Is Adrenal Insufficiency Under-diagnosed in Hospitalized Cirrhosis Patients?
Phase: PHASE3
Conditions: Adrenal Insufficiency, Cirrhosis, Spur Cell Anemia, Lecithin Acyltransferase Deficiency
Sponsor: University of Virginia

Is adrenal insufficiency under-diagnosed in hospitalized cirrhosis patients? 
 
UVA Study Tracking:  
HSR Submission Number: 11963 
Version Date: 11/20/17 
Page Number: 0 of 8 
 
 
 
PI: Zachary Henry, MD 
11-20-2017 
 
University of Virginia 
Is adrenal 
insufficiency under-
diagnosed in 
hospitalized 
cirrhosis patients? 
NCT03368066 

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Version Date: 11-20-17 
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PROTOCOL 
Background 
1. Provide the scientific background, rationale and relevance of this project.   
Answer/Response:  
Cirrhosis is known to cause various end-organ dysfunction, most notably the 
hepatorenal and hepatopulmonary syndromes. However, the increased recognition of adrenal 
insufficiency in cirrhotic patients has led to postulation about the presence of a so-called 
“hepatoadrenal syndrome1.” While originally this syndrome was recognized in cirrhotic patients 
with sepsis and shock states1,2, further investigation has also identified adrenal insufficiency in 
cirrhotic patients with variceal bleeding3 as well as stable patients4. Although there is 
controversy about the best diagnostic test to assess adrenal insufficiency in cirrhotic patients5,6, 
it is well established that cirrhotic patients with adrenal insufficiency have increased mortality, 
particularly as they become more decompensated7. 
In addition to this endocrinological complication, cirrhosis also has a profound effect on 
a patient’s hematologic parameters. In a study of rheologic properties of 38 patients (18 
cirrhotic – mostly of cryptogenic origin, 20 control), cirrhotic patients were noted to have 
increased erythrocyte rigidity and decreased plasma and blood viscosity8.  The authors argued 
that their findings were related to a deficiency of high-density lipoprotein (HDL), although the 
correlation was admittedly weak in their study. A follow-up study by the same authors also 
confirmed an increase in erythrocyte rigidity in patients with alcoholic and post-hepatic 
cirrhosis; importantly, they noted the prior association between alcoholic liver disease and 
development of macrocytosis as a function of lecithin-cholesterol acetyltransferase (LCAT) 
enzyme deficiency9. This enzyme is responsible for esterification of cholesterol and subsequent 
incorporation into normal red cell membranes. Interestingly, its deficiency is directly 
manifested as an altered free cholesterol to phospholipid ratio10, with the increased cholesterol 
fraction causing the membrane rigidity previously described.  
Another abnormality well documented in cirrhotic patients, more frequently in patients 
with advanced disease, is spur cell anemia. Although first described in alcoholic cirrhosis, it is 
seen in all etiologies and is a poor prognostic marker. A spur cell burden of ≥5% was found to 
be associated with a 67% 90-day mortality rate in a recent study11. Furthermore, patients with 
spur cell anemia have decreased red blood cell (RBC) lifespan and are transfusion resistant as 
transfused RBCs quickly acquire the pathologic morphology. Spur cells are thought to occur 
secondary to red cell membrane abnormalities secondary to altered cholesterol composition, 
similar to the abnormal erythrocyte rigidity rheologic property described above. Interestingly, 
the lecithin-cholesterol acetyltransferase enzyme has been investigated as a potential key 
factor in spur cell anemia. A 1972 study by Cooper et al12 measured LCAT enzyme activity by 
way of a surrogate method involving change in serum free cholesterol after 6-hour incubation. 
Lower levels of LCAT were found in patients with spur cells compared to normal controls; 
however, there was only a weak association with red cell lipid content.  
Furthermore, platelet dysfunction in cirrhosis has also been tied to lipid abnormalities. A 
review of dyslipidemia in liver disease by Miller noted that platelets, similar to erythrocytes, 
have an imbalanced cholesterol to phospholipid ratio and associated impaired aggregation13. 
Thus, the deficient primary hemostasis noted in cirrhotic patients may not be just a function of 

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thrombocytopenia itself but also an intrinsic abnormality of platelet function related to a 
disruption in normal cholesterol pathways.   
It has been demonstrated that LCAT is a key enzyme for many homeostatic functions 
within the human body and its deficient production in cirrhosis appears to correlate with 
various hematologic abnormalities. However, it may very well also play an important role in 
adrenal steroidogenesis and help explain the so called “hepatoadrenal” syndrome that has 
begun to permeate through the literature. More importantly, however, is that cholesterol 
esterification levels, a surrogate marker of LCAT enzymatic activity, are directly associated with 
mortality and thus can serve as a prognostic indicator. A recent study by Kaiser et al.14 found 
that the cholesterol esterification fraction was more accurate than INR for estimation of liver 
synthetic capability and both 3-month and 1-year mortality. Thus, if one can quantify this 
deficiency in cirrhotic patients and demonstrate association with several related pathologic 
conditions, it may soon become a useful biomarker to direct treatment and more reliably 
predict mortality. 
Objectives/Hypothesis  
Answer/Response: 
The hepatoadrenal syndrome has been well described in the literature and is known to 
be associated with poorer outcomes in both stable and critically ill cirrhotic patients. In chronic 
liver disease, adrenal (and more specifically cortisol) insufficiency is thought to be a byproduct 
of altered lipid metabolism that results in decreased HDL production and thus decreased 
delivery of cholesterol to the adrenal for subsequent corticosteroid production. Studies to date 
have implicated lecithin-cholesterol acetyltransferase (LCAT) as the key enzyme which is 
deficient in some cirrhotic patients, leading to an impaired ability to esterify cholesterol and 
thus a loss of normal cellular functioning and membrane stability. We seek to quantify this LCAT 
deficiency in a cohort of cirrhotic patients and demonstrate its association with various 
abnormal physiologies associated with chronic liver disease, including spur cell anemia, low HDL 
levels, and adrenal insufficiency. 
 
Study Design: Biomedical 
1.  Will controls be used? 
Answer/Response: No a priori controls (i.e. we will not be including patients without cirrhosis in 
our study). We will compare cirrhotic patients with and without adrenal insufficiency. 
►IF YES, explain the kind of controls to be used. 
Answer/Response: N/A 
 
2. What is the study design?  
Answer/Response: prospective cohort study 
 
3. Does the study involve a placebo? 
Answer/Response: No 
►IF YES, provide a justification for the use of a placebo 
Answer/Response: N/A 

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Human Participants 
Ages: _18-100___ 
Sex: 
_Men and women___ 
Race: _All___ 
 
Subjects- see below 
1.  Provide target # of subjects (at all sites) needed to complete protocol. 
Answer/Response: 100 
 
2.  Describe expected rate of screen failure/ dropouts/withdrawals from all sites.   
Answer/Response: 20 
 
3.  How many subjects will be enrolled at all sites?    
Answer/Response: 120 
 
4.  How many subjects will sign a consent form under this UVa protocol?     
Answer/Response: 120 
Inclusion/Exclusion Criteria 
1.  List the criteria for inclusion  
Answer/Response:  
 age >= 18 years,  
 diagnosis of cirrhosis,  
 admission to hospital 
 
2.  List the criteria for exclusion 
Answer/Response:  
 age <18 years, 
 prior enrollment in study (i.e. readmission), 
  prisoner,  
 prednisone or hydrocortisone use in the past 24 hours, 
 pregnancy 
 
3.  List any restrictions on use of other drugs or treatments. 
Answer/Response: None 
Statistical Considerations 
1. 
Is stratification/randomization involved? 
Answer/Response: No 
►IF YES, describe the stratification/ randomization scheme. 
Answer/Response: N/A 
►IF YES, who will generate the randomization scheme?  
_____ Sponsor 

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_____ UVa Statistician.   Insert name Answer/Response: 
_____ UVa Investigational Drug Service (IDS) 
_____ Other:  Specify   Answer/Response: 
 
2.  What are the statistical considerations for the protocol?  
Answer/Response: 
Subjects will be classified as adrenally sufficient or insufficient on the basis of standard-
dose cortisol stimulation test. Variables of interest for comparison between the groups include 
MELD score, Child-Turcotte-Pugh (CTP) classification, high-density lipoprotein (HDL) levels, 
presence of spur cell anemia, serum cholesterol ester percentage (surrogate for LCAT 
enzymatic activity), cortisol binding globulin levels, and free cortisol levels. Student’s t-test and 
Chi Square tests will be utilized to determine significance; a p-value <0.05 will be used as our 
threshold for significance. If multiple factors are found to be significantly different in a 
univariate fashion between classification groups, a multivariate logistic regression analysis will 
be performed for adjusted analysis. We will also seek to define any correlations between 
variables. Furthermore, we will assess correlation between MELD score and serum cholesterol 
ester percentage, spur cell anemia, HDL levels, cortisol binding globulin levels, and free cortisol 
levels; similar correlate analysis will be done using CTP classification instead of MELD score. 
Prevalence of adrenal insufficiency (AI) in cirrhosis varies widely based on several 
factors, including severity of liver disease (i.e. MELD score or CTP classification) and the testing 
modality used to assess for AI. Given that admitted patients to UVA are typically CTP B or C, we 
expect the incidence to be about 33%. Thus, 120 patients recruited should be sufficient to 
provide a statistical difference in our outcome measures of interest, assuming a 20% dropout 
rate to lead to 100 patients completing study protocol.  
 
3.  Provide a justification for the sample size used in this protocol.   
Answer/Response: 
Given that admitted patients to UVA are typically CTP B or C, we expect the incidence of 
adrenal insufficiency to be about 33%. We feel 100 patients would be adequate to complete a 
pilot study to test for correlation between adrenal insufficiency, low serum cholesterol ester 
percentage, and spur cell anemia. There is no current literature upon which to do a formal 
power calculation (although low levels of cholesterol esterification is a well-established near-
universal metabolic finding in cirrhotic patients), but this sample size would allow for adequate 
size comparator groups and decrease the likelihood of a Type II statistical error.  
 
4.  What is your plan for primary variable analysis? 
Answer/Response:  
Our primary outcome of interest is comparing prevalence of cholesterol esterification 
deficiency and spur cell anemia in cirrhotic patients with and without adrenal insufficiency. 
 
5.  What is your plan for secondary variable analysis?  
Answer/Response: 
- 
Demonstrate association between deficiency in cholesterol esterification and presence 
of spur cell anemia 

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- 
Demonstrate association between deficiency in cholesterol esterification and low HDL 
levels 
- 
Correlate higher MELD score with presence of adrenal insufficiency 
- 
Correlate higher MELD score with presence of spur cell anemia 
- 
Correlate higher MELD score with lower HDL levels 
- 
Correlate higher MELD score with deficiency in cholesterol esterification 
- 
Correlate higher CTP classification with presence of adrenal insufficiency 
- 
Correlate higher CTP classification with presence of spur cell anemia 
- 
Correlate higher CTP classification with lower HDL levels 
- 
Correlate higher CTP classification with deficiency in cholesterol esterification 
- 
Correlate lower cortisol binding globulin levels with higher MELD score and CTP 
classification 
- 
Correlate lower cortisol binding globulin levels with adrenal insufficiency 
- 
Correlate lower free cortisol levels with higher MELD score and CTP classification 
- 
Correlate lower free cortisol levels with adrenal insufficiency 
- 
Multivariate logistic regression if several variables are found to be significant 
 
6. Have you been working with a statistician in designing this protocol? 
Answer/Response: No 
IF YES, what is their name?   
Answer/Response: N/A 
 
7.  Will data from multiple sites be combined during analysis?   
Answer/Response: No 
7(a).  Does the study involve randomization?   
Answer/Response: No 
IF YES, will randomization be done at each site or among sites?   
Answer/Response: N/A 
 
7(b).  Has the sample size calculation considered the variation among sites?  
Answer/Response: N/A 
 
7(c).  When combining the data from multiple sites to assess the study results, is the 
effect of the treatment to be tested (or the association to be tested) assumed to be the 
same across sites or vary among sites? What is the modelling strategy? 
Answer/Response: N/A 
 
7(d). Is there a common protocol used in all sites?  
Answer/Response: N/A 
IF NO, how will differences among sites, such as those related to the 
implementation, inclusion criteria, patient characteristics, or other sites 
characteristics, be considered to assess the study results? 
Answer/Response: N/A 

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Study Procedures-Biomedical Research 
1.  What will be done in this protocol?    
Answer/Response:  
Hospitalized cirrhotic patients at UVA that meet study eligibility criteria will be 
approached by a member of the study team to obtain consent for participation. If a 
patient agrees to become a study subject, women of childbearing potential will be 
administered a urine pregnancy test to exclude pregnancy. Eligible subjects will have an 
approximate total of 35ml of blood drawn by the inpatient phlebotomy team. Lab tests 
to be performed include: peripheral blood smear, lipid panel, free cortisol, cortisol 
binding globulin, serum cholesterol esters (surrogate for LCAT enzyme activity), and a 
standard-dose cortisol stimulation test. The latter involves blood drawn with the initial 
collection, administration of an intravenous 250mcg dose of synthetic ACTH by the 
patient’s nurse, and then repeat small-volume (2 teaspoons) blood draws at 30 minutes 
and 60 minutes later. 
The interventions listed above have the potential to identify subjects with 
adrenal insufficiency, which may be beneficial to their long-term care. Early 
identification of adrenal insufficiency may lead to earlier corticosteroid administration in 
critical illness or GI bleeding, thus improving morbidity and mortality. Otherwise, the 
blood draws themselves pose minimal risk given that it is a routine procedure done 
during hospitalization, often on a daily or even more frequent basis. Very small risks of 
bleeding, bruising, or infection do exist.  
Most patients tolerate intravenous ACTH as part of the cortisol stimulation test, 
however known reactions include nausea, diaphoresis, dizziness, pruritus, palpitations, 
and facial flushing. All patients will be monitored and these side effects, if present, 
typically resolve within a few hours. Any patients identified as having adrenal 
insufficiency will be reported to the primary hepatology team caring for the patient to 
determine whether or not endocrinology consult and/or treatment should be 
considered. 
 
2. If this protocol involves study treatment, explain how a subject will be transitioned from 
study treatment when they have completed their participation in the study.   
Answer/Response: N/A 
 
Subject Compliance with Study Procedures 
1.  Explain how the study team will monitor the subject for compliance with the study 
procedures. 
Answer/Response: study team will communicate with subjects and subjects’ nurses about study 
protocol and will follow-up to be sure labs were drawn and cortisol stimulation test was 
administered. This can be tracked through the electronic medical record as well.  
 

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2.  Describe criteria for when a subject is considered to be non-compliant with study 
procedures.   
Answer/Response: subject refuses study-related blood draw(s) or ACTH administration 
Bibliography 
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review. Hepatology 2012; 55(4): 1282-1291. 
 
2O’Beirne J, Holmes M, Agarwal B, et al. Adrenal insufficiency in liver disease – What is the 
evidence? J Hepatol 2007; 47: 418-423. 
 
3Triantos CK, Marzigie M, Fede G, et al. Critical illness-related corticosteroid insufficiency in 
patients with cirrhosis and variceal bleeding. Clin Gastro and Hepatol 2011; 9:595-601. 
 
4Fede G, Spadaro L, Tomaselli T, et al. Assessment of adrenocortical reserve in stable patients 
with cirrhosis. J Hepatol 2011; 54:243-250. 
 
5Fede G, Spadaro L, Tomaselli T, et al. Comparison of total cortisol, free cortisol, and surrogate 
markers of free cortisol in diagnosis of adrenal insufficiency in patients with stable cirrhosis. Clin 
Gastro and Hepatol 2014; 12:504-512. 
 
6Tan T, Chang L, Woodward A, et al. Characterising adrenal function using directly measured 
plasma free cortisol in stable liver disease. J Hepatol 2010; 53:841-848. 
 
7Jang JY, Kim TY, Sohn JH, et al. Relative adrenal insufficiency in chronic liver disease: Its 
prevalence and effects on long-term mortality. Alimentary Pharm & Ther 2014; 40;819-826. 
 
8Tamer S, Cefle K, Palanduz S, and Vatansever S. Rheologic properties of blood in patients with 
chronic liver disease. Clin Hemorheol and Microcirc 2002; 26:9-14. 
 
9Tame S, Cefle K, Gokkusu C, et al. Comparison of rheologic parameters in patients with post 
hepatic and alcoholic cirrhosis. Clin Hemorheol and Microcirc 2007; 36:247-252. 
 
10Kakimoto H, Imai Y, Kawata S, et al. Altered lipid composition and differential changes in 
activities of membrane-bound enzymes of erythrocytes in hepatic cirrhosis. Metabolism 1995; 
825-832. 
 
11Alexopoulou A, Vasilieva L, Kanellopoulou T, et al. Presence of spur cells as a highly predictive 
factor of mortality in patients with cirrhosis. J Gastro and Hepatol 2013; 29:830-834. 
 
12Cooper RA, Diloy-Puray M, Lando P, and Greenberg MS. An analysis of lipoproteins, bile acids 
and red cell membranes associated with target cells and spur cells in patients with liver disease. 
J Clin Investig 1972; 51:3182-3192. 

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13Miller JP. Dyslipoproteinaemia of liver disease. Balliere’s Clinical Endocrinology and 
Metabolism 1990. 4(4) 807-832. 
 
14Kaiser T, Kinny-Koster B, Bartels M, et al. Cholesterol esterification in plasma as a biomarker 
for liver function and prediction of mortality. BMC Gastroenterology 2017; 17(57):1-7.