the following in the method section: “To eliminate or reduce the carry-over effect, we separated the experimental days by one week.”

We primarily wanted to compare the differences between groups rather than within groups, although we agree that the change from baseline also is a relevant measure of the effect of the intervention. We believe the change from baseline is clearly assessable in our figures, and we have actually done the analysis of change from baseline for each type of measurement confirming a significant change from baseline to peak values in the AAC-group. We chose not to include the analysis since it does not change or add substantially to the conclusions.

Minor points

1. Definition of movement index (p.6): why is denominator time spent in bed, rather than TST? As defined, longer TST could lead to increased movement index.

Authors’ reply:

Thank you for your comment. This was a typing mistake from our side. We have now corrected the denominator. The actual calculations were done by the proprietary ActiGraph software and was not influenced by this mistake. The following is the corrected calculation:

Movement index=(Time with movements during sleep (minutes))/(Total sleep time (minutes))×100

2. Definition of fragmentation index unclear (what is ‘Number of sleep epochs = 1 minute’?).

Authors’ reply:

We have now rephrased the definition slightly:

“Fragmentation index=(Number of periods of one minute of sleep)/(Total sleep time (minutes))×100”

3. Please justify the assumed effect size (0.8 is unusually large).

Authors’ reply:

We based the expectancy of a large effect size on the differences in PIPR seen in other studied groups of subjects: choroideremia vs. healthy subjects, where the ratio of the difference in means vs. standard deviation was approximately 0.8.

4. The distribution of baseline values before AAC and GS respectively should be compared, since similar baselines are need to ensure a fair comparison.

Authors’ reply:

We did compare the baseline values between AAC and GS in our primary analysis as reported in the manuscript. We observed no group differences at baseline.

5. In Fig. 3, are mean and standard deviation displayed? With only 9 subjects, it might be possible and more informative to plot the individual series.

Authors’ reply:

Thank you for your comment. In figure 3 and 4, the error bars represent the median absolute deviation, a robust statistics for non-normally distributed data or small series. The legibility of “spaghetti plots” of our data was not good, which is why we chose summary statistics instead of individual data series.

6. Table 2: IQR should be given as (lower quartile, upper quartile).

Authors’ reply:

Thank you for the suggestion. We have now changed the metric to 25% and 75% quartiles.

7. Throughout Results, exact p-values should be quoted.

Authors’ reply:

Thank you for pointing this out. We have now added exact p-values throughout the manuscript

8. The graphic quality of Fig. 2 is very low, to the point of illegibility.

Authors’ reply:

Thank you for your comment. We have now revised the figure.

Reviewer #2:

This is an interesting, innovative, well presented and honestly discussed piece of research.

The only suggestion I have is to replace commercial names with the actual compounds for the anti-HE drugs listed on page 4 (Hepa-Merz.....)

Authors’ reply

Thank you for the suggestion. We have now changed the names: “lactulose, rifaximin, L-ornithine L-aspartate or branched chain amino acid supplementation”

Reviewer #3:

The authors present an interesting crossover trial of AAC vs GS in nine patients with cirrhosis where oral AAC was associated with increased sleepiness on Karolinska but not with hypothesized pupillary light response. I have a few comments regarding the methodology.

1. Did you consider potential learning effects of repeated PHES and CRT? Typically, PHES is not repeated in such a short interval (i.e. 1-day) and as with any neuropsych evaluation there can be a learning effect. Summarizing the details of PHES results (i.e. each component of the test and score), by #day (1st vs 2nd) and by AAC vs GS can be helpful in understanding how the subjects performed after intervention. The fact that CRT time was reduced after AAC also suggests that the learning effects may have been an important factor.

Authors’ reply:

Thank you for your comment. We do agree this is relevant to consider. We tried to consider it by making a crossover design, even though it is not completely balanced. Further, we used unique PHES test for each test (4 in all). The reduced CRT index means an impairment of the CRT, which we interpret, could be due to tiredness and the ammonia increase. We have now mentioned the potential learning effect in the discussion: “The lack of association between hyperammonemia and PHES could be caused by a potential learning effect of the short period between tests. However, we tried to prevent this by applying four unique PHES test sets per patient.”

2. Please include more details of liver disease in the subjects (MELD score, complications of liver cirrhosis, etc.). The heterogeneity of cirrhosis etiology in a small sample could have contributed to the negative result.

Authors’ reply:

Thank you for your comment. We agree that the heterogeneity in the etiology could have contributed to the negative result, which we have now added in our discussion: “For example, the patients’ different liver etiology could have contributed to the negative result. Further, the population might have been more susceptible if the included patients had more severe liver disease, e.g., Child-Pugh class C and a history of hepatic encephalopathy. By this, the effect of intervention might have been more pronounced. In the design phase of the study, we decided to use the present inclusion criteria anticipating a rather high need for practical cooperation during the time-consuming assessments, in particular the pupillometry.”

Unfortunately, the components for the MELD and further complications to the cirrhosis is not available in our data set. Therefore, we cannot change table 1. Anyhow, Child Pugh Score is a good and reliable instrument to evaluation of the stage of liver disease

3. The figure 2 (schematic representation of pupillary light response) is difficult to read, either due to rendering or due to the font. Please revise to help with readability.

Authors’ reply:

Thank you for your comment. We have now revised the figure.

4. PSQI questions ask sleep quality over the past 1 month. How was this handled? One-time intervention is unlikely to affect the scoring over 1-month interval over 2-day study period.

Authors’ reply:

Thank you for your comment. The PSQI was only completed once before the first experimental day. We have made this more clear in the method section: “Before the first experimental day, the patients completed the validated self-rated assessment tool Pittsburgh Sleep Quality Index (PSQI), which assesses sleep quality and disturbances within the preceding month.”

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21 Jul 2022

The effect of induced hyperammonaemia on sleep and melanopsin-mediated pupillary light response in patients with liver cirrhosis: A single-blinded randomised crossover trial

PLOS ONE

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17 Aug 2022

Response to reviewers

Journal Requirements

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Authors’ reply: Thank you. We have ensured that the reference list is correct. We have not changed any current citations but have added an extra reference according to the reviewer's comments.

Review Comments to the Author

Reviewer #2:

There are no further comments from this reviewer - previous issues raised have been addressed adequately.

Reviewer #3:

1. The authors conducted an interesting, hypothesis-driven experiment with patients with a low-grade cirrhosis to test whether induced hyperammonemia acutely affect the pupillary response, as a surrogate measure of ipRGC function. There is a phase response curve to ipRGC; phase-shift effects response of ipRGC to the same intensity, spectrum, and duration of light will vary depending on the time of day. In addition, prior light history has been shown to affect the sensitivity of ipRGCs. Given the small sample size, it is not feasible to correct for multiple covariates. However, it will be useful to have additional information on the timing (both clock time and “circadian time” relative to the waketime of each subject) of measured pupillary response. It appears that the response was measured at hour 0, 2, and 4. Please include information on the clock/circadian time of these measures. Since ipRGC’s is most sensitive to light in the evening, the authors may consider including this in the discussion for a potential contributor to the lack of difference in the primary outcome.

Authors’ reply: Thank you for this important comment. We have now described it more thoroughly in the method section: “The study assessments were performed at different time points during the inclusion period, as shown in Table 1. The experimental days ran from morning (8.00 am) until afternoon (3.00 pm) to reduce differences in the circadian rhythm between patients.” And in the discussion, we have now included it as a potential contributor to the limited response: “In addition, the daytime assessment may have affected the results, as ipRGCs are more sensitive to light stimulation in the evening.”

2. Page 8. Pupilometer: What was the intensity of the blue light stimulation? Again, the response will depend on duration, intensity, and spectrum of the light.

Authors’ reply: Thank you for this comment. We have now inserted more detailed information about pupilometry: “The duration of light stimulation was 20 seconds, and the illuminance level of both light colors was 100 lux.”

3. Impaired sleep quality by which measure? PSQI? Please provide this information in the Table. (both baseline and after intervention)

Authors’ reply: Thank you for this comment. We measured the baseline self-assessed sleep quality by PSQI, which was not repeated after the intervention, as described in the method section. Besides, we measure the sleep quality during the inclusion period by Actigraph. We have now inserted the number of patients with high vs. low PSQI scores in table 1.

4. Child-Pugh Score an outdated measure of cirrhosis severity because of the subjective scoring. Can you provide MELD or MELD-Na score for the patients?

Authors’ reply: Unfortunately, the components for the MELD and further complications to the cirrhosis are not available in our data set. Therefore, we cannot change table 1. Anyhow, we consider the Child-Pugh Score a good and reliable instrument for evaluating the stage of liver disease.

5. The denominator of the Movement index should be TIB because TST does not include all of the “Time with movements during sleep”, which can be scored as wake during the sleep interval (therefore not included in TST). This likely won’t have a huge impact on the fragmentation index, but I wanted to make a note on it, disagreeing with the first reviewer.

Authors’ reply: Thank you for your comment on this. We have used the definitions provided by ActiGraph, the producer of the sleep evaluation watch. We have considered your note but have decided to keep the definitions suggested by Actigraph to promote reproducibility. Please find definitions in this reference: ActiLife 6 Manual | ActiGraph (actigraphcorp.com), page 62.

6. Discussion paragraph #3: How do you define superficial sleep? Would it be based on PSG study? I would use caution defining deep vs superficial sleep based on actigraphy measures. The subjects had less WASO and higher sleep efficiency after the experiment, regardless the intervention arm. Therefore, I would conclude that they were exhausted from a whole day of activities, which might have resulted in a better sleep.

Authors’ reply: Thank you for your comment. We have now changed the paragraph to clarify this: “We would have expected that sleep after AAC decreased sleep efficiency because Bersagliere et al. [4] found superficial sleep after an equivalent AAC intervention in a similar population. However, differences in the study design could explain the divergence because Bersagliere et al. assessed sleep during a nap in the same afternoon with EEG analysis. Moreover, heavier sleep in our population could be due to exhaustion from a long experimental day rather than the intervention itself.”

Submitted filename: Response to Reviewers.docx

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12 Sep 2022

The effect of induced hyperammonaemia on sleep and melanopsin-mediated pupillary light response in patients with liver cirrhosis: A single-blinded randomized crossover trial

PONE-D-21-00133R2

Dear Dr. Kann,

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Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: No further comments.

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Reviewer #3: Thank you for addressing this reviewer's comments thoroughly. I have no additional comments and accept the manuscript in the current form.

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Reviewer #2: Yes: Sara Montagnese

Reviewer #3: No

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19 Sep 2022

PONE-D-21-00133R2

The effect of induced hyperammonaemia on sleep and melanopsin-mediated pupillary light response in patients with liver cirrhosis: a single-blinded randomized crossover trial

Dear Dr. Kann:

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