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Assessment of adrenal function in cirrhotic patients: Salivary cortisol should be preferred
Research Article
Assessment of adrenal function in cirrhotic patients: Salivary cortisol should be preferred Arnaud Galbois1, Marika Rudler1, Julien Massard1, Yvonne Fulla2, Abdelhai Bennani3, Dominique Bonnefont-Rousselot4, Vincent Thibault5, Stéphanie Reignier1, Anne Bourrier1, Thierry Poynard1, Dominique Thabut1,* 1
Université Pierre et Marie Curie, Service d’hépato-gastroentérologie, Hôpital de la Pitié-Salpêtrière (AP-HP), Paris, France; 2Université Paris V, Laboratoire Hormones et Marqueurs, Médecine Nucléaire, Hôpital Cochin (AP-HP), Paris, France; 3Université Pierre et Marie Curie, Laboratoire de Biochimie Endocrinienne et Oncologique, Hôpital de la Pitié-Salpêtrière (AP-HP), Paris, France; 4EA 3617, Faculté de Pharmacie, Université Paris Descartes, Paris; 5Université Pierre et Marie Curie, ER-1 Virology CERVI, Hôpital de la Pitié-Salpêtrière (AP-HP), Paris, France
Background & Aims: Adrenal insufficiency is a common disorder among cirrhotic patients. Adrenal function is usually assessed with serum total cortisol assays. Free cortisol (active fraction) represents only 10% of serum total cortisol, the remaining 90% being linked to cortisol-binding globulin (CBG) and albumin. In cirrhotic patients, the synthesis of these proteins is reduced, which could lead to an overestimation of the prevalence of adrenal insufficiency. Salivary cortisol assessment adequately reflects free cortisol plasma concentration. However, this method has never been validated in cirrhotic patients. The objectives of this report were to assess the following parameters by a prospective observational study: (1) correlation between salivary, serum total and free cortisol, (2) adrenal insufficiency prevalence using salivary and serum assays, (3) parameters associated with a discrepancy between both tests, and (4) adrenal insufficiency risk factors among cirrhotic patients. Methods: Salivary and serum total cortisol were assessed before and 1 h following an injection of corticotropin (250 lg) in patients hospitalized for cirrhosis complications without shock. CBG was measured and free cortisol was assessed by the Coolens formula. Results: Eighty-eight patients were included in the study (Child– Pugh C: 68.2%). Free cortisol was more strongly correlated with salivary than with serum total cortisol (Spearman coefficient = 0.91 vs. 0.76, respectively, p <0.001). Among included patients, 9.1% had adrenal insufficiency according to salivary cortisol and 33.0% had adrenal insufficiency according to serum total
Keywords: Adrenal insufficiency; Liver cirrhosis; Hepatoadrenal syndrome; Cortisol; Serum total cortisol; Salivary cortisol; Free cortisol. Received 24 September 2009; received in revised form 5 January 2010; accepted 17 January 2010; available online 15 March 2010 * Corresponding author. Address: Université Pierre et Marie Curie, Service d’hépato-gastroentérologie, Hôpital de la Pitié-Salpêtrière (AP-HP), 47-83 bd de l’hôpital, 75013 Paris, France. Tel.: +33 1 42 16 14 54; fax: +33 1 42 16 14 25. E-mail addresses: [email protected], [email protected] (D. Thabut). Abbreviations: AI, adrenal insufficiency; ICU, intensive care unit; FC, free cortisol; STC, serum total cortisol; CBG, cortisol-binding globulin; SC, salivary cortisol; CFC, calculated free cortisol; HDL-c, high-density lipoprotein-cholesterol.
cortisol (p = 0.001). Hypoalbuminemia was the only factor associated with a discrepancy between the results of both tests. Adrenal insufficiency risk factors were ascites and low HDLcholesterol plasma concentration. Conclusion: Using serum total cortisol assays overstate adrenal insufficiency prevalence among cirrhotic patients, mainly because of inaccurate concentrations related to hypoalbuminemia. Salivary cortisol assays should be preferably used in these patients. Ó 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Introduction Association of liver disease and adrenal insufficiency (AI), socalled hepatoadrenal syndrome, seems to be common in patients with severe cirrhosis admitted to intensive care units (ICU) [1–6] or not [7,8]. However, methods used to assess AI could be invalid in patients with cirrhosis. Ninety percent of measured serum total cortisol (STC) is linked to albumin or cortisol-binding globulin (CBG) [9–13]. In cases of hypoalbuminemia or reduced CBG, the STC bound fraction is reduced while free cortisol (FC), responsible for glucocorticoid activity on peripheral organs, remains unchanged. In these conditions, STC assays are no longer consistent with FC concentrations and, thus, with adrenal function [14– 16]. It has been shown that STC assays overestimate AI prevalence in ICU patients with hypoalbuminemia [17–20] and it is now recommended to no longer use STC assays in these patients [21,22]. In patients with cirrhosis, because CBG and albumin plasma concentrations are also reduced [7,23,24], AI prevalence could be overestimated as well. However, FC plasma concentrations are not routinely assessed in cirrhotic patients. Salivary cortisol (SC) concentrations accurately reflect FC plasma concentrations in non-cirrhotic patients [25–32], even in cases of hypoalbuminemia or CBG abnormality [25,29,33–36]. However, no study assessed SC evaluation in cirrhotic patients. The aims of this study were to assess, in cirrhotic patients, the following parameters: (1) the correlation between SC, STC, and FC, (2) AI prevalence using SC and STC assays, (3)
Journal of Hepatology 2010 vol. 52 j 839–845
Research Article parameters associated with a discrepancy between both tests, and (4) AI risk factors.
lipoprotein B. The remaining cholesterol (corresponding to HDL-c) was measured in the supernatant as described above. Plasma concentrations of low-density lipoprotein-cholesterol were calculated by the Friedewald formula [41] when triglyceride concentrations were less than 3.40 g/L; otherwise, they were directly measured by an enzyme kit (KONELAB, Thermoclinical Labsystmes, Cergy-Pontoise, France).
Material and methods Definitions This is a prospective observational study. Patients were included during their hospitalisation in the Hepato-Gastroenterology unit of the Pitié-Salpêtrière hospital (Paris, France) between May 2006 and July 2009. Patients were considered for study during their hospitalisation as they fulfilled the following inclusion criteria: (1) histological proof of cirrhosis or a diagnosis on the basis of clinical and biological criteria, (2) hospitalisation for complications related to cirrhosis, (3) written informed consent. Exclusion criteria were the following: (1) age under 18, (2) pregnancy, (3) history of pituitary or adrenal disease, (4) treatment by steroids or other drugs known to influence cortisol production (e.g., etomidate, ketoconazole, etc.) or CBG production in the preceding 6 months, (5) haemodynamic instability as defined by severe sepsis or septic shock, according to ACCP criteria, [37] or haemorrhagic shock [38], (6) administration of albumin, fresh frozen plasma, terlipressin or glypressin infusion during the hospitalisation prior to inclusion, (7) presence of blood in the mouth. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institution’s human research committee. Study protocol: On the day of inclusion, a clinical examination of each patient was performed. The investigator paid particular attention to the absence of blood in the mouth. Patients were instructed to refrain from brushing their teeth, smoking, eating or drinking anything but water at least 60 min prior to sampling. Samples were obtained at least 15 min after the insertion of the indwelling intravenous catheter to avoid stress-induced raises in cortisol. A biological assessment including standard biology, lipid fractions, CBG, STC, and SC was performed at 8 AM (T0) immediately prior to and 60 min following (T60) an intravenous injection of 250 lg of corticotropin (tétracosactide: SynacthèneÒ, Novartis Pharma SAS, Rueil-Malmaison, France). Saliva was collected through dedicated kits (Plain Salivette, Starstedt, Newton, NC). The provided cotton was chewed for 2–3 min by each patient and then collected in a plastic tube according to supplier’s instructions. Infection was determined by blood, ascites and urine cultures, chest X-ray and, if necessary, other assays.
Adrenal insufficiency according to STC assays and our laboratory standards: basal value (T0) <9 lg/dl or a concentration 1 h after corticotropin injection (T60) <18 lg/dl or an increase between T0 and T60 (D) <9 lg/dl. Adrenal insufficiency according to SC assays and our laboratory standards as well as published works: basal value (T0) <1.8 ng/ml or a concentration 1 h after corticotropin injection (T60) <12.7 ng/ml or an increase between T0 and T60 (D) <3 ng/ml [36,42–44]. Acute alcoholic hepatitis was defined by a Maddrey score P 32 and histological proof. SIRS and sepsis were defined according to ACCP criteria [37,45]. Statistical methods Considering that there is a reported overestimation of AI by STC assays in about 40% of ICU patients without cirrhosis due to hypoproteinemia [17], and the high prevalence of hypoproteinemia in patients with cirrhosis as well as the AI prevalence using STC assays in previously published studies [7,8], we hypothesised that STC assays overstate AI prevalence by 50% when compared to SC assays in patients with cirrhosis. Eighty-five patients had to be studied to confirm this hypothesis. Continuous variables were expressed as mean and standard deviation when the distribution was normal and median and confidence interval when the distribution was not. Continuous variables were compared by Student’s t test or the Mann–Whitney test. Categorical variables were expressed in numerical values (n) and percentages, and compared with the Chi-squared test or Fisher’s exact test. Risk factors were assessed in multivariate analysis by logistic regression. Continuous variable correlation was assessed by the Spearman coefficient; CI and p values were assessed by bootstrap resampling. Concordance between both tests was assessed by the kappa coefficient. Statistical analyses were performed with NCSS software for Windows (NCSS 2007).
Biological assays STC assays: STC determinations were performed using the chemoluminescent immunoenzymology method with Immulite 2000 Cortisol kit (Siemens Medical Diagnostics Solutions, Erlangen, Germany). Analytical sensitivity was 0.20 lg/dl. Inter-assay variations (CV) were 5.5%, 4.4%, 4.3%, and intra-assay variations (CV) were 4.5%, 3.8%, and 4.2% at 2.2, 8.5, and 26.9 lg/dl, respectively. SC assays: Once transported to the laboratory, saliva was collected by centrifugation. SC determinations were performed by the radioimmunoassay method with Coat-a-Count kit (Diagnostic Products, Los Angeles, CA) according to the protocol supplied by the manufacturer. Calibrators were prepared in different dilutions in buffer (0, 0.36, 1.45, 7.25, and 36.23 ng/ml). Patients’ samples and calibrators (150 ll) were analysed in duplicate. A cortisol tracer (500 ll) was added to each reaction. All tubes were mixed, covered with paraffin film and incubated for 30 min at 37 °C. Tubes were decanted and washed once with 1 ml of distilled water and then decanted again. The radioactivity of each tube was counted using a gamma counter. Measurement range was 0.36–36.23 ng/ ml. Automatic results were obtained using a spline function curve. Analytical sensitivity was 0.29 ng/ml. Inter and intra-assay variations (CV) were 7.0% and 6.0% respectively. CBG assays: CBG assays were performed using radioimmunoassay protocols (Biosource; Lifescreen, Watford, Herts, UK). Calculated basal free cortisol (CFC) assessment: Basal CFC was assessed by the Coolens formula [23] which allows the calculation of FC using STC and CBG assays, taking into account the affinity of cortisol for CBG and albumin: p CFC ¼ ðZ 2 þ 0:0122 STCÞ Z Z ¼ 0:0167 þ 0:182ðCBG STCÞ Lipid fractions: Total cholesterol was determined by enzymatic testing in the KONELAB 30i analyser (KONELAB, Thermo Clinical Labsystems, Cergy-Pontoise, France). Triglycerides were measured by an automated enzymatic technique (Biomérieux, Marcy l’Etoile, France). Lipoprotein modifications in patients with cirrhosis make routine determination of HDL-c concentrations difficult [39,40]. HDL-c plasma concentrations were determined through a reactive phosphotungstate of sodium/MgCl2 (Boehringer Mannheim) to precipitate lipoproteins containing apo-
840
Results Patients’ characteristics Ninety-eight patients were included in this study. The data acquired from ten patients were not analysed, as the collected amount of saliva was insufficient. Thus, final analysis was completed for 88 patients. Their clinical and biological characteristics at inclusion are shown in Table 1. Reasons for hospitalisation were: digestive haemorrhage in 57%, hepatic encephalopathy in 13%, ascites in 11%, renal failure in 7%, and other reasons in 12% of cases. CBG plasma concentrations CBG plasma concentrations were 22.36 ± 8.84 lg/ml and decreased with the severity of cirrhosis according to the Child– Pugh score (28.60 ± 6.76 lg/ml in Child–Pugh A cirrhosis, 21.84 ± 8.84 lg/ml in Child–Pugh B cirrhosis and 20.80 ± 8.32 lg/ ml in Child–Pugh C cirrhosis, p <0.05). The CBG plasma concentrations were not correlated to the albumin plasma concentrations [Spearman coefficient 0.23 (0.01 to 0.45)]. Correlation between basal SC, STC and CFC The observed correlation between the basal SC at T0 and CFC was excellent (Spearman coefficient = 0.91 (0.87–0.95), p <0.001) Correlation between STC and CFC was lower (Spearman
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JOURNAL OF HEPATOLOGY Table 1. Patients characteristics at inclusion.
54 ± 10 64 (72.7) 4.1 ± 2.5 5.1 ± 2.4 4.9 ± 2.6 2.6 ± 1.6
$
63 (71.6) 14/63 (22.2) 13 (14.8) 8 (9.1) 4 (4.5)
30.0
20.0
10.0
0.0
29 (33.0) 25 (28.4) 34 (38.6) 32 (36.4) 10 ± 2 4 (4.6)/24 (27.3)/60 (68.2) 16 ± 8 8 (9.1) 31 (35.2) 38 (43.2) 24 (27.3) 13 6 3 2 14 (15.9) 46 ± 15 28 ± 5 74 (54–104) 8.2 ± 4.2 9.6 ± 1.8 87.5 (69.0–95.0) 76 (64–90) 41 (35–46) 64 (59–70) 0.89 (0.70–1.03) 0.48 (0.42–0.56) 0.25 (0.16–0.35) 0.36 (0.22–0.51)
Salivary cortisol (ng/ml)
Age, years* Male sexe, n (%) Time between admission and inclusion, days* - Admission for digestive haemorrhage - Admission for encephalopathy - Other reasons of admission Causes of cirrhosis, n (%) - Alcohol: - Weaned from alcohol for >6+ months: - Viral hepatitis: - Mixed: - Other: Ascites, n (%) - Absence - Medically controlled - Poorly controlled ascites, n (%) Hepatic encephalopathy, n (%) Child–Pugh score* - Class A/B/C, n (%) MELD score* Hepatocellular carcinoma, n (%) Acute alcoholic hepatitis, n (%) SIRS, n (%) Infection, n (%) Localisation, n: - Ascites: - Bacteriemia: - Urine tract: - Other: Sepsis, n (%) Prothrombin time ratio, %* Albumin, g/L* Total serum bilirubin, lmol/L$ Leukocyte count, G/L* Hemoglobin, g/dl* Platelet, G/L$ ASAT, UI/ml ALAT, UI/ml$ Serum creatinine, lmol/L$ Cholesterol, g/L$ Triglyceride, g/L$ HDL-cholesterol, g/L$ Apolipoprotein A1, g/L$
Serum total cortisol (µg/dl)
Data
*
50.0
40.0
Variable
37.5
25.0
12.5
0.0
0.0
1.0
2.0
3.0
4.0
0.0
Free cortisol (µg/dl) Spearman coefficient = 0.76, p <0.001
1.0
2.0
3.0
4.0
Free cortisol (µg/dl) Spearman coefficient = 0.91, p <0.001
Fig. 1. Correlation between calculated free cortisol and salivary cortisol concentrations and between calculated free cortisol and serum total cortisol concentrations at T0. Whereas this correlation was excellent between calculated free cortisol and salivary cortisol, it was significantly lower between calculated free cortisol and serum total cortisol (p = 0.001).
than according to SC (8/88: 9.1%) (p = 0.001). Five patients fit at least 2 biological criteria used to define AI according to SC assays. Ten patients satisfied at least 2 biological criteria used to define AI according to STC assays (Table 3). Correlation between STC and SC assays (Fig. 2) Correlations between STC and SC assays for T0, T60 and D were strong in patients with albumin concentrations > 25 g/L (Spearman coefficient = 0.69, 0.71 and 0.62, respectively, all p = 0.0001) whereas, in patients with albumin 625 g/L, the correlation was strong at T0 (Spearman coefficient = 0.62, p = 0.0007), poor at T60 (Spearman coefficient = 0.31, p = 0.12) and absent for D (Spearman coefficient = 0.0004, p = 0.93) (Fig 2). To avoid data-driven results, in the protocol of this study, the serum albumin cut-off (25 g/L) was defined a priori, according to Hamrahian data [17]. Discrepancy between salivary and serum tests for AI diagnosis
Mean ± standard deviation. Median (confidence interval).
coefficient = 0.76 (0.6–0.85), p <0.001). The difference between these two correlations was significant (p <0.001) (Fig. 1). Adrenal insufficiency prevalence The results of STC and SC measurements are reported in Table 2. The AI prevalence was higher according to STC (29/88: 33.0%)
Agreement between the salivary and serum tests was low (kappa = 0.28 [0.08–0.49]). Twenty-two patients had AI according to STC assays, but normal adrenal function according to salivary assays. One patient had AI according to salivary assays, but normal adrenal function according to serum assays. Patient characteristics were compared between the group with concordant tests (n = 65) and the group with discordant tests (n = 23). Univariate analysis identified candidate factors for discordant tests: male gender
Table 2. Plasma and salivary cortisol concentrations. All the patients (n = 88)
Adrenal insufficiency according to salivary cortisol (n = 8)
No adrenal insufficiency according to salivary cortisol (n = 80)
p Value
Serum total cortisol T0 (lg/dl) T60 (lg/dl) D (lg/dl)
15.4 (14.1–18.1) 29.6 (27.5–32.5) 12.5 (11.4–13.9)
3.5 (1.3–10.8) 13.7 (4.4–21.7) 7.9 (0.6–11.5)
15.7 (14.3–18.6) 30.2 (27.6–32.6) 12.9 (11.5–14.4)
0.001 0.0002 0.02
Salivary cortisol T0 (ng/ml) T60 (ng/ml) D (ng/ml)
8.9 (6.5–10.0) 34.3 (30.5–37.5) 22.5 (19.4–24.8)
1.4 (0.3–2.7) 13.2 (2.1–27.8) 11.8 (1.1–27.5)
9.4 (7.1–11.7) 35.1 (31.3–38.8) 23.0 (20.3–25.8)
0.00004 0.00009 0.003
Values are given in median (confidence interval).
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Research Article Table 3. Adrenal insufficiency prevalence according to serum total cortisol and salivary cortisol assays during corticotropin test. Adrenal insufficiency according to salivary cortisol
Adrenal insufficiency according to serum total cortisol 12 10 19 29/88 (33.0%)
Patients with albumin >25 g/L (n = 61)
Patients’ characteristics according to their adrenal function assessed by SC assays and univariate analysis are reported in Table 4. Several models have been considered in the multivariate analysis; we report here the model with total bilirubin, prothrombin time, total cholesterol, HDL-c, and ascites. In multivariate analysis, only ascites and lower HDL-c plasma concentrations remained independently associated with AI (p = 0.01 for both).
842
35.0
Salivary cortisol (ng/ml)
Risk factors for AI
30.0
20.0
10.0
26.3
17.5
8.8
0.0
0.0 0.0
15.0
30.0
45.0
60.0
0.0
Serum total cortisol (µg/dl) Spearmann coefficient = 0.69, p = 0.0001
17.5
26.3
35.0
Salivary cortisol (ng/ml)
50.0
60.0
40.0
20.0
0.0 0.0
20.0
40.0
60.0
40.0
30.0
20.0
10.0 15.0
80.0
Serum total cortisol (µg/dl) Spearmann coefficient = 0.71, p = 0.0001
22.5
30.0
37.5
45.0
Serum total cortisol (µg/dl) Spearmann coefficient = 0.31, p = 0.12
50.0
45.0
Salivary cortisol (ng/ml)
Salivary cortisol (ng/ml)
8.8
Serum total cortisol (µg/dl) Spearmann coefficient = 0.62, p = 0.0007
80.0
T60
This study shows that: (1) SC assays are more correlated with CFC (active fraction) than STC assays and, thus, are a better reflection of adrenal function in patients with cirrhosis; (2) STC assays overstate AI prevalence in patients with cirrhosis; (3) the only risk factor for AI misdiagnosis by STC assays is hypoalbuminemia; (4) risk factors for AI in cirrhotic patients were ascites and low HDL-c plasma concentration. In previously published studies using STC assays in cirrhotic patients without shock, AI prevalence was high and correlated with the Child–Pugh score [7,8]. The influence of STC assay results on cirrhotic patients’ outcomes in the ICU is controversial because AI determined by STC assays has been associated not only with mortality [5] but also with survival [1]. The same controversy exists in ICU patients without cirrhosis [46,47], resulting in a recommendation to no longer use STC assays in these patients [21,22]. Indeed, it has been shown that STC assays overestimate AI prevalence in ICU patients without cirrhosis but who have albuminemia 6 25 g/L [17]. Patients admitted to the ICU and patients with cirrhosis both have a frequent decrease in CBG and albumin plasma concentrations because of hepatocellular insufficiency and inflammation [7,24,48–51]. We confirmed that CBG plasma concentrations decreased with the severity of
Patients with albumin <25 g/L (n = 27)
40.0
T0
Discussion
0.001
cirrhosis. However, there was no correlation between albumin plasma concentrations and those of CBG. Moreover, lower albuminemia was associated with a discrepancy between salivary and serum tests for AI, whereas a lower CBG plasma concentration was not. These results could be explained by an earlier decrease in CBG concentrations than in albumin levels in patients
Salivary cortisol (ng/ml)
(91% vs. 68%, p = 0.03), cirrhosis not due to alcohol (35% vs. 14%, p = 0.03), absence of acute alcoholic hepatitis (87% vs. 61%, p = 0.02), hepatocellular carcinoma (17% vs. 6%, p = 0.11), presence of ascites (78% vs. 62%, p = 0.17), lower albuminemia (26 ± 4 g/L vs. 29 ± 5 g/L, p = 0.01), lower leukocyte count (6.9 ± 4.0 g/L vs. 8.5 ± 4.3 g/L, p = 0.15), lower ASAT plasma concentration (67 [46– 84] IU/ml vs. 87 [67–116] IU/ml, p = 0.07) and lower triglyceride plasma concentration (0.46 [0.27–0.56] g/L vs. 0.56 [0.40–0.64] g/L, p = 0.19). There was no correlation between plasma concentrations of CBG and discordant tests (22.36 ± 8.32 lg/ml vs. 21.84 ± 10.40 lg/ml, p = 0.85). In multivariate analysis in 2 different models, the only factor associated with discordant tests was lower serum albumin levels (p = 0.01 and 0.009). Profound hypoalbuminemia (serum albumin level 6 25 g/L) was more frequent in patients of which STC assays, but not SC assays, indicated AI (54%) than in others (23%, p = 0.006).
6 3 3 8/88 (9.1%)
Salivary cortisol (ng/ml)
Adrenal insufficiency according to T0, n Adrenal insufficiency according to T60, n Adrenal insufficiency according to D, n Total number of patients with adrenal insufficiency, n (%)
p Value
37.5
25.0
12.5
0.0 0.0
10.0
20.0
30.0
40.0
Serum total cortisol (µg/dl) Spearmann coefficient = 0.62 p = 0.0001
36.3
27.5
18.8
10.0 5.0
10.0
15.0
20.0
25.0
Serum total cortisol (µg/dl) Spearmann coefficient = 0.0004 p = 0.93
Fig. 2. Correlation between total serum cortisol and salivary cortisol concentrations at T0, T60 and for D values (T60–T0) in patients with albumin >25 g/L and in patients with albumin 625 g/L. Whereas this correlation was very good in patients with albumin >25 g/L, it was good at T0, bad at T60 and null for D values in patients with albumin 625g/L.
Journal of Hepatology 2010 vol. 52 j 839–845
JOURNAL OF HEPATOLOGY with cirrhosis. Indeed, even if CBG and albumin plasma concentrations decrease with cirrhosis severity, CBG is already much decreased in patients with Child–Pugh A cirrhosis, whereas albumin remains closed to normal range. In patients with cirrhosis, serum total cortisol would overstate AI only when albumin plasma concentrations decrease, which is later in the evolution of liver disease. Thus, plasma albumin concentration, a routinely performed assay, should be used to identify cirrhotic patients who should be assessed via a salivary cortisol assay rather than a serum total cortisol assay. We have identified risk factors for AI in cirrhotic patients. Ascites was associated with higher AI prevalence. Other studies have already found a correlation between the liver disease severity and AI [7,8], but as these studies used STC assays, it could be argued that low CBG and albumin plasma concentrations could explain these results. Using SC assays allowed this bias to be avoided for the first time, and we confirmed the relationship between severe liver disease and AI. A low HDL-c plasma concentration was also associated with AI. These results confirm those of several studies in patients with and without cirrhosis [1,2,52]. HDL-c brings cholesterol into adrenal glands where it is necessary for cortisol synthesis [53]. This result adds the potential beneficial effect of increasing HDL-c plasma concentrations in patients with cirrhosis in addition to limiting the lipopolysaccharide
induced pro-inflammatory response as previously reported [54,55]. This study has several limitations. First, most of the cirrhosis cases were due to alcohol (71.6%) which is common in France (22.2% had been weaned from alcohol for more than 6 months). In previous studies, alcoholic patients were excluded [7] or weaned for more than 3 months [8]. In non-weaned patients, alcohol can stimulate cortisol synthesis through the adrenal glands [56] which could explain the relatively low AI prevalence in our study. Second, in our study, 27.3% of patients were infected. Infection can increase cortisol concentrations and reduce AI prevalence. However, infection was always controlled by antibiotics and no patient had haemodynamic instability in the days preceding inclusion. However, this cannot have influenced the main result of this study, which is a high overestimation of AI prevalence by STC assays in cirrhotic patients. Moreover, most cirrhosis complications were digestive haemorrhage (57%). This could be explained by the fact that our Hepatology unit contains a 10-bed intermediate care unit where there are a large number of hospitalisations for cirrhotic patients with digestive haemorrhage from mobile care units and emergency units of other hospitals in the Paris area. However, salivary assays were performed only 4.9 ± 2.5 days after admission following confirmation of the absence of blood in the mouth. Reasons for
Table 4. Risk factors of adrenal insufficiency, defined by salivary assays, in univariate analysis. Adrenal insufficiency according to salivary cortisol assays Variable
Yes (n = 8)
No (n = 80)
p Value
Age, years* Male sexe, n (%) Cirrhosis due to alcohol, n (%) Weaning from alcohol, n (%) Hepatocellular carcinoma, n (%) Child-Pugh score* Child-Pugh score class C, n (%) MELD score* Ascites, n (%) Encephalopathy, n (%) Reason of hospitalization, n (%) - Digestive haemorrhage - Hepatic encephalopathy - Ascites - Renal failure Acute alcoholic hepatitis, n (%) SIRS, n (%) Infection, n (%) Sepsis, n (%) Prothrombine time ratio, %* Albumin, g/L* Total serum bilirubin, lmol/L$ Leukocyte count, G/L* Hemoglobin, g/dl* Platelet, G/L$ ASAT, UI/ml$ ALAT, UI/ml$ Serum creatinine, lmol/L$ Cholesterol, g/L$ Triglyceride, g/L$ HDL cholesterol, g/L$ Apolipoprotein A1, g/L$ STC: TO, lg/dl$ STC: T60, lg/dl$ STC: D, lg/dl$
56 ± 11 6 (75) 7 (88) 3 (43) 0 (0) 12.0 ± 1.7 8 (100) 11 ± 5 8 (100) 3 (37)
54 ± 10 59 (74) 64 (80) 29 (33) 78 (10) 10.2 ± 2.3 52 (65) 8±1 50 (64) 26 (33)
0.71 0.94 0.61 0.59 0.34 0.04 0.04 0.02 0.04 0.79 0.03
2 (25.0) 1 (12.5) 4 (50.0) 1 (12.5) 4 (50.0) 2 (25.0) 2 (25.0) 2 (25.0) 40 ± 15 29 ± 4 155 (37–254) 7.7 ± 3.7 9.2 ± 1.7 71 (38–114) 91 (38–114) 48 (27–81) 174 (39–118) 0.45 (0.30–0.70) 0.54 (0.26–0.60) 0.09 (0–0.17) 0.17 (0.10–0.20) 3.5 (1.3–10.8) 13.7 (4.4–21.7) 7.9 (0.6–11.5)
52 (65.0) 11 (13.8) 9 (11.3) 4 (5.0) 24 (30.0) 36 (45.6) 22 (28) 12 (15.0) 47 ± 15 28 ± 4 70 (54–99) 8.1 ± 4.3 9.7 ± 1.3 88 (70–99) 74 (64–95) 41 (35–48) 62 (59–71) 0.97 (0.50–1.01) 0.72 (0.52–0.84) 0.31 (0.21–0.37) 0.58 (0.19–0.66) 15.7 (14.3–18.6) 30.2 (27.6–32.6) 12.9 (11.5–14.4)
0.25 0.26 0.88 0.47 0.23 0.58 0.19 0.81 0.53 0.65 0.96 0.87 0.40 0.08 0.29 0.04 0.04 0.001 0.0002 0.02
STC: Serum total cortisol. Mean ± standard deviation. $ Median (confidence interval).
*
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Research Article hospitalisation were not associated with AI in multivariate analysis. Finally, we included only haemodynamically stable patients, while AI diagnosis seems crucial in patients with sepsis or septic shock. This is a pilot study and, even if it has been shown that SC was still highly correlated with FC in patients with shock [57,58], SC threshold values remain to be assessed in these patients.
Conclusion This study shows that STC assays overstate AI prevalence in patients with cirrhosis and hypoalbuminemia. Determination of SC is an easily available and more accurate method to assess adrenal function in these patients.
Conflicts of interest The Authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Acknowledgements The work was carried out at Service d’hépato-gastroentérologie, hôpital de la Pitié-Salpêtrière (AP-HP), 47-83 bd de l’hôpital, 75013 Paris. Financial support: This study has been supported by an ARMHV grant (Association pour la Recherche sur les Maladies Hépatiques Virales). References [1] Marik PE, Gayowski T, Starzl TE. The hepatoadrenal syndrome: a common yet unrecognized clinical condition. Crit Care Med 2005;33:1254–1259. [2] Marik PE. Adrenal-exhaustion syndrome in patients with liver disease. Intensive Care Med 2006;32:275–280. [3] Harry R, Auzinger G, Wendon J. The clinical importance of adrenal insufficiency in acute hepatic dysfunction. Hepatology 2002;36: 395–402. [4] Fernandez J, Escorsell A, Zabalza M, Felipe V, Navasa M, Mas A, et al. Adrenal insufficiency in patients with cirrhosis and septic shock: effect of treatment with hydrocortisone on survival. Hepatology 2006;44:1288–1295. [5] Tsai MH, Peng YS, Chen YC, Liu NJ, Ho YP, Fang JT, et al. Adrenal insufficiency in patients with cirrhosis, severe sepsis and septic shock. Hepatology 2006;43:673–681. [6] Thierry S, Giroux Leprieur E, Lecuyer L, Brocas E, Van de Louw A. Echocardiographic features, mortality, and adrenal function in patients with cirrhosis and septic shock. Acta Anaesthesiol Scand 2008;52:45–51. [7] McDonald JA, Handelsman DJ, Dilworth P, Conway AJ, McCaughan GW. Hypothalamic–pituitary adrenal function in end-stage non-alcoholic liver disease. J Gastroenterol Hepatol 1993;8:247–253. [8] Zietz B, Lock G, Plach B, Drobnik W, Grossmann J, Scholmerich J, 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;15:495–501. [9] Dunn JF, Nisula BC, Rodbard D. Transport of steroid hormones: binding of 21 endogenous steroids to both testosterone-binding globulin and corticosteroid-binding globulin in human plasma. J Clin Endocrinol Metab 1981;53:58–68. [10] Ekins R. Measurement of free hormones in blood. Endocr Rev 1990;11:5–46. [11] Mendel CM. The free hormone hypothesis: a physiologically based mathematical model. Endocr Rev 1989;10:232–274.
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[48] Savu L, Zouaghi H, Carli A, Nunez EA. Serum depletion of corticosteroid binding activities, an early marker of human septic shock. Biochem Biophys Res Commun 1981;102:411–419. [49] Pugeat M, Bonneton A, Perrot D, Rocle-Nicolas B, Lejeune H, Grenot C, et al. Decreased immunoreactivity and binding activity of corticosteroid-binding globulin in serum in septic shock. Clin Chem 1989;35:1675–1679. [50] Bernier J, Jobin N, Emptoz-Bonneton A, Pugeat MM, Garrel DR. Decreased corticosteroid-binding globulin in burn patients: relationship with interleukin-6 and fat in nutritional support. Crit Care Med 1998;26:452–460. [51] Tsigos C, Kyrou I, Chrousos GP, Papanicolaou DA. Prolonged suppression of corticosteroid-binding globulin by recombinant human interleukin-6 in man. J Clin Endocrinol Metab 1998;83:3379. [52] van der Voort PH, Gerritsen RT, Bakker AJ, Boerma EC, Kuiper MA, de Heide L. HDL-cholesterol level and cortisol response to synacthen in critically ill patients. Intensive Care Med 2003;29:2199–2203. [53] Borkowski AJ, Levin S, Delcroix C, Mahler A, Verhas V. Blood cholesterol and hydrocortisone production in man: quantitative aspects of the utilization of circulating cholesterol by the adrenals at rest and under adrenocorticotropin stimulation. J Clin Invest 1967;46:797–811. [54] Thabut D, Tazi KA, Bonnefont-Rousselot D, Aller M, Farges O, Guimont MC, et al. High-density lipoprotein administration attenuates liver proinflammatory response, restores liver endothelial nitric oxide synthase activity, and lowers portal pressure in cirrhotic rats. Hepatology 2007;46:1893–1906. [55] Galbois A, Thabut D, Tazi KA, Rudler M, Mohammadi MS, BonnefontRousselot D, et al. Ex vivo effects of high-density lipoprotein exposure on the lipopolysaccharide-induced inflammatory response in patients with severe cirrhosis. Hepatology 2009;49:175–184. [56] Kirkman S, Nelson DH. Alcohol-induced pseudo-Cushing’s disease: a study of prevalence with review of the literature. Metabolism 1988;37:390–394. [57] Cohen J, Venkatesh B, Galligan J, Thomas P. Salivary cortisol concentration in the intensive care population: correlation with plasma cortisol values. Anaesth Intensive Care 2004;32:843–845. [58] Arafah BM, Nishiyama FJ, Tlaygeh H, Hejal R. Measurement of salivary cortisol concentration in the assessment of adrenal function in critically ill subjects: a surrogate marker of the circulating free cortisol. J Clin Endocrinol Metab 2007;92:2965–2971.
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Assessment of adrenal function in cirrhotic patients: Salivary cortisol should be preferred Arnaud Galbois1, Marika Rudler1, Julien Massard1, Yvonne Fulla2, Abdelhai Bennani3, Dominique Bonnefont-Rousselot4, Vincent Thibault5, Stéphanie Reignier1, Anne Bourrier1, Thierry Poynard1, Dominique Thabut1,* 1
Université Pierre et Marie Curie, Service d’hépato-gastroentérologie, Hôpital de la Pitié-Salpêtrière (AP-HP), Paris, France; 2Université Paris V, Laboratoire Hormones et Marqueurs, Médecine Nucléaire, Hôpital Cochin (AP-HP), Paris, France; 3Université Pierre et Marie Curie, Laboratoire de Biochimie Endocrinienne et Oncologique, Hôpital de la Pitié-Salpêtrière (AP-HP), Paris, France; 4EA 3617, Faculté de Pharmacie, Université Paris Descartes, Paris; 5Université Pierre et Marie Curie, ER-1 Virology CERVI, Hôpital de la Pitié-Salpêtrière (AP-HP), Paris, France
Background & Aims: Adrenal insufficiency is a common disorder among cirrhotic patients. Adrenal function is usually assessed with serum total cortisol assays. Free cortisol (active fraction) represents only 10% of serum total cortisol, the remaining 90% being linked to cortisol-binding globulin (CBG) and albumin. In cirrhotic patients, the synthesis of these proteins is reduced, which could lead to an overestimation of the prevalence of adrenal insufficiency. Salivary cortisol assessment adequately reflects free cortisol plasma concentration. However, this method has never been validated in cirrhotic patients. The objectives of this report were to assess the following parameters by a prospective observational study: (1) correlation between salivary, serum total and free cortisol, (2) adrenal insufficiency prevalence using salivary and serum assays, (3) parameters associated with a discrepancy between both tests, and (4) adrenal insufficiency risk factors among cirrhotic patients. Methods: Salivary and serum total cortisol were assessed before and 1 h following an injection of corticotropin (250 lg) in patients hospitalized for cirrhosis complications without shock. CBG was measured and free cortisol was assessed by the Coolens formula. Results: Eighty-eight patients were included in the study (Child– Pugh C: 68.2%). Free cortisol was more strongly correlated with salivary than with serum total cortisol (Spearman coefficient = 0.91 vs. 0.76, respectively, p <0.001). Among included patients, 9.1% had adrenal insufficiency according to salivary cortisol and 33.0% had adrenal insufficiency according to serum total
Keywords: Adrenal insufficiency; Liver cirrhosis; Hepatoadrenal syndrome; Cortisol; Serum total cortisol; Salivary cortisol; Free cortisol. Received 24 September 2009; received in revised form 5 January 2010; accepted 17 January 2010; available online 15 March 2010 * Corresponding author. Address: Université Pierre et Marie Curie, Service d’hépato-gastroentérologie, Hôpital de la Pitié-Salpêtrière (AP-HP), 47-83 bd de l’hôpital, 75013 Paris, France. Tel.: +33 1 42 16 14 54; fax: +33 1 42 16 14 25. E-mail addresses: [email protected], [email protected] (D. Thabut). Abbreviations: AI, adrenal insufficiency; ICU, intensive care unit; FC, free cortisol; STC, serum total cortisol; CBG, cortisol-binding globulin; SC, salivary cortisol; CFC, calculated free cortisol; HDL-c, high-density lipoprotein-cholesterol.
cortisol (p = 0.001). Hypoalbuminemia was the only factor associated with a discrepancy between the results of both tests. Adrenal insufficiency risk factors were ascites and low HDLcholesterol plasma concentration. Conclusion: Using serum total cortisol assays overstate adrenal insufficiency prevalence among cirrhotic patients, mainly because of inaccurate concentrations related to hypoalbuminemia. Salivary cortisol assays should be preferably used in these patients. Ó 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Introduction Association of liver disease and adrenal insufficiency (AI), socalled hepatoadrenal syndrome, seems to be common in patients with severe cirrhosis admitted to intensive care units (ICU) [1–6] or not [7,8]. However, methods used to assess AI could be invalid in patients with cirrhosis. Ninety percent of measured serum total cortisol (STC) is linked to albumin or cortisol-binding globulin (CBG) [9–13]. In cases of hypoalbuminemia or reduced CBG, the STC bound fraction is reduced while free cortisol (FC), responsible for glucocorticoid activity on peripheral organs, remains unchanged. In these conditions, STC assays are no longer consistent with FC concentrations and, thus, with adrenal function [14– 16]. It has been shown that STC assays overestimate AI prevalence in ICU patients with hypoalbuminemia [17–20] and it is now recommended to no longer use STC assays in these patients [21,22]. In patients with cirrhosis, because CBG and albumin plasma concentrations are also reduced [7,23,24], AI prevalence could be overestimated as well. However, FC plasma concentrations are not routinely assessed in cirrhotic patients. Salivary cortisol (SC) concentrations accurately reflect FC plasma concentrations in non-cirrhotic patients [25–32], even in cases of hypoalbuminemia or CBG abnormality [25,29,33–36]. However, no study assessed SC evaluation in cirrhotic patients. The aims of this study were to assess, in cirrhotic patients, the following parameters: (1) the correlation between SC, STC, and FC, (2) AI prevalence using SC and STC assays, (3)
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Research Article parameters associated with a discrepancy between both tests, and (4) AI risk factors.
lipoprotein B. The remaining cholesterol (corresponding to HDL-c) was measured in the supernatant as described above. Plasma concentrations of low-density lipoprotein-cholesterol were calculated by the Friedewald formula [41] when triglyceride concentrations were less than 3.40 g/L; otherwise, they were directly measured by an enzyme kit (KONELAB, Thermoclinical Labsystmes, Cergy-Pontoise, France).
Material and methods Definitions This is a prospective observational study. Patients were included during their hospitalisation in the Hepato-Gastroenterology unit of the Pitié-Salpêtrière hospital (Paris, France) between May 2006 and July 2009. Patients were considered for study during their hospitalisation as they fulfilled the following inclusion criteria: (1) histological proof of cirrhosis or a diagnosis on the basis of clinical and biological criteria, (2) hospitalisation for complications related to cirrhosis, (3) written informed consent. Exclusion criteria were the following: (1) age under 18, (2) pregnancy, (3) history of pituitary or adrenal disease, (4) treatment by steroids or other drugs known to influence cortisol production (e.g., etomidate, ketoconazole, etc.) or CBG production in the preceding 6 months, (5) haemodynamic instability as defined by severe sepsis or septic shock, according to ACCP criteria, [37] or haemorrhagic shock [38], (6) administration of albumin, fresh frozen plasma, terlipressin or glypressin infusion during the hospitalisation prior to inclusion, (7) presence of blood in the mouth. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institution’s human research committee. Study protocol: On the day of inclusion, a clinical examination of each patient was performed. The investigator paid particular attention to the absence of blood in the mouth. Patients were instructed to refrain from brushing their teeth, smoking, eating or drinking anything but water at least 60 min prior to sampling. Samples were obtained at least 15 min after the insertion of the indwelling intravenous catheter to avoid stress-induced raises in cortisol. A biological assessment including standard biology, lipid fractions, CBG, STC, and SC was performed at 8 AM (T0) immediately prior to and 60 min following (T60) an intravenous injection of 250 lg of corticotropin (tétracosactide: SynacthèneÒ, Novartis Pharma SAS, Rueil-Malmaison, France). Saliva was collected through dedicated kits (Plain Salivette, Starstedt, Newton, NC). The provided cotton was chewed for 2–3 min by each patient and then collected in a plastic tube according to supplier’s instructions. Infection was determined by blood, ascites and urine cultures, chest X-ray and, if necessary, other assays.
Adrenal insufficiency according to STC assays and our laboratory standards: basal value (T0) <9 lg/dl or a concentration 1 h after corticotropin injection (T60) <18 lg/dl or an increase between T0 and T60 (D) <9 lg/dl. Adrenal insufficiency according to SC assays and our laboratory standards as well as published works: basal value (T0) <1.8 ng/ml or a concentration 1 h after corticotropin injection (T60) <12.7 ng/ml or an increase between T0 and T60 (D) <3 ng/ml [36,42–44]. Acute alcoholic hepatitis was defined by a Maddrey score P 32 and histological proof. SIRS and sepsis were defined according to ACCP criteria [37,45]. Statistical methods Considering that there is a reported overestimation of AI by STC assays in about 40% of ICU patients without cirrhosis due to hypoproteinemia [17], and the high prevalence of hypoproteinemia in patients with cirrhosis as well as the AI prevalence using STC assays in previously published studies [7,8], we hypothesised that STC assays overstate AI prevalence by 50% when compared to SC assays in patients with cirrhosis. Eighty-five patients had to be studied to confirm this hypothesis. Continuous variables were expressed as mean and standard deviation when the distribution was normal and median and confidence interval when the distribution was not. Continuous variables were compared by Student’s t test or the Mann–Whitney test. Categorical variables were expressed in numerical values (n) and percentages, and compared with the Chi-squared test or Fisher’s exact test. Risk factors were assessed in multivariate analysis by logistic regression. Continuous variable correlation was assessed by the Spearman coefficient; CI and p values were assessed by bootstrap resampling. Concordance between both tests was assessed by the kappa coefficient. Statistical analyses were performed with NCSS software for Windows (NCSS 2007).
Biological assays STC assays: STC determinations were performed using the chemoluminescent immunoenzymology method with Immulite 2000 Cortisol kit (Siemens Medical Diagnostics Solutions, Erlangen, Germany). Analytical sensitivity was 0.20 lg/dl. Inter-assay variations (CV) were 5.5%, 4.4%, 4.3%, and intra-assay variations (CV) were 4.5%, 3.8%, and 4.2% at 2.2, 8.5, and 26.9 lg/dl, respectively. SC assays: Once transported to the laboratory, saliva was collected by centrifugation. SC determinations were performed by the radioimmunoassay method with Coat-a-Count kit (Diagnostic Products, Los Angeles, CA) according to the protocol supplied by the manufacturer. Calibrators were prepared in different dilutions in buffer (0, 0.36, 1.45, 7.25, and 36.23 ng/ml). Patients’ samples and calibrators (150 ll) were analysed in duplicate. A cortisol tracer (500 ll) was added to each reaction. All tubes were mixed, covered with paraffin film and incubated for 30 min at 37 °C. Tubes were decanted and washed once with 1 ml of distilled water and then decanted again. The radioactivity of each tube was counted using a gamma counter. Measurement range was 0.36–36.23 ng/ ml. Automatic results were obtained using a spline function curve. Analytical sensitivity was 0.29 ng/ml. Inter and intra-assay variations (CV) were 7.0% and 6.0% respectively. CBG assays: CBG assays were performed using radioimmunoassay protocols (Biosource; Lifescreen, Watford, Herts, UK). Calculated basal free cortisol (CFC) assessment: Basal CFC was assessed by the Coolens formula [23] which allows the calculation of FC using STC and CBG assays, taking into account the affinity of cortisol for CBG and albumin: p CFC ¼ ðZ 2 þ 0:0122 STCÞ Z Z ¼ 0:0167 þ 0:182ðCBG STCÞ Lipid fractions: Total cholesterol was determined by enzymatic testing in the KONELAB 30i analyser (KONELAB, Thermo Clinical Labsystems, Cergy-Pontoise, France). Triglycerides were measured by an automated enzymatic technique (Biomérieux, Marcy l’Etoile, France). Lipoprotein modifications in patients with cirrhosis make routine determination of HDL-c concentrations difficult [39,40]. HDL-c plasma concentrations were determined through a reactive phosphotungstate of sodium/MgCl2 (Boehringer Mannheim) to precipitate lipoproteins containing apo-
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Results Patients’ characteristics Ninety-eight patients were included in this study. The data acquired from ten patients were not analysed, as the collected amount of saliva was insufficient. Thus, final analysis was completed for 88 patients. Their clinical and biological characteristics at inclusion are shown in Table 1. Reasons for hospitalisation were: digestive haemorrhage in 57%, hepatic encephalopathy in 13%, ascites in 11%, renal failure in 7%, and other reasons in 12% of cases. CBG plasma concentrations CBG plasma concentrations were 22.36 ± 8.84 lg/ml and decreased with the severity of cirrhosis according to the Child– Pugh score (28.60 ± 6.76 lg/ml in Child–Pugh A cirrhosis, 21.84 ± 8.84 lg/ml in Child–Pugh B cirrhosis and 20.80 ± 8.32 lg/ ml in Child–Pugh C cirrhosis, p <0.05). The CBG plasma concentrations were not correlated to the albumin plasma concentrations [Spearman coefficient 0.23 (0.01 to 0.45)]. Correlation between basal SC, STC and CFC The observed correlation between the basal SC at T0 and CFC was excellent (Spearman coefficient = 0.91 (0.87–0.95), p <0.001) Correlation between STC and CFC was lower (Spearman
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JOURNAL OF HEPATOLOGY Table 1. Patients characteristics at inclusion.
54 ± 10 64 (72.7) 4.1 ± 2.5 5.1 ± 2.4 4.9 ± 2.6 2.6 ± 1.6
$
63 (71.6) 14/63 (22.2) 13 (14.8) 8 (9.1) 4 (4.5)
30.0
20.0
10.0
0.0
29 (33.0) 25 (28.4) 34 (38.6) 32 (36.4) 10 ± 2 4 (4.6)/24 (27.3)/60 (68.2) 16 ± 8 8 (9.1) 31 (35.2) 38 (43.2) 24 (27.3) 13 6 3 2 14 (15.9) 46 ± 15 28 ± 5 74 (54–104) 8.2 ± 4.2 9.6 ± 1.8 87.5 (69.0–95.0) 76 (64–90) 41 (35–46) 64 (59–70) 0.89 (0.70–1.03) 0.48 (0.42–0.56) 0.25 (0.16–0.35) 0.36 (0.22–0.51)
Salivary cortisol (ng/ml)
Age, years* Male sexe, n (%) Time between admission and inclusion, days* - Admission for digestive haemorrhage - Admission for encephalopathy - Other reasons of admission Causes of cirrhosis, n (%) - Alcohol: - Weaned from alcohol for >6+ months: - Viral hepatitis: - Mixed: - Other: Ascites, n (%) - Absence - Medically controlled - Poorly controlled ascites, n (%) Hepatic encephalopathy, n (%) Child–Pugh score* - Class A/B/C, n (%) MELD score* Hepatocellular carcinoma, n (%) Acute alcoholic hepatitis, n (%) SIRS, n (%) Infection, n (%) Localisation, n: - Ascites: - Bacteriemia: - Urine tract: - Other: Sepsis, n (%) Prothrombin time ratio, %* Albumin, g/L* Total serum bilirubin, lmol/L$ Leukocyte count, G/L* Hemoglobin, g/dl* Platelet, G/L$ ASAT, UI/ml ALAT, UI/ml$ Serum creatinine, lmol/L$ Cholesterol, g/L$ Triglyceride, g/L$ HDL-cholesterol, g/L$ Apolipoprotein A1, g/L$
Serum total cortisol (µg/dl)
Data
*
50.0
40.0
Variable
37.5
25.0
12.5
0.0
0.0
1.0
2.0
3.0
4.0
0.0
Free cortisol (µg/dl) Spearman coefficient = 0.76, p <0.001
1.0
2.0
3.0
4.0
Free cortisol (µg/dl) Spearman coefficient = 0.91, p <0.001
Fig. 1. Correlation between calculated free cortisol and salivary cortisol concentrations and between calculated free cortisol and serum total cortisol concentrations at T0. Whereas this correlation was excellent between calculated free cortisol and salivary cortisol, it was significantly lower between calculated free cortisol and serum total cortisol (p = 0.001).
than according to SC (8/88: 9.1%) (p = 0.001). Five patients fit at least 2 biological criteria used to define AI according to SC assays. Ten patients satisfied at least 2 biological criteria used to define AI according to STC assays (Table 3). Correlation between STC and SC assays (Fig. 2) Correlations between STC and SC assays for T0, T60 and D were strong in patients with albumin concentrations > 25 g/L (Spearman coefficient = 0.69, 0.71 and 0.62, respectively, all p = 0.0001) whereas, in patients with albumin 625 g/L, the correlation was strong at T0 (Spearman coefficient = 0.62, p = 0.0007), poor at T60 (Spearman coefficient = 0.31, p = 0.12) and absent for D (Spearman coefficient = 0.0004, p = 0.93) (Fig 2). To avoid data-driven results, in the protocol of this study, the serum albumin cut-off (25 g/L) was defined a priori, according to Hamrahian data [17]. Discrepancy between salivary and serum tests for AI diagnosis
Mean ± standard deviation. Median (confidence interval).
coefficient = 0.76 (0.6–0.85), p <0.001). The difference between these two correlations was significant (p <0.001) (Fig. 1). Adrenal insufficiency prevalence The results of STC and SC measurements are reported in Table 2. The AI prevalence was higher according to STC (29/88: 33.0%)
Agreement between the salivary and serum tests was low (kappa = 0.28 [0.08–0.49]). Twenty-two patients had AI according to STC assays, but normal adrenal function according to salivary assays. One patient had AI according to salivary assays, but normal adrenal function according to serum assays. Patient characteristics were compared between the group with concordant tests (n = 65) and the group with discordant tests (n = 23). Univariate analysis identified candidate factors for discordant tests: male gender
Table 2. Plasma and salivary cortisol concentrations. All the patients (n = 88)
Adrenal insufficiency according to salivary cortisol (n = 8)
No adrenal insufficiency according to salivary cortisol (n = 80)
p Value
Serum total cortisol T0 (lg/dl) T60 (lg/dl) D (lg/dl)
15.4 (14.1–18.1) 29.6 (27.5–32.5) 12.5 (11.4–13.9)
3.5 (1.3–10.8) 13.7 (4.4–21.7) 7.9 (0.6–11.5)
15.7 (14.3–18.6) 30.2 (27.6–32.6) 12.9 (11.5–14.4)
0.001 0.0002 0.02
Salivary cortisol T0 (ng/ml) T60 (ng/ml) D (ng/ml)
8.9 (6.5–10.0) 34.3 (30.5–37.5) 22.5 (19.4–24.8)
1.4 (0.3–2.7) 13.2 (2.1–27.8) 11.8 (1.1–27.5)
9.4 (7.1–11.7) 35.1 (31.3–38.8) 23.0 (20.3–25.8)
0.00004 0.00009 0.003
Values are given in median (confidence interval).
Journal of Hepatology 2010 vol. 52 j 839–845
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Research Article Table 3. Adrenal insufficiency prevalence according to serum total cortisol and salivary cortisol assays during corticotropin test. Adrenal insufficiency according to salivary cortisol
Adrenal insufficiency according to serum total cortisol 12 10 19 29/88 (33.0%)
Patients with albumin >25 g/L (n = 61)
Patients’ characteristics according to their adrenal function assessed by SC assays and univariate analysis are reported in Table 4. Several models have been considered in the multivariate analysis; we report here the model with total bilirubin, prothrombin time, total cholesterol, HDL-c, and ascites. In multivariate analysis, only ascites and lower HDL-c plasma concentrations remained independently associated with AI (p = 0.01 for both).
842
35.0
Salivary cortisol (ng/ml)
Risk factors for AI
30.0
20.0
10.0
26.3
17.5
8.8
0.0
0.0 0.0
15.0
30.0
45.0
60.0
0.0
Serum total cortisol (µg/dl) Spearmann coefficient = 0.69, p = 0.0001
17.5
26.3
35.0
Salivary cortisol (ng/ml)
50.0
60.0
40.0
20.0
0.0 0.0
20.0
40.0
60.0
40.0
30.0
20.0
10.0 15.0
80.0
Serum total cortisol (µg/dl) Spearmann coefficient = 0.71, p = 0.0001
22.5
30.0
37.5
45.0
Serum total cortisol (µg/dl) Spearmann coefficient = 0.31, p = 0.12
50.0
45.0
Salivary cortisol (ng/ml)
Salivary cortisol (ng/ml)
8.8
Serum total cortisol (µg/dl) Spearmann coefficient = 0.62, p = 0.0007
80.0
T60
This study shows that: (1) SC assays are more correlated with CFC (active fraction) than STC assays and, thus, are a better reflection of adrenal function in patients with cirrhosis; (2) STC assays overstate AI prevalence in patients with cirrhosis; (3) the only risk factor for AI misdiagnosis by STC assays is hypoalbuminemia; (4) risk factors for AI in cirrhotic patients were ascites and low HDL-c plasma concentration. In previously published studies using STC assays in cirrhotic patients without shock, AI prevalence was high and correlated with the Child–Pugh score [7,8]. The influence of STC assay results on cirrhotic patients’ outcomes in the ICU is controversial because AI determined by STC assays has been associated not only with mortality [5] but also with survival [1]. The same controversy exists in ICU patients without cirrhosis [46,47], resulting in a recommendation to no longer use STC assays in these patients [21,22]. Indeed, it has been shown that STC assays overestimate AI prevalence in ICU patients without cirrhosis but who have albuminemia 6 25 g/L [17]. Patients admitted to the ICU and patients with cirrhosis both have a frequent decrease in CBG and albumin plasma concentrations because of hepatocellular insufficiency and inflammation [7,24,48–51]. We confirmed that CBG plasma concentrations decreased with the severity of
Patients with albumin <25 g/L (n = 27)
40.0
T0
Discussion
0.001
cirrhosis. However, there was no correlation between albumin plasma concentrations and those of CBG. Moreover, lower albuminemia was associated with a discrepancy between salivary and serum tests for AI, whereas a lower CBG plasma concentration was not. These results could be explained by an earlier decrease in CBG concentrations than in albumin levels in patients
Salivary cortisol (ng/ml)
(91% vs. 68%, p = 0.03), cirrhosis not due to alcohol (35% vs. 14%, p = 0.03), absence of acute alcoholic hepatitis (87% vs. 61%, p = 0.02), hepatocellular carcinoma (17% vs. 6%, p = 0.11), presence of ascites (78% vs. 62%, p = 0.17), lower albuminemia (26 ± 4 g/L vs. 29 ± 5 g/L, p = 0.01), lower leukocyte count (6.9 ± 4.0 g/L vs. 8.5 ± 4.3 g/L, p = 0.15), lower ASAT plasma concentration (67 [46– 84] IU/ml vs. 87 [67–116] IU/ml, p = 0.07) and lower triglyceride plasma concentration (0.46 [0.27–0.56] g/L vs. 0.56 [0.40–0.64] g/L, p = 0.19). There was no correlation between plasma concentrations of CBG and discordant tests (22.36 ± 8.32 lg/ml vs. 21.84 ± 10.40 lg/ml, p = 0.85). In multivariate analysis in 2 different models, the only factor associated with discordant tests was lower serum albumin levels (p = 0.01 and 0.009). Profound hypoalbuminemia (serum albumin level 6 25 g/L) was more frequent in patients of which STC assays, but not SC assays, indicated AI (54%) than in others (23%, p = 0.006).
6 3 3 8/88 (9.1%)
Salivary cortisol (ng/ml)
Adrenal insufficiency according to T0, n Adrenal insufficiency according to T60, n Adrenal insufficiency according to D, n Total number of patients with adrenal insufficiency, n (%)
p Value
37.5
25.0
12.5
0.0 0.0
10.0
20.0
30.0
40.0
Serum total cortisol (µg/dl) Spearmann coefficient = 0.62 p = 0.0001
36.3
27.5
18.8
10.0 5.0
10.0
15.0
20.0
25.0
Serum total cortisol (µg/dl) Spearmann coefficient = 0.0004 p = 0.93
Fig. 2. Correlation between total serum cortisol and salivary cortisol concentrations at T0, T60 and for D values (T60–T0) in patients with albumin >25 g/L and in patients with albumin 625 g/L. Whereas this correlation was very good in patients with albumin >25 g/L, it was good at T0, bad at T60 and null for D values in patients with albumin 625g/L.
Journal of Hepatology 2010 vol. 52 j 839–845
JOURNAL OF HEPATOLOGY with cirrhosis. Indeed, even if CBG and albumin plasma concentrations decrease with cirrhosis severity, CBG is already much decreased in patients with Child–Pugh A cirrhosis, whereas albumin remains closed to normal range. In patients with cirrhosis, serum total cortisol would overstate AI only when albumin plasma concentrations decrease, which is later in the evolution of liver disease. Thus, plasma albumin concentration, a routinely performed assay, should be used to identify cirrhotic patients who should be assessed via a salivary cortisol assay rather than a serum total cortisol assay. We have identified risk factors for AI in cirrhotic patients. Ascites was associated with higher AI prevalence. Other studies have already found a correlation between the liver disease severity and AI [7,8], but as these studies used STC assays, it could be argued that low CBG and albumin plasma concentrations could explain these results. Using SC assays allowed this bias to be avoided for the first time, and we confirmed the relationship between severe liver disease and AI. A low HDL-c plasma concentration was also associated with AI. These results confirm those of several studies in patients with and without cirrhosis [1,2,52]. HDL-c brings cholesterol into adrenal glands where it is necessary for cortisol synthesis [53]. This result adds the potential beneficial effect of increasing HDL-c plasma concentrations in patients with cirrhosis in addition to limiting the lipopolysaccharide
induced pro-inflammatory response as previously reported [54,55]. This study has several limitations. First, most of the cirrhosis cases were due to alcohol (71.6%) which is common in France (22.2% had been weaned from alcohol for more than 6 months). In previous studies, alcoholic patients were excluded [7] or weaned for more than 3 months [8]. In non-weaned patients, alcohol can stimulate cortisol synthesis through the adrenal glands [56] which could explain the relatively low AI prevalence in our study. Second, in our study, 27.3% of patients were infected. Infection can increase cortisol concentrations and reduce AI prevalence. However, infection was always controlled by antibiotics and no patient had haemodynamic instability in the days preceding inclusion. However, this cannot have influenced the main result of this study, which is a high overestimation of AI prevalence by STC assays in cirrhotic patients. Moreover, most cirrhosis complications were digestive haemorrhage (57%). This could be explained by the fact that our Hepatology unit contains a 10-bed intermediate care unit where there are a large number of hospitalisations for cirrhotic patients with digestive haemorrhage from mobile care units and emergency units of other hospitals in the Paris area. However, salivary assays were performed only 4.9 ± 2.5 days after admission following confirmation of the absence of blood in the mouth. Reasons for
Table 4. Risk factors of adrenal insufficiency, defined by salivary assays, in univariate analysis. Adrenal insufficiency according to salivary cortisol assays Variable
Yes (n = 8)
No (n = 80)
p Value
Age, years* Male sexe, n (%) Cirrhosis due to alcohol, n (%) Weaning from alcohol, n (%) Hepatocellular carcinoma, n (%) Child-Pugh score* Child-Pugh score class C, n (%) MELD score* Ascites, n (%) Encephalopathy, n (%) Reason of hospitalization, n (%) - Digestive haemorrhage - Hepatic encephalopathy - Ascites - Renal failure Acute alcoholic hepatitis, n (%) SIRS, n (%) Infection, n (%) Sepsis, n (%) Prothrombine time ratio, %* Albumin, g/L* Total serum bilirubin, lmol/L$ Leukocyte count, G/L* Hemoglobin, g/dl* Platelet, G/L$ ASAT, UI/ml$ ALAT, UI/ml$ Serum creatinine, lmol/L$ Cholesterol, g/L$ Triglyceride, g/L$ HDL cholesterol, g/L$ Apolipoprotein A1, g/L$ STC: TO, lg/dl$ STC: T60, lg/dl$ STC: D, lg/dl$
56 ± 11 6 (75) 7 (88) 3 (43) 0 (0) 12.0 ± 1.7 8 (100) 11 ± 5 8 (100) 3 (37)
54 ± 10 59 (74) 64 (80) 29 (33) 78 (10) 10.2 ± 2.3 52 (65) 8±1 50 (64) 26 (33)
0.71 0.94 0.61 0.59 0.34 0.04 0.04 0.02 0.04 0.79 0.03
2 (25.0) 1 (12.5) 4 (50.0) 1 (12.5) 4 (50.0) 2 (25.0) 2 (25.0) 2 (25.0) 40 ± 15 29 ± 4 155 (37–254) 7.7 ± 3.7 9.2 ± 1.7 71 (38–114) 91 (38–114) 48 (27–81) 174 (39–118) 0.45 (0.30–0.70) 0.54 (0.26–0.60) 0.09 (0–0.17) 0.17 (0.10–0.20) 3.5 (1.3–10.8) 13.7 (4.4–21.7) 7.9 (0.6–11.5)
52 (65.0) 11 (13.8) 9 (11.3) 4 (5.0) 24 (30.0) 36 (45.6) 22 (28) 12 (15.0) 47 ± 15 28 ± 4 70 (54–99) 8.1 ± 4.3 9.7 ± 1.3 88 (70–99) 74 (64–95) 41 (35–48) 62 (59–71) 0.97 (0.50–1.01) 0.72 (0.52–0.84) 0.31 (0.21–0.37) 0.58 (0.19–0.66) 15.7 (14.3–18.6) 30.2 (27.6–32.6) 12.9 (11.5–14.4)
0.25 0.26 0.88 0.47 0.23 0.58 0.19 0.81 0.53 0.65 0.96 0.87 0.40 0.08 0.29 0.04 0.04 0.001 0.0002 0.02
STC: Serum total cortisol. Mean ± standard deviation. $ Median (confidence interval).
*
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Research Article hospitalisation were not associated with AI in multivariate analysis. Finally, we included only haemodynamically stable patients, while AI diagnosis seems crucial in patients with sepsis or septic shock. This is a pilot study and, even if it has been shown that SC was still highly correlated with FC in patients with shock [57,58], SC threshold values remain to be assessed in these patients.
Conclusion This study shows that STC assays overstate AI prevalence in patients with cirrhosis and hypoalbuminemia. Determination of SC is an easily available and more accurate method to assess adrenal function in these patients.
Conflicts of interest The Authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Acknowledgements The work was carried out at Service d’hépato-gastroentérologie, hôpital de la Pitié-Salpêtrière (AP-HP), 47-83 bd de l’hôpital, 75013 Paris. Financial support: This study has been supported by an ARMHV grant (Association pour la Recherche sur les Maladies Hépatiques Virales). References [1] Marik PE, Gayowski T, Starzl TE. The hepatoadrenal syndrome: a common yet unrecognized clinical condition. Crit Care Med 2005;33:1254–1259. [2] Marik PE. Adrenal-exhaustion syndrome in patients with liver disease. Intensive Care Med 2006;32:275–280. [3] Harry R, Auzinger G, Wendon J. The clinical importance of adrenal insufficiency in acute hepatic dysfunction. Hepatology 2002;36: 395–402. [4] Fernandez J, Escorsell A, Zabalza M, Felipe V, Navasa M, Mas A, et al. Adrenal insufficiency in patients with cirrhosis and septic shock: effect of treatment with hydrocortisone on survival. Hepatology 2006;44:1288–1295. [5] Tsai MH, Peng YS, Chen YC, Liu NJ, Ho YP, Fang JT, et al. Adrenal insufficiency in patients with cirrhosis, severe sepsis and septic shock. Hepatology 2006;43:673–681. [6] Thierry S, Giroux Leprieur E, Lecuyer L, Brocas E, Van de Louw A. Echocardiographic features, mortality, and adrenal function in patients with cirrhosis and septic shock. Acta Anaesthesiol Scand 2008;52:45–51. [7] McDonald JA, Handelsman DJ, Dilworth P, Conway AJ, McCaughan GW. Hypothalamic–pituitary adrenal function in end-stage non-alcoholic liver disease. J Gastroenterol Hepatol 1993;8:247–253. [8] Zietz B, Lock G, Plach B, Drobnik W, Grossmann J, Scholmerich J, 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;15:495–501. [9] Dunn JF, Nisula BC, Rodbard D. Transport of steroid hormones: binding of 21 endogenous steroids to both testosterone-binding globulin and corticosteroid-binding globulin in human plasma. J Clin Endocrinol Metab 1981;53:58–68. [10] Ekins R. Measurement of free hormones in blood. Endocr Rev 1990;11:5–46. [11] Mendel CM. The free hormone hypothesis: a physiologically based mathematical model. Endocr Rev 1989;10:232–274.
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