Comparison of Total Cortisol, Free Cortisol, and Surrogate Markers of Free Cortisol in Diagnosis of Adrenal Insufficiency in Patients With Stable Cirrhosis

Accepted Manuscript Comparison of Total Cortisol, Free Cortisol, and Surrogate Markers of Free Cortisol in Diagnosis of Adrenal Insufficiency in Patients with Stable Cirrhosis Giuseppe Fede, Luisa Spadaro, Tania Tomaselli, Graziella Privitera, Roberto Scicali, Panayota Vasianopoulou, Evangelos Thalassinos, Nicholas Martin, Michael Thomas, Francesco Purrello, Andrew Kenneth Burroughs PII: DOI: Reference:

S1542-3565(13)01230-5 10.1016/j.cgh.2013.08.028 YJCGH 53472

To appear in: Clinical Gastroenterology and Hepatology Accepted Date: 10 August 2013 Please cite this article as: Fede G, Spadaro L, Tomaselli T, Privitera G, Scicali R, Vasianopoulou P, Thalassinos E, Martin N, Thomas M, Purrello F, Burroughs AK, Comparison of Total Cortisol, Free Cortisol, and Surrogate Markers of Free Cortisol in Diagnosis of Adrenal Insufficiency in Patients with Stable Cirrhosis, Clinical Gastroenterology and Hepatology (2013), doi: 10.1016/j.cgh.2013.08.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in Clinical Gastroenterology and Hepatology are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.

ACCEPTED MANUSCRIPT

Comparison of Total Cortisol, Free Cortisol, and Surrogate Markers of Free Cortisol in Diagnosis of Adrenal Insufficiency in Patients with Stable Cirrhosis Giuseppe Fede1-2, Luisa Spadaro2, Tania Tomaselli2, Graziella Privitera2, Roberto Scicali2, Panayota Vasianopoulou1, Evangelos Thalassinos1, Nicholas Martin3, Michael Thomas3, Francesco

1

RI PT

Purrello2, Andrew Kenneth Burroughs1

The Royal Free Sheila Sherlock Liver Centre and University Department of Surgery - University

College London and Royal Free Hospital, London, UK

Department of “Biomedicina clinica e molecolare”, University of Catania – Garibaldi Hospital,

SC

2

Catania, Italy

Department of Clinical Biochemistry, Royal Free Hospital, London, UK

M AN U

3

Contributorship statement:

Fede Giuseppe: conception and design, acquisition of data, analysis and interpretation of data, drafting the article; Luisa Spadaro: revising the article critically for important intellectual content;

TE D

Tania Tomaselli: analysis and interpretation of data; Graziella Privitera: analysis and interpretation of data; Roberto Scicali: analysis and interpretation of data; Panayota Vasianopoulou: acquisition of data; Evangelos Thalassinos: acquisition of data; Nicholas Martin: acquisition of data; Michael Thomas: revising the article critically for important intellectual content; Francesco Purrello:

EP

revising the article critically for important intellectual content; Andrew Kenneth Burroughs: revising the article critically for important intellectual content and final approval of the version to

AC C

be published.

Corrisponding Authors:

G Fede, MD. Department of “Biomedicina clinica e molecolare”, University of Catania – Garibaldi Hospital, Via Palermo 636, 95122 Catania, Italy; e-mail: [email protected] fax 0039- 095 7598401 AK Burroughs, FRCP FMedSci, Professor of Hepatology, The Sheila Sherlock Liver Centre, Royal Free Hospital, Pond Street, Hampstead, NW3 2QG London (UK); e-mail : [email protected] fax: 0044-02074726226

1

ACCEPTED MANUSCRIPT

List of abbreviations: Adrenal Insufficiency (AI), hypothalamus-pituitary-adrenal (HPA), short Synacthen test (SST), critical illness-related corticosteroid insufficiency (CIRCI), low dose short Synacthen test (LDSST), total serum cortisol (TC), free serum cortisol (FC), corticosteroid binding globulin (CBG), salivary cortisol (SC), calculated free cortisol (cFC) and free cortisol index (FCI),

RI PT

coefficients of variability (CVs), receiver operating characteristic (ROC). Conflict of interest/Study support:

AC C

EP

TE D

M AN U

SC

This study did not receive financial support and there are no potential competing interests.

2

ACCEPTED MANUSCRIPT

ABSTRACT

RI PT

Background and Aims: Measurements of serum levels of total cortisol can overestimate the prevalence of adrenal dysfunction in patients with cirrhosis, because they have low concentrations of cortisol-binding globulin (CBG) and albumin. We used measurements of serum total cortisol and serum free cortisol after the low-dose short Synacthen test (LDSST) to assess adrenal dysfunction.

SC

Methods: We studied 79 patients with stable cirrhosis; adrenal dysfunction was defined by a peak concentrations of total cortisol ≤494 mmol/L and/or peak concentrations of free cortisol ≤33 nmol/L after the LDSST. We determined free cortisol index (FCI) scores and calculated free cortisol (cFC) levels using Coolens’ equation. The Cox regression model was used to assess the relationship between adrenal dysfunction and outcomes (death or liver transplant).

TE D

M AN U

Results: Based on measurement of total cortisol, 34% of patients had adrenal dysfunction, and based on measurement of free cortisol, 29% had adrenal dysfunction. There was agreement between total cortisol and free cortisol levels in 22% of patients; in 13%, adrenal dysfunction was diagnosed only based on total cortisol, and in 6% only based on free cortisol (κ-coefficient 0.56, P<.01). Low concentrations of CBG (21 vs 54 ug/ml, P<.01) led to an overestimation of adrenal dysfunction based on measurement of total cortisol. Measurements of cFC constantly overestimated free cortisol concentrations, with variations as large as 87% for baseline values and up to 84% after stimulation. Adrenal insufficiency, defined by FCI scores<12, was detected in 30% of patients; among them 23% had also a subnormal peak levels of free cortisol (κ-coefficient, 0.70; P<.001). Adrenal dysfunction was not significantly associated with patient outcomes, based on Cox model analysis.

EP

Conclusions: Adrenal insufficiency, defined by LDSST, is frequent in patients with stable cirrhosis, based on measurements of total and free cortisol. FCI scores are better than measurement of total cortisol in assessing adrenal function in patients with cirrhosis. We did not associate adrenal dysfunction with outcome, but further studies are needed.

AC C

KEY WORDS: Adrenal function, liver disease, test, diagnosis, diagnostic assay

INTRODUCTION

Adrenal Insufficiency (AI) is frequent in patients with liver disease (1), and has been described with the term “hepato-adrenal syndrome” (2). The clinical presentation of AI may be insidious in the absence of adrenal crisis, with nonspecific symptoms such as tiredness, fatigue, weakness, which are common in cirrhosis (3). The prevalence of AI in patients with liver disease varies widely according to the study population: critically ill patients (33%-92%) (2, 4-7), stable cirrhosis (31%60%) (8-13) or decompensated cirrhosis, such as variceal bleeding (30%-48%) (14, 15) and ascites (26%-64%) (16). 3

ACCEPTED MANUSCRIPT

The short Synacthen test (SST), using 250 µg of cosyntropin (Synacthen®), is currently the recommended test to diagnose AI in critically ill patients, best described as critical illness-related corticosteroid insufficiency (CIRCI) (17). The low dose short Synacthen test (LDSST), using 1 µg of cosyntropin, is a more sensitive marker of sub-optimal adrenal function in non-critically ill

RI PT

patients (18). However there are no clear recommendations to assess adrenal function in patients with liver disease (1).

A relevant issue in the assessment of AI in cirrhosis is the measurement of total serum cortisol (TC) rather than serum free cortisol (FC). Normally, 70% of circulating cortisol is bound to corticosteroid

SC

binding globulin (CBG), 20% is bound to albumin, whereas the remaining 10% is free or unbound. Only the latter is the biologically active fraction (19). In patients with stable cirrhosis the TC overestimates the prevalence of AI compared to salivary cortisol assessment (9), which is surrogate

M AN U

marker of FC, and also with respect to directly measured FC (11, 20). Thus concerns have been expressed about the clinical relevance of AI associated with cirrhosis (21). The aim of this study was to assess adrenal function in cirrhosis using total cortisol and free cortisol measurement after LDSST. Secondly to evaluate the usefulness of estimated methods of free cortisol, ie calculated free cortisol (cFC) and free cortisol index (FCI), comparing them with directly measured free cortisol. Lastly we evaluated the potential prognostic value of adrenal

TE D

function assessment in terms of composite outcome (death/liver transplant). METHODS Patients

EP

This prospective observational study was performed at the Royal Free Sheila Sherlock Liver Centre (London, UK). The study protocol was approved by the local ethics committee. Patients with cirrhosis, referred for evaluation for liver transplant or for decompensation of cirrhosis (ascites,

AC C

variceal bleeding, jaundice, encephalopathy), if they gave informed consent, were consecutively enrolled. Exclusion criteria were: sepsis or hemodynamic instability (mean arterial pressure < 60 mmHg or vasopressor dependency), previous history of HPA axis disease, current or recent (within the preceding 3 months) history of corticosteroid therapy or other drugs that could impair the HPA axis, younger than 18 years, pregnancy. Laboratory Measurements In all patients the LDSST was performed by two authors (GF and VP), between 8.00 and 9.00 AM, following an overnight fast. Blood samples were obtained immediately before, 20 minutes and 30 minutes after intravenous injection of 1 µg of Synacthen. Low-dose Synacthen was prepared by 4

ACCEPTED MANUSCRIPT

adding 250 µg of Synacthen in 250 ml saline in a plastic bottle; 1 ml of this solution (containing 1 µg of Synacthen) was injected immediately, as previously described (22). Serum CBG concentrations were measured using a commercial RIA kit (Diasource, Nivelles, Belgium), intra-assay coefficients of variability (CVs): 3.9-8.6%; inter-assay CVs: 4.8-10.8%.

RI PT

Normal ranges for CBG (based on 2.5% and 97.5% percentiles) were: men 22-55 ug/L, women 40154 ug/L. TC concentrations were measured using an electrochemiluminescence immunoassay on a Roche modular E170 analyser (Roche Diagnostics, Mannheim, Germany), intra-assay CVs < 1.8% (assessed at 129 nmol/L, 352 nmol/L and 717 nmol/L); inter-assay CVs < 4.0% (assessed at 68 nmol/L, 516 nmol/L and 832 nmol/L). Plasma ACTH concentrations, assessed in a sub-set of 30

SC

consecutive patients, were measured at the University College London Hospital using a chemiluminescence immunoassay on an Immulite 2500 analyser (Siemens Medical Solutions

M AN U

Diagnostics Limited, Llanberis, UK) (intra-assay CVs < 4.7%; inter-assay CVs < 6.4%). FC was measured at the University Hospital of South Manchester as previously described (23). Briefly, 0.5 mL samples of serum were centrifuged in Ultrafiltration devices (Amicon Ultra-4 10 kDa; Millipore, Bedford, MA), and free cortisol was measured in the ultrafiltrate by LC-MS/MS. FCI was calculated as ratio between TC (nmol/L) and CBG (mg/L) (24). cFC was derived using the Coolens’ equation (supplementary material) (19).

TE D

Definition of adrenal insufficiency

For the purpose of this study, AI was defined by a peak-TC below 494 nmol/L at 20 or 30 minutes after stimulation (19, 25). Moreover a sub-set analysis was done using a baseline-TC < 138 nmol/L

EP

and a delta-TC (difference between peak and basal cortisol ) < 250 nmol/L (18). A low FC response after LDSST was defined by a peak-FC < 33 nmol/L after stimulation (19, 26). A sub-set analysis was done using a peak-FC < 25 nmol/L (27). Lastly, a FCI > 12 represented

AC C

sufficient adrenal response (24). Statistical Analysis

Data are expressed as means with standard error (SE), frequency (percentage) or medians with interquartile range (IQR) as appropriate. Comparisons between patients with and without adrenal dysfunction were performed using χ2 test (for categorical variables), Student’s t-test or the MannWhitney U test (for continuous variables, as appropriate). Correlation analysis were performed using the Spearman correlation coefficient. A Bland-Altman plot was used to compare the agreement between cFC and FC (28).

5

ACCEPTED MANUSCRIPT

Prognostic analysis for time to death or liver transplantation was performed by the Cox regression model; candidate prognostic variables were established a priori and included in the study protocol. Receiver operating characteristic (ROC) curves were used to evaluate the levels with the highest sensitivity and specificity of variables associated with prognosis in the simple Cox regression

RI PT

analysis. Cumulative survival curves were generated by the Kaplan–Meier approach and the two patients groups (dead/transplanted or alive without liver transplantation) compared with the logrank test. Variables that achieved a p value < 0.2 in simple Cox regression model were included in a multiple Cox regression modeling by backward elimination.

All statistical tests were two-tailed, and the significance level was set at p=0.05 or less. All

SC

statistical analyses were done using the SPSS 13.0 software for Windows.

Clinical characteristics of patients

M AN U

RESULTS

Seventy-nine patients with cirrhosis were enrolled: viral cirrhosis in 14 patients, alcohol in 44 (21 active drinkers), other aetiologies in 21 (supplementary material-table S1). The most frequent reason for hospital admission was transplant work up (61%), and decompensation of cirrhosis in the remainder 39% of patients (new onset or worsening ascites in 11 patients, variceal bleeding in 7,

TE D

encephalopathy in 7, and jaundice in 6). Assessment of adrenal function

Serum CBG: Serum CBG was related to Child-Pugh score (R:-0.28, p<0.05), and INR (R:-0.40,

EP

p<0.01), but there was no correlation with serum albumin. Moreover serum CBG was related to baseline-TC (R:+0.30, p<0.001) and peak-TC (R:+0.43, p<0.001), but not to baseline-FC and peak-

AC C

FC.

Plasma ACTH: Baseline plasma ACTH, measured in a sub-set of 30 patients, was not significantly different between patients with and without adrenal dysfunction defined either by TC (median 11 vs. 9 pmol/L, p=ns) or by FC (6 vs 12 pmol/L, p=ns). No patient had an ACTH value > 100 pmol/L, which is the threshold suggesting primary AI (29). Total serum cortisol: Delta-TC was related to Child-Pugh score (R:-0.25, p<0.01), whereas basalTC (R:-0.06, p=0.29) and peak-TC (R:-0.19, p=0.10) were not. Serum free cortisol: Basal-FC was related to severity of liver disease, scored by Child-Pugh score (R:+0.27, p<0.05), whereas peak-FC was not (R:+0.21, p=0.08). There was a significant correlation 6

ACCEPTED MANUSCRIPT

between TC and FC in baseline (R:+0.67, p<0.001), and after stimulation (peak-values: R=+0.66; delta-values: R=+0.61; all p<0.001) (Figure 1). Total Cortisol and Free cortisol response after LDSST

RI PT

AI defined by TC was present in 27/79 patients (34%). A basal-TC < 138 nmol/L was present in 10/79 patients (13%), and 56 patients (71%) had a delta-TC < 250 nmol/L (Table 1).

A peak-FC < 33 nmol was present in 22/79 (28%) patients (Table 1), whereas 15/79 (19%) had a

SC

peak-FC < 25 nmol (supplementary material-table S2).

Adrenal Function in patients with hypoalbuminemia and ascites

M AN U

We stratified our population into two subgroups according to albumin concentrations: Alb≤25 g/L (7 patients) and Alb>25 g/L (72 patients). The prevalence of adrenal dysfunction was similar in the two groups either using TC criteria (peak-TC<494 nm/L: 43% vs. 32%, p=ns) or FC criteria (peakFC<33 nm/L: 29% vs. 28%; all p=ns) (supplementary material–table S3). Patients with ascites (46%) had lower serum sodium (p=0.001), serum albumin (p=0.035) and CBG

TE D

(p=0.046) levels, and a more severe liver disease (Child-Pugh: p < 0.001; MELD scores: p=0.037). Moreover they showed significantly higher FC concentrations both in baseline (p=0.003) and after stimulation (peak FC: p=0.001; delta FC: p=0.043), probably due to the hypoalbuminemia present

EP

in these patients (supplementary material–table S4).

AC C

Comparison between total cortisol and free cortisol criteria in assessing adrenal function Among the 27 patients diagnosed with AI by TC measurement (peak-TC<494 nm/L), 17 (63%) had also a subnormal peak-FC, whereas 10 (37%) showed a normal peak-FC. Furthermore 5/79 (6%) patients showed a subnormal FC but normal TC response. The Kappa-coefficient between the two sets of criteria was 0.56, p<0.001. Patients that were diagnosed as having AI by TC but with a normal FC response, had lower CBG concentrations (21 vs. 54 ug/ml, p<0.01), and this explains the discordance between TC and FC concentrations. Other factors, such as severity of liver disease did not explain the discordance (Table 2, supplementary material-table S5). 7

ACCEPTED MANUSCRIPT

Comparison between measured serum free cortisol, calculated serum free cortisol, and free cortisol index

RI PT

Calculated serum free cortisol (cFC): There was a significant correlation between FC and cFC both at baseline (R:+0.87, p<0.001) and after stimulation (R:+0.90, p<0.001) (Figure S1). However cFC constantly overestimated FC, with a variation up to 87% (mean variation 55%) for baseline value and up to 84% (mean variation 57%) after stimulation.

SC

Bland-Altman plot showed a systematic bias in which the cFC exceeded the measured FC values both for baseline (mean percent bias of 81%, limits of agreement from 16 to 146) and after

M AN U

stimulation (mean percent bias of 82%, limits of agreement from 38 to 125) (Figure 2). Free Cortisol Index (FCI): FCI was significantly related to FC both at baseline (R:+0.84, p<0.001) and after stimulation (R:+0.66, p<0.001) (Figure S1). Among patients with FCI < 12 (n=24), 18 had also a subnormal peak-FC. The Kappa-coefficient between the two sets of criteria was 0.70 (p<0.001), showing good agreement.

TE D

Prognostic analysis for time to death or liver transplantation During a median follow-up of 8 (2-11) months 16 patients were transplanted, 14 died, and 1 was lost to follow-up. Causes of death were gastrointestinal bleeding (n=2), sepsis (n=2), end-stage liver

EP

failure (n=6), unknown (n=4) (supplementary material-table S6). In simple Cox regression analysis the following variables were studied as candidate prognostic

AC C

variables: free cortisol levels (baseline, post-stimulated and delta values); Child-Pugh score; serum creatinine; serum sodium. Lower serum sodium (median 136vs.139 mmol/L, p<0.05) and higher Child-Pugh score (median 9 vs.8, p<0.05) were associated with a worse prognosis (Table 3). Using the ROC curves serum sodium value ≤ 136 mmol/L was able to discriminate patients with worse prognosis (AUC 0.64, p = 0.04), whereas AUC for Child Score (AUC 0.63, p = 0.06) was no significant. In multiple Cox regression model only serum sodium resulted significant prognostic factor (Table 3). In a sub-set analysis MELD score, serum total cortisol levels, and cause of admission were analyzed as potential prognostic factor, but none of them were significantly associated with prognosis in the simple Cox regression analysis (supplementary material-table S7).

8

ACCEPTED MANUSCRIPT

The Kaplan-Meier curve showed similar transplant free survival between patients with and without AI defined by TC (10.7 vs. 13.5 months, p=ns) or by FC (11.9 vs. 12.9 months, p=ns). Additionally, no significant differences were observed in the composite endpoint of mortality/liver transplantation: AI defined by TC (8.7 vs. 11.1 months, p=ns) or by FC (10.6 vs. 10.2 months,

RI PT

p=ns). We subdivided our study population according to the tertile distribution of TC and FC levels (supplementary material-table S7). The Kaplan-Meier curves showed no significant differences between groups, however patients with low basal FC (≤ 13.5 nmol/L) showed a better outcome

SC

(supplementary material-figure S2).

DISCUSSION

M AN U

Our study suggests that in stable cirrhosis using both serum total cortisol and serum free cortisol criteria the prevalence of adrenal dysfunction is increased, although there was discordance between the two diagnostic criteria. We also showed that CBG concentration is the main factor explaining the discordance between the two diagnostic criteria, and that the free cortisol index has a good level of agreement with free cortisol.

In our study the prevalence of AI using TC measurement was 34%, similar to that reported

TE D

previously (8-12). Moreover, a high proportion of our patients (29%) had a peak-FC less than 33 nmol/L, which is the lowest published limit of FC response after SST in healthy subjects (26, 30). Using a more restrictive cutoff (peak-FC < 25 nmol/L) (27), the prevalence of adrenal dysfunction was still high (19%). Tan et al (11) using the same threshold for peak-FC (less than 33 nmol/L) to

EP

assess adrenal function in 43 patients with stable cirrhosis found adrenal dysfunction in only 12% of patients. The higher prevalence of adrenal dysfunction in our study could be explained by the use of LDSST, which is more sensitive than SST in assessing adrenal function in non-critically ill patients

AC C

(18). Moreover the group of patients with low peak-FC had also a significantly lower basal-FC (and basal-TC), confirming the existence of adrenal dysfunction in these patients independently from the stimulation test. This is an important finding of our study, and confirms the existence of adrenal dysfunction in cirrhosis even taking in account free cortisol rather than total cortisol measurement. Basal free cortisol was positively related to severity of liver disease scored by Child-Pug score. This is in accordance with previous data (13), and may suggest that more severely ill patients have a basal activated adrenal axis, which could lead to an “adrenal-exhaustion syndrome” (31). Moreover patients with ascites showed significantly higher free cortisol concentrations, similarly to that

9

ACCEPTED MANUSCRIPT

reported by Thevenot et al.(13), and this is probably due to the lower CBG and albumin concentrations in these patients. In 19% of patients there was discordance between total cortisol and free cortisol criteria in diagnosing AI. This discordance was explained by CBG, whereas albumin concentrations, or other

RI PT

factors associated with severity of liver disease (ie MELD and Child-Pugh score) did not explain the discordance. This suggests that CBG concentration has a very important role in the cortisol binding in cirrhosis. However, previous data showed that a concomitant CBG and albumin deficiency had a synergistic addictive effect on free cortisol concentration (32). In our study cohort,

SC

only 7 (9%) patients had hypoalbuminemia.

The Coolens’ equation estimates free cortisol from total cortisol and CBG levels, and this method

M AN U

has been validated in critically ill patients (19). In our study the cFC using the Coolens’ equation consistently overestimated free cortisol with variations up to 87%, and there was poor agreement shown by the Bland-Altman analysis, similarly to that shown by Tan et al (11). Coolens’ equation is probably inappropriate in cirrhosis because it does not account for the contribution of hypoalbuminemia (32), and for the non-linear binding properties of CBG following cosyntropin stimulation (33).

TE D

The FCI, ratio between total cortisol and CBG, has been used as marker of free cortisol in previous studies (11, 24). In our study FCI correlated well with both basal-FC (R = 0.84) and peak-FC (R=0.66). Moreover there was a good level of agreement between free cortisol and FCI to diagnose adrenal insufficiency. This is an interesting finding and, if confirmed by further studies, could have

EP

important clinical implications, because the FCI is a simple method to estimate FC. Conversely, free cortisol assays is complex and expensive and is not routinely available in clinical practice;

AC C

secondly diagnostic cut-off values have not been clearly defined. Primary adrenal insufficiency is characterized by high levels of basal ACTH (higher than 100 pg/ml) and it is ruled out by normal ACTH values (29). We measured the baseline plasma ACTH in a sub-set of 30 patients: no patient had ACTH value greater than 100 pg/ml. Furthermore ACTH concentrations were not significantly different between patients with and without AI. This could suggest a secondary rather than a primary adrenal insufficiency. However the lack of other tests to study the HPA axis, does not allow firm conclusions to be drawn. The prognostic relevance of adrenal insufficiency in non-critically ill patients with liver disease has not been defined (1). In the study of Tan et al (11), adrenal dysfunction defined by a stimulated-FC < 33 nmol/L was significantly associated with higher mortality. In contrast, Thevenot et al.(13) 10

ACCEPTED MANUSCRIPT

found that a higher stimulated-FC was associated with lower survival in a cohort of 95 patients with stable cirrhosis. Lastly, in a recent study (34) assessing 143 non-critically ill patients with cirrhosis, low total delta cortisol values have been associated with higher incidence of severe sepsis, type 1 hepatorenal syndrome, and higher short-term mortality. However in this study direct measurement

RI PT

of free cortisol was not performed. In our study we did not find an association between adrenal insufficiency, defined either by total or by free cortisol, and outcomes (death or liver transplant). Patients with the lowest basal FC level, according to the tertile distribution, showed a better outcome, albeit there was not statistical significance. This may suggest that high free cortisol levels

SC

in cirrhosis may be a marker of “adrenal stress state” (13), and worse prognosis.

Our study was not powered to perform a prognostic analysis. Further studies are urgently needed to assess the association between adrenal function and prognosis in patients with cirrhosis. These

M AN U

studies, adequately powered and with a long follow-up, should serially assess adrenal function to investigate the incidence of adrenal dysfunction in cirrhosis and its correlation not only with survival, but also with other outcomes such as new onset of ascites, encephalopathy, bleeding, infections/sepsis and shock. In fact, in stable cirrhosis adrenal insufficiency could be a sub-clinical condition triggered by acute events such as infections or other acute illness, leading to a worse prognosis (8). Moreover, standardized criteria are needed to define adrenal insufficiency in

TE D

cirrhosis.

The peak-FC threshold used in this study (25 and 33 nmol/L) to define adrenal dysfunction reflexes the data, albeit scare, investigating serum free cortisol in healthy subjects (26, 27). There is a need

EP

to define clinically relevant cut-off values of free cortisol for future clinical studies. In conclusion, this study shows a high prevalence of adrenal dysfunction in patients with cirrhosis independently of cortisol assays (free or total cortisol), cutoffs of peak-FC (25 or 33 nmol/L), and

AC C

stimulation test (both basal and peak cortisol were lower in patients diagnosed with adrenal insufficiency). The FCI < 12, as previously established (24), was closely correlated to a suboptimal free cortisol response after LDSST. This suggests that FCI should have a place in the assessment of adrenal function in patients with liver disease. Our findings with free cortisol assays should be validated in other studies. A crucial issue remains to establish if adrenal insufficiency has clinical consequences in cirrhosis, and if so, if this can be prevented and/or remedied by cortisol supplementation therapy.

11

ACCEPTED MANUSCRIPT

ACKNOWLEDGEMENTS We would like to thank Laura Owen for organizing the serum free cortisol measurements, Filippo Palermo for helping with statistics, and Gennaro D’Amico for helping with statistics and prognostic

RI PT

analysis.

FIGURE LEGENDS

Figure 1: Relationship between serum total cortisol and plasma free cortisol: Spearman

stimulation (Panel B), and for delta values (Panel C).

SC

correlation between serum total cortisol and plasma free cortisol in baseline (Panel A), after

AC C

EP

TE D

M AN U

Figure 2: Agreement between measured and calculated free cortisol: Bland-Altman plot comparing measured (FC) and calculated (cFC) free cortisol in baseline (Panel A) and after stimulation (Panel B). The average of the measured and calculated free cortisol values are shown on the x-axis and the percent (%) prediction bias, which is the percent of the difference between calculated and measured free cortisol, is shown on the y-axis. The heavy solid line represents the mean percent bias between the two set of values, the dash lines represent the 95% limits of agreement, the dotted line represents the zero percent bias shown as reference. The plots show a systematic bias in which the calculated free cortisol exceeded the measured values both at baseline (mean percent bias of 81%, limits of agreement from 16 to 146) (Panel A) and after stimulation (mean percent bias of 82%, limits of agreement from 38 to 125) (panel B).

12

ACCEPTED MANUSCRIPT

Reference List

(1) Fede G, et al. Adrenocortical dysfunction in liver disease: a systematic review. Hepatology 2012 Apr;55(4):1282-1291.

RI PT

(2) Marik PE, et al. The hepatoadrenal syndrome: a common yet unrecognized clinical condition. Crit Care Med 2005 Jun;33(6):1254-1259.

(3) Tomaselli T, et al. Corticosteroid replacement therapy in hepatoadrenal syndrome: case report with a review of the literature. Eur J Gastroenterol Hepatol 2012 Jul 30.

SC

(4) Fernandez J, et al. Adrenal insufficiency in patients with cirrhosis and septic shock: Effect of treatment with hydrocortisone on survival. Hepatology 2006 Nov;44(5):1288-1295.

M AN U

(5) Harry R, et al. The clinical importance of adrenal insufficiency in acute hepatic dysfunction. Hepatology 2002 Aug;36(2):395-402. (6) Tsai MH, et al. Adrenal insufficiency in patients with cirrhosis, severe sepsis and septic shock. Hepatology 2006 Apr;43(4):673-681. (7) Arabi YM, et al. Low-dose hydrocortisone in patients with cirrhosis and septic shock: a randomized controlled trial. CMAJ 2010 Dec 14;182(18):1971-1977. (8) Fede G, et al. Assessment of adrenocortical reserve in stable patients with cirrhosis. J Hepatol 2011 Feb;54(2):243-250.

TE D

(9) Galbois A, et al. Assessment of adrenal function in cirrhotic patients: salivary cortisol should be preferred. J Hepatol 2010 Jun;52(6):839-845. (10) McDonald JA, et al. Hypothalamic-pituitary adrenal function in end-stage non-alcoholic liver disease. J Gastroenterol Hepatol 1993 May;8(3):247-253.

EP

(11) Tan T, et al. Characterising adrenal function using directly measured plasma free cortisol in stable severe liver disease. J Hepatol 2010 Nov;53(5):841-848.

AC C

(12) Zietz B, et al. Dysfunction of the hypothalamic-pituitary-glandular axes and relation to Child-Pugh classification in male patients with alcoholic and virus-related cirrhosis. Eur J Gastroenterol Hepatol 2003 May;15(5):495-501. (13) Thevenot T, et al. High Serum Levels of Free Cortisol Indicate Severity of Cirrhosis in Hemodynamically Stable Patients. J Gastroenterol Hepatol 2012 May 31. (14) Graupera J, et al. Incidence and prognostic significance of relative adrenal insufficiency in cirrhotic patients with severe variceal bleeding. 2011. p. 121A-319A. (15) Triantos C, et al. Critical illness related corticosteroid insufficiency (CIRCI) in patients with cirrhosis and variceal bleeding. Clinical Gastroenterology and Hepatology 2011;9:595-601. (16) Risso A, et al. Adrenal dysfunction in nonseptic cirrhotic patients with ascites: Impact on survival., 43 ed 2011. p. S74. 13

ACCEPTED MANUSCRIPT

(17) Marik PE, et al. Recommendations for the diagnosis and management of corticosteroid insufficiency in critically ill adult patients: consensus statements from an international task force by the American College of Critical Care Medicine. Crit Care Med 2008 Jun;36(6):1937-1949.

RI PT

(18) Kazlauskaite R, et al. Corticotropin tests for hypothalamic-pituitary- adrenal insufficiency: a metaanalysis. J Clin Endocrinol Metab 2008 Nov;93(11):4245-4253. (19) Coolens JL, et al. Clinical use of unbound plasma cortisol as calculated from total cortisol and corticosteroid-binding globulin. J Steroid Biochem 1987 Feb;26(2):197-202. (20) Thevenot T, et al. Assessment of adrenal function in cirrhotic patients using concentration of serum-free and salivary cortisol. Liver Int 2011 Mar;31(3):425-433.

SC

(21) Thevenot T, et al. Assessing adrenal function in cirrhotic patients: is there a reliable test? Gastroenterol Clin Biol 2009 Jun;33(6-7):584-588.

M AN U

(22) Gonc EN, et al. Significance of low-dose and standard-dose ACTH tests compared to overnight metyrapone test in the diagnosis of adrenal insufficiency in childhood. Horm Res 2003;60(4):191-197. (23) Perogamvros I, et al. Salivary cortisone is a potential biomarker for serum free cortisol. J Clin Endocrinol Metab 2010 Nov;95(11):4951-4958. (24) Vincent RP, et al. Serum total cortisol and free cortisol index give different information regarding the hypothalamus-pituitary-adrenal axis reserve in patients with liver impairment. Ann Clin Biochem 2009 Nov;46(Pt 6):505-507.

TE D

(25) Wiest R, et al. Uncoupling of sympathetic nervous system and hypothalamic-pituitaryadrenal axis in cirrhosis. J Gastroenterol Hepatol 2008 Dec;23(12):1901-1908. (26) Vogeser M, et al. Dialyzable free cortisol after stimulation with Synacthen. Clin Biochem 2002 Oct;35(7):539-543.

EP

(27) Limor R, et al. Serum free cortisol as an ancillary tool in the interpretation of the low-dose 1-mug ACTH test. Clin Endocrinol (Oxf) 2011 Sep;75(3):294-300.

AC C

(28) Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986 Feb 8;1(8476):307-310. (29) Oelkers W. Adrenal insufficiency. N Engl J Med 1996;335:1206-1212. (30) Lewis JG, et al. Plasma free cortisol fraction reflects levels of functioning corticosteroidbinding globulin. Clin Chim Acta 2005 Sep;359(1-2):189-194. (31) Marik PE. Adrenal-exhaustion syndrome in patients with liver disease. Intensive Care Med 2006 Feb;32(2):275-280. (32) Dorin RI, et al. Validation of a simple method of estimating plasma free cortisol: role of cortisol binding to albumin. Clin Biochem 2009 Jan;42(1-2):64-71. (33) Hamrahian AH, et al. Measurements of serum free cortisol in critically ill patients. N Engl J Med 2004 Apr 15;350(16):1629-1638. 14

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

(34) Acevedo J, et al. Relative adrenal insufficiency in decompensated cirrhosis. relationship to short-term risk of severe sepsis, hepatorenal syndrome and death. Hepatology 2013 May 31.

15

ACCEPTED MANUSCRIPT

TABLES

Table 1. Clinical characteristics of patients with and without adrenal insufficiency diagnosed according total cortisol and free cortisol criteria after Low Dose Short Sinacthen Test peak TC ≥

494 nmol/L 494 nmol/L

peak FC < p value

33 nmol/L

(n: 27/79)

(n: 52/79)

(22/79)

Age (years)

44 (39-54)

57 (51-61)

ns

41 (39-53)

Gender (male/female)

18/9

35/17

ns

14/8

Viral (%)

15

19

ns

27

Alcohol/active drinkers* (%)

56/26

56/27

ns/ns

50/23

Other (%)

29

25

ns

23

Ascites (%)

33

52

ns

23

A/B/C

5/17/5

14/26/12

ns

Encephalopaty (%)

19

17

ns

115 (99-

120 (100-

Systolic BP (mmHg)

129)

140)

Diastolic BP (mmHg)

60 (50-67)

HR (bpm) MAP (mmHg) Creatinine (µmol/L)

51 (44-105) 64 (51-95)

33 nmol/L

p value

(57/79)

56 (51-61)

<0.01

39/18

ns

14

ns

58/28

ns/ns ns < 0.01

5/14/3

14/29/14

ns

18

18

ns

119 (99-

115 (100-

ns

129)

138)

ns

70 (57-70)

ns

60 (50-66)

68 (58-70)

ns

80 (65-93)

85 (80-90)

ns

80 (72-95)

83 (77-90)

ns

78 (71-84)

80 (70-93)

ns

69 (65-86)

80 (73-93)

ns

ns

45 (43-83)

67 (54-102) ns

140 (138-

140 (130-

144)

142)

3.9 (3.7-

4.5 (3.7-

4.4)

5.0)

Child-Pugh class

M AN U

28

54

TE D

SC

Aetiology

peak FC ≥

RI PT

peak TC<

140 (138-

140 (127-

143)

142)

4.1 (3.7-

4.1 (3.7-

4.6)

4.9)

3.7 (2.9-

6.2 (4.3-

3.5 (2.9-

6.2 (4.6-

5.3)

8.4)

0.01

4.4)

8.0)

<0.01

38 (32-46)

26 (12-38)

ns

37 (30-40)

29 (14-55)

ns

32 (28-36)

32 (26-38)

ns

32 (26-37)

32 (26-37)

ns

Total Cholesterol (mmol/L)

3.3 ±1.2

2.8 ±0.6

ns

3.6 ±1.6

2.7 ±0.6

ns

HDL (mmol/L)

1.2 ± 0.8

0.8 ± 0.6

ns

1.3 ± 0.8

0.8 ± 0.5

ns

LDL (mmol/L)

1.8 ± 0.5

1.6 ± 0.5

ns

1.9 ± 0.5

1.5 ± 0.5

ns

1.6 (1.3-

1.5 (1.3-

1.6 (1.3-

1.6 (1.3-

Sodium (mmol/L)

Urea (mmol/L) Total Bilirubin (mmol/L)

AC C

Albumin (g/L)

EP

Potassium (mmol/L)

ns

ns

ns

ns

1.8)

1.7)

0.01

1.6)

2.1)

ns

Child-Pugh Score

8 (6-9)

8 (6-9)

ns

6 (6-9)

9 (6-10)

ns

MELD Score

12 (11-17)

14 (11-24)

ns

13 (11-13)

14 (11-14)

ns

CBG (ug/ml)

33 ± 11

42 ± 19

0.01

36 ± 9

39 ± 20

ns

ACTH (pmol/L)

11 (5-23)

9 (5-30)

ns

6.3 (5-27)

12 (5-27)

ns

113 (67-

586 (315-

Basal TC (nmol/L)

179)

623)

<0.01

97 (48-302) 622)

<0.01

Peak TC (nmol/L)

305 (224-

719 (594-

<0.01

301 (173-

<0.01

INR

532 (302-

719 (588-

#

ACCEPTED MANUSCRIPT

869)

350)

867)

201 (126-

243 (220-

198 (104-

246 (225-

229)

283)

214)

282)

4.6 (2.2-

27 (17.7-

3.0 (1.6-

24 (15.1-

9.6)

43.8)

7.2)

42.4)

19.6 (8.6-

64.5 (47.5-

15.3 (6.5-

61.7 (50-

33.7)

91.5)

24.7)

88.5)

14.8 (6.4-

35.5 (22.2-

Delta FC (nmol/L)

24.5)

47.6)

Basal cFC (nmol/L)

28 (11-42)

76 (34-120) <0.01

76.8)

148 (126-

12.3 (4.8-

Delta TC (nmol/L)

Basal FC (nmol/L)

Peak FC (nmol/L)

<0.01

<0.01

<0.01

5.9 (2.97.6)

<0.01

53.6 (30.5-

Peak FCI +

Deaths (%) Liver Transplent

+

(%)

186)

7.6 (4.3-

12.4 (7.5-

10.0)

17.1)

12.6 (8.1-

17.9 (13.9-

17.5)

25.0)

19

17

22

19

<0.01

17.5)

<0.001

<0.01

<0.01

12.3 (8.617.2)

<0.01

136.8 (110180)

<0.01

40.1 (25.547.4)

SC

Basal FCI

64 (38-96)

5.9 (2.9-

12.2 (8.6-

7.6)

17.2)

9.8 (7.9-

19.2 (15.2-

M AN U

Peak cFC (nmol/L)

0.02

RI PT

398)

<0.01

<0.01

<0.001

11.6)

25.4)

<0.01

ns

9

21

ns

ns

14

25

ns

BP: blood pressure; TC: serum total cortisol; FC: serum free cortisol; cFC: calculated free cortisol by Coolens’ equation; FCI: free cortisol index. Mann-Whitney test

TE D

#

*number of patients with alcohol related cirrhosis active drinking at LDSST date

EP

+ One patients was lost to follow-up

Table 2. Comparison between patients with and without adrenal dysfunction assessed by total

AC C

cortisol and free cortisol response after low dose Synacthen test. Low FC

Low TC

Low TC

Normal FC

And

And

and

and

Normal TC

Normal FC

Low FC

Normal TC

(n = 5)

(n = 10)

(n = 17)

(n = 47)

#

p value

Age (years)

54 (45-68)

54 (52-65)

49 (40-57)

57 (51-65)

ns

Gender (male/female)

3/2

7/3

11/6

32/15

ns

Viral (%)

40

0

23.5

17

ns

Alcohol/active drinkers* (%)

20/20

50/30

59/24

60/28

ns/ns

Other (%)

40

50

17.5

23

ns

Aetiology

20

50

23.5

55

ns

A/B/C

1/2/2

1/5/4

4/12/1

13/24/10

ns

Encephalopaty (%)

20

20

17.6

17

ns

Total Bilirubin (mmol/L)

50 (25-65)

54 (38-113)

37 (29-69)

33 (21-69)

ns

Albumin (g/L)

38 (34-40)

32 (28-34)

32 (27-37)

32 (29-37)

ns

Total Cholesterol (mmol/L)

3.6 ± 0.4

2.0 ±1.3

3.2 ±1.4

3.4 ± 1.3

ns

HDL (mmol/L)

1.1 ± 0.1

0.7 ± 0.6

1.2 ± 0.8

0.9 ± 06

ns

LDL (mmol/L)

2.1 ± 0.3

1.0 ± 0.6

1.6 ± 0.7

2.1 ± 1.1

ns

INR

1.5 (1.1-1.6)

1.7 (1.5-2.1)

1.6 (1.4-1.8)

1.4 (1.3-1.6)

ns

Child-Pugh Score

7 (6-9)

9 (8-10)

8 (7-9)

7 (6-9)

ns

MELD Score

23 (15-33)

14 (12-19)

14 (11-18)

16 (13-21)

ns

CBG (ug/ml)

54 ± 16

21 ± 7

33 ± 12

40 ± 20

<0.01

Basal TC (nmol/L)

304 (125-406) 219(164-288)

SC

ACCEPTED MANUSCRIPT

Ascites (%)

174 (94-245)

460 (335-587) ns

Peak TC (nmol/L)

531 (510-688) 423 (380-440) 276 (235-410) 678 (588-802) <0.01

Delta TC (nmol/L)

304 (230-397) 163 (116-247) 113 (64-179)

217 (150-294) <0.05

Basal FC (nmol/L)

4.4 (2.7-11)

16.9 (14-32)

9.4 (3.8-15)

26.3(15-47)

<0.05

Peak FC (nmol/L)

27 (19-31)

50 (44-58)

21.3 (13-25)

62.6 (45-85)

<0.01

Delta FC (nmol/L)

20 (12.7-24.9) 27 (21-42)

11.6 (6-15)

35.6 (23-45)

<0.01

M AN U

RI PT

Child-Pugh class

#

TE D

Low FC: peak free cortisol < 33 nm/L, Normal FC: peak free cortisol > 33 nm/L, Low TC: peak total cortisol < 494 nm/L, Normal TC: peak total cortisol > 494 nm/L. Kruskal-Wallis test

EP

*number of patients with alcohol related cirrhosis active drinking at LDSST date

Table 3. Cox regression analysis of variables associated with death or liver transplant.

AC C

SIMPLE COX REGRESSION Hazard

Continuous Variables

Ratio

p value

(95%CI)

Creatinine (µmol/L)

1.00 (0.99-1.01) 0.42

Sodium (mmol/L)

0.92 (0.87-0.97) 0.002

Child-Pugh Score

1.36 (1.10-1.67) 0.004

Basal FC* (nmol/L)

1.01 (0.99-1.03) 0.55

Peak FC* (nmol/L)

1.01 (0.99-1.02) 0.09

Delta FC* (nmol/L)

1.00 (0.98-1.03) 0.7

ACCEPTED MANUSCRIPT

MULTIPLE COX REGRESSION Variables

Hazard ratio

P values

Serum sodium

0.91 (0.86-0.96) < 0.001

Child-Pugh Score

1.18 (0.93-1.50) 0.18

RI PT

(95%CI)

SC

Peak FC* (nmol/L) 1.00 (0.99-1.02) 0.76

M AN U

FC: serum free cortisol

* Free cortisol levels (baseline, post-stimulated and delta values) were included as markers of adrenal function,

due to the correlation between total and free cortisol in our cohorts, and

AC C

EP

TE D

considering the higher reliability of the latter as marker of adrenal function in cirrhosis.

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

SUPPLEMENTARY FIGURE LEGENDS Figure S1: Spearman correlation between measured free cortisol and calculated free cortisol (Panel A), and between measured free cortisol and free cortisol index (Panel B). Figure S2: Kaplan-Meier curves showing the prevalence of death/liver transplantation according to

RI PT

the tertile distribution of baseline (Panel A and D), stimulated (Panel B) and delta (Panel C) free

AC C

EP

TE D

M AN U

SC

cortisol.

ACCEPTED MANUSCRIPT

SUPPLEMENTARY MATERIAL Table S1. Demographic data of the 79 patients enrolled in the study

Age (years)

55 (50-63)

Gender (male/female)

53/26

RI PT

Median (IQR)

Aetiology Viral (%) Alcohol/active drinkers (%)

56/27

Other (%)

26

Ascites (%)

46

SC

18 #

(A/B/C)

19/43/17

Encephalopaty (%)

18

HCC (%)

15

SBP (%)

5

Infections other than SBP 9

Systolic BP (mmHg)

120 (102-134)

Diastolic BP (mmHg)

67 (60-72)

HR (bpm)

80 (70-90)

MAP (mmHg)

83 (76-93)

TE D

and sepsis* (%)

EP

Serum Creatinine (µmol/L)

Serum Sodium (mmol/L)

AC C

Serum Potassium

M AN U

Child-Pugh class

71 (56-88)

138 (135-140)

(mmol/L)

4.1 (3.8-4.5)

Serum Urea (mmol/L)

4.8 (3.5-6.7)

Serum total Bilirubin (mmol/L)

37 (25-70)

Serum Albumin (g/L)

32 (29-37)

Total Cholesterol (mmol/L)

3.4 (2.6-4.5)

HDL (mmol/L)

1.1 (0.5-1.6)

ACCEPTED MANUSCRIPT

1.9 (1.2-2.5)

INR

1.5 (1.4-1.7)

Child-Pugh Score

8 (7-9)

MELD Score

15 (13-20)

#

RI PT

LDL (mmol/L)

number of patients with alcohol related cirrhosis active drinking at LDSST date

*Infections: Chest infections in 5 patients, urinary tract infection in 1, and skin infection in 1.

SC

Table S2. Clinical characteristics of patients with low peak FC response (< 25 nmol/L) after LDSST compared to normal responders. peak FC ≥ 25

nmol/L

nmol/L

p

(64/79)

value#

(15/79)

M AN U

peak FC < 25

Age (years)

50 (41-54)

55 (51-64)

<0.03

Gender (male/female)

10/5

43/21

ns

Viral (%)

20

17

ns

Alcohol/active drinkers* (%)

67/33

53/25

ns/ns

Other (%)

13

30

ns

Ascites (%)

27

50

ns

Child-Pugh class

TE D

Aetiology

3/9/3

16/34/14

ns

Encephalopaty (%)

20

17

ns

Systolic BP (mmHg)

120 (103-130) 120 (101-139)

ns

60 (51-72)

69 (60-71)

ns

80 (70-98)

80 (70-90)

ns

80 (71-87)

82 (77-93)

ns

Creatinine (µmol/L)

56 (45-74)

74 (59-94)

ns

Sodium (mmol/L)

138 (137-140) 138 (134-141)

ns

Potassium (mmol/L)

4.1 (3.9-4.2)

4.1 (3.7-4.4)

ns

Urea (mmol/L)

3.5 (2.7-4.5)

5.1 (3.9-8.1)

<0.01

Total Bilirubin (mmol/L)

37 (28-72)

38 (25-70)

ns

Albumin (g/L)

32 (30-38)

32 (29-37)

ns

Total Cholesterol (mmol/L)

3.6 (3-4.2)

3.2 (2.4-3.7)

ns

HDL (mmol/L)

1.3 (0.9-1.9)

1 (0.3-1.2)

ns

LDL (mmol/L)

1.8 (1.4-2.6)

1.8 (1.1-2.2)

ns

INR

1.6 (1.5-1.8)

1.5 (1.4-1.6)

ns

Diastolic BP (mmHg) HR (bpm)

AC C

MAP (mmHg)

EP

A/B/C

Child-Pugh Score

8 (7-9)

8 (7-9)

ns

MELD Score

15 (12-24)

15 (13-21)

ns

CBG (ug/ml)

34 (22-45)

34 (25-45)

ns

ACTH (pmol/L)

17 (5.3-28)

14 (5-30)

ns

Basal TC (nmol/L)

130 (90-214)

399 (282-549)

<0.01

Peak TC (nmol/L)

300 (233-430) 643 (516-743)

<0.01

Delta TC (nmol/L)

113 (69-198)

206 (136-290)

0.02

21.8 (14.2-41.4)

<0.01

6.67 (2.713.7)

Basal FC (nmol/L)

19.9 (10.922.6)

54.7 (41.9-75.9)

<0.01

Delta FC (nmol/L)

9.8 (4.5-13.1)

28.5 (21.4-43.8)

<0.01

61 (33.6-111)

<0.01

SC

Peak FC (nmol/L)

26.7) 44.9 (24.2-

M AN U

13.4 (4.2Basal cFC (nmol/L)

Peak cFC (nmol/L)

62.4)

134 (107-179)

<0.01

Basal FCI

4.7 (2.0-7.1)

11.8 (7.8-17.1)

<0.01

Peak FCI

9.1 (6.6-11)

18.2 (13.9-25.1)

<0.01

7

20

ns

13

22

(%)

Liver Transplent

+

(%)

ns

TE D

Deaths

+

RI PT

ACCEPTED MANUSCRIPT

SBP: spontaneous bacterial peritonitis; BP: blood pressure; TC: serum total cortisol; FC: plasma free cortisol; cFC: calculated free cortisol by Coolens’ equation; FCI: free cortisol index. #

Mann-Whitney test

One patients was lost to follow-up

AC C

+

EP

*number of patients with alcohol related cirrhosis active drinking at LDSST date

Table S3. Adrenal function in patients with and without hypoalbuminemia (albumina < 25 g/L) Alb < 25 g/L

Alb > 25 g/L

P

(7 patients)

(72 patients)

value

CBG (ug/ml)

34 (22-39)

35 (25-46)

ns

ACTH (pmol/L)

25 (20-30)

11 (5-29)

ns

Basal TC (nmol/L)

575 (178-650)

346 (211-479)

ns

Peak TC (nmol/L)

700 (276-732)

579 (434-720)

ns

Delta TC (nmol/L)

117 (276-732)

202 (135-289)

< 0.01

#

ACCEPTED MANUSCRIPT

Basal FC (nmol/L)

39 (10-81)

17 (11-32)

ns

Peak FC (nmol/L)

58 (25-98)

46 (31-65)

ns

Delta FC (nmol/L)

18 (16-39)

27 (16-42)

ns

43

32

ns

29

28

ns

14

19

ns

N. of patients with peak TC < 494 nmol/L (%)

< 33 nmol/L (%) N. of patients with peak FC < 25 nmol/L (%)

Mann-Whitney test

M AN U

#

SC

CBG: cortisol binding globulin; TC: total serum cortisol; FC: serum free cortisol

Table S4. Clinical characteristics in patients with and without ascites Pat. with

Pat. without

ascites (36/79)

ascites

p

(43/79)

value

55 (50-62)

55 (50-64)

ns

Gender (male/female)

24/12

29/14

ns

Aetiology Viral (%)

6

Alcohol/active drinkers* (%) Other (%)

ns

69/31

44/23

ns

25

28

ns

2/22/12

17/22/4

<0.001

22

14

ns

73 (59-107)

71 (55-87)

ns

EP

AC C

Creatinine (µmol/L)

#

28

Child-Pugh class

Encephalopaty (%)

TE D

Age (years)

A/B/C

RI PT

N. of patients with peak FC

Sodium (mmol/L)

136 (128-140) 139 (137-142)

0.001

Potassium (mmol/L)

4 (3.7-4.7)

4.1 (3.9-4.4)

ns

Urea (mmol/L)

5 (3.4-9.3)

4.7 (3.5-6.3)

ns

Albumin (g/L)

32 (28-35)

35 (31-39)

0.035

INR

1.5 (1.4-1.7)

1.5 (1.3-1.6)

ns

Child-Pugh Score

9 (8-10)

7 (6-8)

<0.001

MELD Score

14 (11-19)

17 (14-24)

0.037

CBG (ug/ml)

32 (21-40)

37 (27-48)

0.046

Basal TC (nmol/L)

366 (288-484) 304 (171-488)

ns

Peak TC (nmol/L)

611 (474-714) 531 (390-727)

ns

Delta TC (nmol/L)

187 (130-267) 197 (113-304)

ns

ACCEPTED MANUSCRIPT

Basal FC (nmol/L)

25 (17-47)

14 (8-25)

0.003

Peak FC (nmol/L)

62 (46-81)

41 (23-53)

0.001

Delta FC (nmol/L)

35 (18-45)

21 (15-36)

0.043

Peak FCI

18 (14-28)

14 (10-20)

0.002

#

RI PT

TC: serum total cortisol; FC: plasma free cortisol; FCI: free cortisol index. Mann-Whitney test

SC

*number of patients with alcohol related cirrhosis active drinking at LDSST date

Table S5. Comparison between patients with and without adrenal dysfunction assessed by total cortisol and free cortisol response after low dose Synacthen test.

And Normal TC (n = 2)

Low TC

Low TC

Normal FC

And

and

and

p

Normal FC

Low FC

Normal TC

value#

(n = 14)

(n = 13)

(n = 50)

M AN U

Low FC

Age (years)

52 (50-54)

55 (52-63)

48 (40-56)

57 (51-65)

ns

Gender (male/female)

1/1

9/5

9/4

34/16

ns

Viral (%)

0

7

23

20

ns

Alcohol/active drinkers* (%)

50/50

43/21

69/30

56/26

ns/ns

Other (%)

50

50

8

24

ns

Ascites (%)

50

43

23

52

ns

0/0/2

2/8/4

3/9/1

14/26/10

ns

50

21

15

16

ns

50 (30-70)

54 (35-87)

37 (27-70)

35 (22-66)

ns

36 (32-40)

32 (26-39)

32 (29-37)

33 (30-37)

ns

Total Cholesterol (mmol/L)

3.8 (3.7-3.9)

2.10 (0.75-3.4)

3.6 (2.7-4.5)

3.2 (2.6-4.2)

ns

HDL (mmol/L)

1.2 (1.1-1.3)

0.4 (0.2-1.2)

1.4 (0.8-2.1)

1 (0.4-1.2)

ns

LDL (mmol/L)

2.3 (2.2-2.4)

1.3 (0.3-1.8)

1.5 (1.4-2.3)

1.9 (1.1-2.2)

ns

INR

1.6 (1.4-1.7)

1.7 (1.4-2)

1.6 (1.5-1.9)

1.4 (1.3-1.6)

ns

Child-Pugh Score

10 (10-10)

9 (8-10)

8 (7-9)

7 (6-9)

ns

MELD Score

23 (15-30)

14 (12-19)

15 (11-20)

16 (13-21)

ns

CBG (ug/ml)

51 (46-57)

24 (17-32)

32 (22-43)

37 (27-48)

<0.01

Basal TC (nmol/L)

214 (110-318)

233 (164-288)

130 (88-203)

460 (323-586) ns

Peak TC (nmol/L)

510 (508-512)

423 (330-440)

263 (230-350) 678 (586-801) <0.01

A/B/C Encephalopaty (%) Total Bilirubin (mmol/L)

AC C

Albumin (g/L)

EP

Child-Pugh class

TE D

Aetiology

ACCEPTED MANUSCRIPT

Delta TC (nmol/L)

296 (190-402)

148 (116-240)

98 (64-193)

222 (158-306) <0.05

Basal FC (nmol/L)

3.6 (2.7-4.4)

17 (13-23)

7.4 (2.8-14.2)

25.4 (14-46)

<0.05

Peak FC (nmol/L)

18.5 (14-23)

45 (31-53)

19.9 (11-22)

58.7 (44-81)

<0.01

Delta FC (nmol/L)

15 (9.8-20.1)

22 (16-40)

8.9 (2.6-12)

35.4 (23-45)

<0.01

RI PT

Low FC: peak free cortisol < 25 nm/L, Normal FC: peak free cortisol > 25 nm/L, Low TC: peak total cortisol < 494 nm/L, Normal TC: peak total cortisol > 494 nm/L. TC: serum total cortisol; FC: plasma free cortisol; FCI: free cortisol index. #

Kruskal-Wallis test

SC

*number of patients with alcohol related cirrhosis active drinking at LDSST date

M AN U

Table S6. Clinical characteristics in patients dead or transplanted and in patients alive without liver transplantation

Age (years) Gender (male/female) Aetiology Viral (%)

TE D

patients dead or transplanted (n: 30) 55 (50-62) 67

13

Other (%) Ascites (%)

A/B/C

AC C

Child-Pugh class

EP

Alcohol/active drinkers* (%) 50/20

p value#

patients alive without liver transplantation (n: 48) 54 (50-65)

0.56

69

1.0 0.23

20 58/29

37

20

50

42

0.49 0.057

6/14/10

13/30/5

Encephalopaty (%)

23

15

0.38

Systolic BP (mmHg)

120 (101-135)

120 (101-135)

0.84

Diastolic BP (mmHg)

65 (54-70)

68 (60-73)

0.32

MAP (mmHg)

80 (76-92)

83 (75-95)

0.36

Creatinine (µmol/L)

78 (64-95)

64 (53-87)

0.10

Sodium (mmol/L)

136 (129-140)

139 (136-140)

0.04

Urea (mmol/L)

4.9 (4.1-7.6)

4.7 (3.2-7.3)

0.41

ACCEPTED MANUSCRIPT

51 (29-113)

36 (24-55)

0.13

Albumin (g/L)

31 (28-37)

34 (31-37)

0.086

Total Cholesterol (mmol/L) 3.1 (2.2-4.6)

3.2 (2.6-3.6)

0.87

HDL (mmol/L)

0.9 (0.3-1.2)

1.0 (0.6-1.6)

0.082

LDL (mmol/L)

1.9 (1.1-3.2)

1.5 (1.2-2.0)

0.30

INR

1.5 (1.4-1.7)

1.5 (1.4-1.6)

Child-Pugh Score

9 (7-10)

8 (6-9)

MELD Score

16 (14-24)

15 (11-19)

CBG (ug/ml)

34 (19-48)

36 (26-42)

ACTH (pmol/L)

18 (5-30)

14 (5-27)

0.88

Basal TC < 138 nmol/L (%)

10

15

0.73

Peak TC < 494 nmol/L (%)

37

31

Delta TC < 250 nmol/L (%)

67

Peak FC < 25 nmol/L (%)

10

Peak FC < 33 nmol/L (%)

17

FCI < 12 (%)

20

Basal TC (nmol/L) Peak TC (nmol/L)

Basal FC (nmol/L) Peak FC (nmol/L)

AC C

Peak FCI

SC

M AN U

0.187 0.59

0.63

25

0.14

35

0.12

33

0.30

301 (191-460)

366 (220-499)

0.40

562 (428-684)

589 (433-743)

0.59

210 (119-289)

189 (127-277)

0.70

17 (14-39)

19 (8-37)

0.38

51 (37-61)

46 (24-74)

0.43

27 (18-44)

22 (13-42)

0.40

16 (11-22)

0.54

16 (13-24)

TC: serum total cortisol; FC: plasma free cortisol; FCI: free cortisol index. #

0.055

0.62

EP

Delta FC (nmol/L)

0.69

73

TE D

Delta TC (nmol/L)

RI PT

Total Bilirubin (mmol/L)

Mann-Whitney test

*number of patients with alcohol related cirrhosis active drinking at LDSST date

ACCEPTED MANUSCRIPT

Table S7. Cox regression analysis of variables associated with death or liver transplant. SIMPLE COX REGRESSION Hazard Ratio (95%CI) 1.10 (0.45-2.32) 0.82

MELD Score

1.02 (0.98-1.05) 0.36

Basal TC (nmol/L)

1.00 (0.99-1.01) 0.70

Peak TC (nmol/L)

1.00 (0.99-1.12) 0.95

Delta TC (nmol/L)

0.99 (0.90-1.00) 0.64

SC

Cause of admission *

RI PT

Continuous Variables

p value

M AN U

TC: serum total cortisol * transplant work-up vs. decompensation of cirrhosis

Table S8. Tertile distribution of total cortisol (TC) and free cortisol (FC) in baseline and after ACTH stimulation. Basal TC (mmol/L)

Stimulated TC (mmol/L)

Del ta TC (mmol/L)

Basal FC (mmol/L)

Stimulated FC (mmol/L)

Delta FC (mmol/L)

33.3

277.7

463

147.3

13.5

36.9

18.2

66.6

455.7

673

244.3

26.1

58.5

36.1

AC C

EP

TE D

Percentile

COOLENS’ EQUATION

U2K (1+N) + U[1 + N + K(T-C)]-C = 0 U represents the molar concentration of unbound cortisol, C the molar concentration of total cortisol, T the concentration of CBG, and K is the affinity of CBG for cortisol at 37 °C. N is the ratio of albumin bound to free cortisol, and 1.74 is the value conventionally used [19].