Albumin Mass Balance in Liver Transplantation, an Open Prospective Cohort Study
Conditions: Liver Transplant; Complications, Fluid Overload
Sponsor: Ake Norberg

        
 
 
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ALBUMIN MASS BALANCE IN LIVER 
TRANSPLANTATION 
 
An open exploratory study  
 
 
 
 
 
 
 
Product: 
None 
 
Substance: 
None 
 
NCT-number: 
NCT xxxxx 
 
Karolinska University Hospital 
number: 
K2017-4774 
 
Karolinska Institutet number: 
4-3344/2017 
 
Investigator: 
Åke Norberg 
Karolinska University Hospital, Huddinge 
Perioperative Medicine and Intensive Care 
 
Study type:  
Open observational physiological study 
 
Sponsor study number: 
7 
 
Version: 
1.01 
 
Date:  
2018-02-15 
 
 
 
 
 

        
 
 
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1 TABLE OF CONTENTS 
 
1 
TABLE OF CONTENTS ....................................................................................................................2 
2 
SUMMARY ........................................................................................................................................4 
3 
ABBREVIATIONS .............................................................................................................................5 
4 
ADMINISTRATIVE INFORMATION ..................................................................................................6 
4.1 
PRIMARY INVESTIGATOR ................................................................................................................6 
4.2 
CO-INVESTIGATORS AND OTHER PARTICIPATING RESEARCHERS ......................................................6 
5 
BACKGROUND .................................................................................................................................6 
5.1 
ALBUMIN IN VOLUME RESUSCITATION .............................................................................................6 
5.2 
ALBUMIN IN LIVER TRANSPLANTATION ............................................................................................7 
5.3 
ALBUMIN MASS BALANCE CALCULATIONS ........................................................................................8 
5.4 
ALBUMIN DE NOVO SYNTHESIS .......................................................................................................8 
5.5 
MARKERS OF ENDOTHELIAL INJURY AND INFLAMMATION ..................................................................8 
6 
STUDY AIMS .....................................................................................................................................9 
6.1 
MAIN STUDY .................................................................................................................................9 
6.2 
PLANNED SUB STUDIES .................................................................................................................9 
7 
RESEARCH QUESTION ...................................................................................................................9 
7.1 
PRIMARY QUESTION ......................................................................................................................9 
7.2 
SECONDARY QUESTIONS ...............................................................................................................9 
8 
ENDPOINTS ................................................................................................................................... 10 
8.1 
PRIMARY ENDPOINT ................................................................................................................... 10 
8.2 
SECONDARY ENDPOINTS ............................................................................................................ 10 
9 
STUDY DESIGN ............................................................................................................................. 10 
10 
STUDY PROCEDURES ................................................................................................................. 11 
10.1 
SCREENING, INFORMATION AND CONSENT ................................................................................... 11 
10.2 
FIRST STUDY DAY ...................................................................................................................... 11 
10.3 
POSTOPERATIVE STUDY DAY 1-21 OR TO HOSPITAL DISCHARGE ................................................... 11 
10.4 
POSTOPERATIVE STUDY DAY 4 -21 IN ICU .................................................................................... 11 
11 
METHODS AND ANALYSIS .......................................................................................................... 12 
11.1 
ALBUMIN MASS BALANCE ............................................................................................................ 12 
11.2 
ALBUMIN CONCENTRATION ......................................................................................................... 12 
11.3 
ALBUMIN  SYNTHESIS RATE ......................................................................................................... 12 
11.4 
SAMPLE HANDLING .................................................................................................................... 12 
12 
SUBJECTS ..................................................................................................................................... 12 
12.1 
INCLUSION CRITERIA .................................................................................................................. 12 
12.2 
EXCLUSION CRITERIA ................................................................................................................. 13 
13 
CRITERIA FOR DISCONTINUATION............................................................................................ 13 
13.1 
PATIENT RELATED CRITERIA ....................................................................................................... 13 
13.2 
INVESTIGATORS CRITERIA AND EVALUABILITY .............................................................................. 13 
13.3 
SUBJECT LOG ............................................................................................................................ 13 

        
 
 
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14 
TREATMENT .................................................................................................................................. 13 
15 
ASSESSMENT OF SAFETY AND EFFICACY .............................................................................. 13 
15.1 
ASSESSMENT OF ANALYSIS RESULTS .......................................................................................... 13 
15.2 
ASSESSMENT OF PATIENT SAFETY .............................................................................................. 14 
15.3 
ASSESSMENT OF ANALYSIS RESULTS .......................................................................................... 14 
16 
MANAGEMENT AND REPORTING OF ADVERSE EVENTS ...................................................... 14 
17 
STATISTICS AND DATA MANAGEMENT .................................................................................... 14 
17.1 
DATA MANAGEMENT ................................................................................................................... 14 
17.2 
STATISTICAL ANALYSIS ............................................................................................................... 15 
17.3 
DETERMINATION OF THE NUMBER OF SUBJECTS .......................................................................... 15 
18 
ACCESS TO SOURCE DATA ....................................................................................................... 16 
19 
ETHICS ........................................................................................................................................... 16 
19.1 
ETHICAL REVIEW BOARD ............................................................................................................ 16 
19.2 
ETHICAL CONDUCT OF THE STUDY .............................................................................................. 16 
20 
DATA MANAGEMENT AND ARCHIVE......................................................................................... 16 
20.1 
CASE REPORT FORMS ................................................................................................................ 16 
20.2 
ARCHIVE ................................................................................................................................... 17 
21 
FINANCIAL SUPPORT AND INSURANCE ................................................................................... 17 
22 
PUBLICATION OF RESULTS ....................................................................................................... 17 
23 
REFERENCES ............................................................................................................................... 18 
24 
SIGNATURE ................................................................................................................................... 20 
25 
ATTACHMENTS ............................................................................................................................. 21 
25.1 
FLOW CHART ............................................................................................................................. 21 
 
 
 

        
 
 
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2 SUMMARY 
 
PROTOCOL IDENTITY AND AIM 
NCT-number 
NCT xxxxx 
Protocol title:  
Albumin mass balance in liver transplantation. 
Study aim:  
Assess the losses of albumin from circulation during and after liver 
transplantation and identify predictors of albumin loss 
METHODOLOGY 
 
Study design: 
Open explorative physiological study 
Primary research question: 
Is cumulative perioperative albumin shift from plasma positive at 
postoperative day 7? 
Effect parameter: 
Albumin mass balance  
Safety parameters: 
Vital parameters 
INVESTIGATED POPULATION 
 
Study subjects: 
Patients undergoing liver transplantation  
Number: 
240 
TIME PLAN 
 
First patient included: 
Q1 2018 
Last patient included: 
Q1 2021 
Last patient finalized: 
Q2 2021 
 
 
 
 

        
 
 
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3 ABBREVIATIONS 
 
Abbreviation 
Unit 
Explanation 
ASR 
mg/kg/day 
Absolute Synthesis Rate 
Bq 
 
Becquerel, radiation dose  
CPAS 
g 
Cumulative perioperative albumin loss 
CRF 
 
Case report form  
D5-Phe 
 
Deuterium labeled phenylalanine 
FSR 
%/day 
Fractional Synthesis Rate 
MELD 
 
Model for End-Stage Liver Disease 
MPE 
 
Mol percent excess 
POD 
 
Post operative day 
 
 
 

        
 
 
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4 ADMINISTRATIVE INFORMATION 
  
4.1 
PRIMARY INVESTIGATOR 
 
Åke Norberg, M.D., Ph.D., Ass. Professor 
Karolinska Institutet, CLINTEC, Dept of Anesthesiology 
Perioperative Medicine and Intensive Care 
Karolinska University Hospital, Huddinge B31 
141 86 Stockholm, Sweden 
Phone: 08-585 809 41 
Cell: 073-966 11 52 
Fax: 08-779 54 24 
E-mail: ake.norberg@sll.se 
 
4.2 
CO-INVESTIGATORS AND OTHER PARTICIPATING RESEARCHERS 
 
Mariam Amouzandeh, M.D. 
Perioperative Medicine and Intensive Care 
Karolinska University Hospital, Huddinge B31 
141 86 Stockholm, Sweden 
 
Greg Nowak, M.D., Ph.D. Associate Professor 
Department of Transplant Surgery 
Karolinska University Hospital, Huddinge K63 
141 86 Stockholm, Sweden 
 
Anna Januszkiewicz, M.D., Ph.D. 
Perioperative Medicine and Intensive Care 
Karolinska University Hospital, Huddinge B31 
141 86 Stockholm, Sweden  
 
Jan Wernerman, M.D., Ph.D., Professor 
Perioperative Medicine and Intensive Care 
Karolinska University Hospital, Huddinge B31 
141 86 Stockholm, Sweden 
 
Olav Rooyackers, M.Sc., Ph.D., Professor 
Perioperative Medicine and Intensive Care 
Karolinska University Hospital, Huddinge B31 
141 86 Stockholm, Sweden 
5 BACKGROUND 
5.1 
ALBUMIN IN VOLUME RESUSCITATION 
Albumin is a major constituent of plasma proteins, contributing to the colloid osmotic pressure 
in the capillaries [Kaysen 1998, Margarson 2002]. The association between massive volume 
overload and poor outcome in surgery and critical illness has contributed to the popularity of 
colloid solutions for volume resuscitation for the last 50 years. It might seem logic to use 
exogenous albumin in situations of circulatory instability, in particular when plasma albumin 
concentration is low. The background is that volume resuscitation employing a colloid like 

        
 
 
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albumin may be achieved with less than half the fluid volume as compared to isotonic 
crystalloid solutions, which has been demonstrated repeatedly in healthy subjects [Groeneveld 
2011]. In situations characterized by low blood pressure, the immediate effect upon blood 
pressure of a colloid like albumin is also very convincing in a short time perspective 
[Margarson 2002]. However, in blinded randomized studies of patients with severe sepsis or 
septic shock, the volume needed to resuscitate is not dramatically different between groups 
given a colloid based regimen as compared to a crystalloid based regimen [Annane 2013, 
Finfer 2004, Myburgh 2012, Perner 2012]. In addition, there is no consistent outcome 
advantage for the patients randomized to be given a colloid containing protocol [Delaney 
2011, Dellinger 2013, Perel 2013]. 
The explanation sought for this discrepancy between the situation in healthy subjects and 
patients with an assumed increase in capillary leakage on one hand, and the short term 
obvious effect seen on for example blood pressure after colloid administration, has been 
efforts to select the patients that are most compromised in terms of circulatory shock [Caironi 
2014]. 
Today, after the recommendation against the use of hydroxyethyl starches as a synthetic 
colloid of the European Medical Agency for safety reasons, albumin containing solutions 
remain the only alternative when the use of a colloid is preferred. This is also reflected in 
existing international guidelines that recommend considering the use of albumin containing 
solutions when resuscitation with crystalloid solutions only seems insufficient [Rhodes 2017]. 
This is despite the absence of solid evidence for this treatment strategy in long term 
outcomes. 
5.2 
ALBUMIN IN LIVER TRANSPLANTATION  
End-stage liver failure is associated with high mortality, unless treated by liver transplantation. 
Almost 100 liver transplantations are performed yearly at Karolinska University Hospital 
Huddinge. Of these, 5-10 recipients are children, and 10-20% of the patients will stay in the 
ICU for more than 3 days after the liver transplantation. Some of these patients have large 
losses in drains and in children these losses can amount to 10-30% of total body weight per 
day. There is very sparse literature or evidence for guidance in the treatment of these patients. 
At our unit, fluid losses are largely treated by infusions containing albumin, but there is little 
scientific support for this routine. A more systematic assessment of these patients would be of 
great value for future guidelines.   
In a recent pilot study on selected patients undergoing liver transplantation (EPN D-nr 2015-
1048-31/2), we found a cumulated loss of albumin until the 3rd post-operative day (POD3) of 
48 ± 33 g. There was a wide variation but in contrast to patients undergoing major abdominal 
surgery [Norberg 2016] the loss seemed to persist from end of surgery to end of study. 
Furthermore, albumin synthesis rate was recovered at POD3 in this cohort of patients. This 
pilot study demonstrated the feasibility of a concept using albumin mass balance and  
measurement of synthesis rate of liver export proteins (albumin and fibrinogen) to characterize 
albumin losses from the plasma compartment and albumin physiology. The pilot study 
excluded patients with major peroperative bleeding, re-operations and other risk factors for a 
complicated post-operative course. Therefore, in order to extend both the internal and external 
validity of our observational study, we plan to recruit consecutive patients undergoing liver 
transplantation and to prolong the albumin mass balance part of the study primarily until 
POD7, but if applicable to POD21 or hospital discharge.  
Isotopic analysis of albumin synthesis rate will be performed in the above mentioned patients 
that are in the ICU at POD4 and might be repeated after 3-10 days.   Although albumin 
synthesis increases in connection with inflammation, liver transplantation represents a special 
case in several ways. Some degree of ischemia and liver cell injury is always present after 

        
 
 
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surgery, possibly affecting synthesis capacity for some time. Bleeding and fluid shifts are 
sometimes great, and losses in drains after surgery might be profound. The use of 
immunosuppressive drugs bound to albumin makes interpretation of drug concentrations 
difficult if plasma albumin varies too much over time. In our hospital a plasma albumin of 25 
g/L is routinely used as target post-operatively.  
5.3 
ALBUMIN MASS BALANCE CALCULATIONS 
By rigorous measurement of losses and gains of albumin during and after surgery, i.e. 
assessing bleeding, losses in drains, urine etc and measuring hemoglobin and albumin in 
plasma samples and in all blood products given, it is possible to calculate the loss of albumin 
from plasma that is not accounted for by other means. This is the cumulative perioperative 
albumin shift (CPAS) that represents albumin lost from the blood by capillary leakage or 
catabolism.  
In major gynecological cancer surgery protein loss was reported as 49 g [Rehm 1998], and 
after major abdominal surgery cumulative perioperative albumin shift was 19 g (range 2-61) 
[Norberg 2015]. In liver transplantation the perioperative albumin loss was 37 ± 17 g (EPN D-
nr 2015-1048-31/2, preliminary results) at end of surgery. 
Blood sampling outside clinical routine for albumin mass balance calculations will not exceed 
40 ml during a 21 day period. For children there is no study specific blood sampling at all for 
the mass balance analysis. 
5.4 
ALBUMIN DE NOVO SYNTHESIS 
Albumin is synthesized in the liver at a rate of 150 mg/kg/day, or 13 g/24 h [Barle 2002]. This 
corresponds to a fractional synthesis rate of 6-8 %/24 h in a healthy subject with a plasma 
volume of 3 liters and 120 g of albumin in the intravascular space [Kaysen 1998]. In addition 
there is another 180 g of albumin in the interstitial space. These two compartments are in 
equilibrium by capillary leakage of 5 % per hour that is returned to the vascular space through 
the lymph flow. In states of an increased capillary leakage or circulatory instability, these 
figures may be dramatically altered [Margarson 2002]. 
Albumin synthesis rate is determined by incorporation of isotopically labeled amino acids into 
albumin [Ballmer 1990]. Today the most common technique is to use deuteriated 
phenylalanine given as a flooding dose. De novo synthesis of albumin is stimulated by food 
intake, while it is decreased in states of liver insufficiency [Caso 2007, Ballmer 1996]. 
The blood sampling volume for one measurement of albumin synthesis will be 20 ml. 
5.5 MARKERS OF ENDOTHELIAL INJURY AND INFLAMMATION  
The mechanism of albumin loss is not well uncovered but increased capillary leakage in 
connection with fluid overload and inflammation are likely to contribute. Several substances 
have been suggested as markers of endothelial injury. Commercial enzyme-linked 
immunoabsorbant assay (ELISA) kits are available for syndecan-1, hyaluron, heparansulphate 
etc. They represent components of the glycocalyx that are suggested to be flaking in 
inflammation or injury. Heparin binding protein has been suggested as a mediator of capillary 
leakage and is also measured by ELISA. Interleukins 6, 8 and 10 are early markers of 
inflammation and are likewise measured by ELISA. CRP is a too slow parameter to be of 
relevance in this context, but will be routinely measured and presented. 
The field is rapidly developing and possible new ELISAs in the field of glycocalyx function, 
injury or inflammation might be considered for this investigation.  

        
 
 
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The blood sampling volume for one measurement by ELISA will be 4 mL for adults and 2 ml 
for children. This is the only blood sampling in children. 
6 STUDY AIMS 
6.1 MAIN STUDY 
The primary aim of the main study is to measure if the cumulative perioperative albumin shift 
is normalized on POD 7 in patients undergoing liver transplantation at Karolinska Huddinge. 
 
Secondary aims are to compare the loss at POD 7 with that at EOS and POD 3, the albumin 
loss at hospital discharge, and to explore predictors of increased albumin shift from plasma.  
6.2 PLANNED SUB STUDIES 
Within this heterogeneous group of patients we plan to do subgroup analyses in some pre-
defined groups. In addition we plan to study synthesis rate of albumin, the capillary leakage 
and lymphatic return as secondary outcomes.  
Sub study A, Children < 18 years  
The aim is to find if cumulative perioperative loss of albumin from plasma is positive at EOS, 
POD3 and POD7 respectively, after liver transplantation. 
Sub study B, Adults in need of prolonged ICU stay 
The aim is to investigate if cumulative perioperative loss of albumin from plasma is different at 
EOS, POD3 and POD7, respectively, in adults in need of prolonged ICU stay compared to 
adults not in need of prolonged ICU stay after liver transplantation. 
7 RESEARCH QUESTION 
7.1 PRIMARY QUESTION  
Is cumulative perioperative loss of albumin from plasma positive at post-
operative day 7 (4-10) after liver transplantation? 
7.2 SECONDARY QUESTIONS  
1. 
Does perioperative loss on POD7 (4-10) correlate to losses at EOS and 
POD3? 
2. 
Is cumulative perioperative loss of albumin from plasma still positive at 
hospital discharge (or POD 21) following liver transplantation? 
3. 
Dichotomize patients in two equally sized groups depending on the 
amount of cumulative loss of albumin from plasma on EOS, POD3 and 
POD7 respectively, i.e. to a “leaky” and a “less leaky” group. Which factors 
predict “leakiness” (demographics, bleeding, fluid balance, ascites, 
ELISAs etc)? 

        
 
 
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4. 
Is cumulative perioperative loss of albumin from plasma positive at EOS, 
POD3 and POD7 (4-10), respectively, after liver transplantation in 
children? This is the primary question in the children substudy . 
5. 
Does perioperative loss on POD7 (4-10) correlate to losses at EOS and 
POD3 in children after liver transplantation?  
6. 
Which factors characterize children with large postoperative drain losses 
after liver transplantation? 
7. 
Does cumulative perioperative loss of albumin from plasma at EOS, POD3 
and POD7 (4-10), respectively, differ between adults in need or not in 
need of prolonged ICU stay, respectively, after liver transplantation? This 
is the primary question in the prolonged ICU substudy. 
8. 
Which factors predict “need of prolonged ICU stay” after liver 
transplantation (demographics, bleeding, fluid balance, ascites etc)? 
9. 
How does synthesis rate of albumin develop during prolonged ICU stay?   
8 ENDPOINTS 
8.1 PRIMARY ENDPOINT 
Cumulative perioperative albumin shift (CPAS) by mass balance at POD 7 (4-
10). 
8.2 SECONDARY ENDPOINTS  
A.  
Cumulated albumin mass balance at EOS, POD 3, and at hospital 
discharge (POD21) 
B. 
Indices of inflammation and endothelial dysfunction (such as IL6, 
syndecan, HBP)  
C. 
Albumin synthesis rate on POD 4-6 and on POD 7-14 after liver 
transplantation in adult patients (>18 years) in need of a prolonged ICU 
stay. 
 
9 STUDY DESIGN  
The study is a prospective exploratory study of patient physiologic parameters during and after 
liver transplantation.  
Albumin mass balance is measured during liver transplantation and up to hospital discharge or 
21 days after end of surgery, by keeping close track of gains and losses (all subjects). Albumin 
synthesis is measured by incorporation of 5D-phenylalanine during 90 minutes (adults).  
Samples for ELISA will be obtained from all patient groups.  

        
 
 
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All other factors are routinely measured and calculated within the liver transplantation protocol.  
10 STUDY PROCEDURES 
10.1 SCREENING, INFORMATION AND CONSENT 
All patients accepted for liver transplantation will be approached when accepted for the 
operation. They will then be informed orally and in writing about the study and the study-
specific procedures by the investigator or co-investigator. Preliminary oral informed consent 
will be noted in the patient record. The written informed consent will be obtained from the 
patient (or parent) in connection with the liver transplantation. Preliminary recruitment will likely 
outnumber the anticipated number or patients that are actually studied due to the emergency 
nature of the procedure, and the very variable time from acceptance to the operation. Albumin 
mass balance will start in the operating room and continue until hospital discharge or POD21.  
Three different versions of the study information will be used.  
Adults can participate in all measurements  
Children can only participate in albumin mass balance and ELISAs, and parents and children 
over 12 years will have two different study informations. Children below the age of twelve will 
only be represented by their parents. 
10.2 FIRST STUDY DAY 
The procedure will be an emergency, except for living donors which occurs mainly in children. 
The patients arrive at the operating room at Perioperative Medicine and Intensive Care at 
Karolinska University Hospital Huddinge. Venous and arterial lines will be routinely inserted in 
connection with anesthesia and used for blood sampling. Mass balance of albumin will be 
achieved by keeping track of all losses (bleeding and drains) and infusions containing albumin 
or hemoglobin during and after the surgical procedure and repeated sampling of P-albumin. 
Mass balance sampling and calculation will continue after the surgical procedure.  
In children there is no study specific blood sampling during the surgical procedure. Samples 
will only be taken from suction bottles, drains, urine, and administrated blood products. 
10.3 POSTOPERATIVE STUDY DAY 1-21 OR TO HOSPITAL DISCHARGE 
Mass balance sampling and calculation will continue up to hospital discharge or at most for 21 
days after the surgical procedure. POD 1 ELISAs will be taken in all study patients. POD 4 
also in adults not in the ICU. 
10.4 POSTOPERATIVE STUDY DAY 4 -21 IN ICU 
If an adult patient is in the ICU after POD4 measurement of albumin synthesis rate will be 
performed by the flooding method. The exact time point will be to the discretion of an 
investigator. In the semi-recumbent position measurements of albumin synthesis rate (11.3) is 
performed over 90 minutes. Also a blood sample for ELISAs will be taken. The procedure can 
be repeated once or twice for measurement of albumin synthesis rate but only within the 
permitted total sampling blood volume (100 ml). 
Children in ICU after POD 4 can be subject to ELISAs (2+2 ml).   

        
 
 
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11 METHODS AND ANALYSIS 
11.1 ALBUMIN MASS BALANCE 
During preparations before surgery and during the surgical procedure blood samples for 
albumin concentration determination will be taken at the same time intervals as routine blood 
gases. Thereafter sampling interval will be 1-2 times per day. In parallel all fluid losses will be 
collected, measured and documented, sampled and saved for analysis. Also, infusions 
containing albumin or hemoglobin will be monitored. 
11.2 ALBUMIN CONCENTRATION 
Albumin concentrations in plasma will be measured employing nephelometry at Studiecenter 
at Karolinska University Hospital.  Albumin in other fluids (X-alb) will be analyzed at Karolinska 
Stable Isotope Core Facility at KFC Novum. 
11.3 ALBUMIN  SYNTHESIS RATE 
To measure albumin synthesis rate an infusion of D5-phenylalanine 20 mg/mL10 MPE, 0.45 
g/kg will be given over 10 minutes. A 90-minute blood sampling protocol for determination of 
the ratio D5-phenylalanine/phenylalanine and the incorporation of D5-phenylalanine into 
albumin will be applied as illustrated in Figure 1. Analysis is performed by 
gaschromatography-masspectrometry (GC-MS) at Karolinska Stable Isotope Core Facility at 
KFC Novum. The fractional synthesis rate (FSR) is expressed as %/24 hours of albumin in 
plasma, while the absolute synthesis rate (ASR) expressed as mg/kg/24 hours, can be 
calculated if also plasma volume (from anthropometry) and plasma albumin are known.  
 
0
80
70
B lo o d  s a m p lin g  fo r 5d -P h e , 5d -P he -A lb um in and 5d -P h e -fib rin o g e n
a t th e  a rro w s
10
20
30
50
60
40
90
  
11.4 SAMPLE HANDLING 
Samples for GC-MS and ELISA analysis are anonymized and kept in Stockholms Medicinska 
Biobank reg nr 914 at Perioperative Medicine and Intensive Care at Karolinska University 
Hospital Huddinge, at -80oC pending finalization of study to be analyzed in one sequence to 
optimize precision in accord with our local routines. Samples for albumin analysis will likewise 
be marked in a non-identifiable way and kept in the biobank at -20oC pending finalization of 
study to be analyzed in one sequence to optimize precision in accord with our local routines.  
 
Dr Åke Norberg will be the responsible person for sample handling, anonymization and 
destruction after publication. The responsible organization for personal data is Stockholm City 
Council, Box 22550, 104 22 Stockholm.  
12 SUBJECTS  
12.1 INCLUSION CRITERIA 
Patients undergoing liver transplantation: adult males and females, and children 0-17 
years. 

        
 
 
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Written informed consent. In children below 12 years consent is only obtained from the 
parents.  
A. 
Adults (mass balance, albumin synthesis and ELISAs) 
B. 
Children (only assessment of losses and gains, no blood sampling except for 
ELISAs) 
12.2 EXCLUSION CRITERIA   
Circumstance that makes the investigator judge patient participation unsuitable, mainly 
inability to understand study information (language difficulties or encephalopathy) 
13 CRITERIA FOR DISCONTINUATION 
13.1 PATIENT RELATED CRITERIA   
• 
The patient may, at any time during the study, chose to end his/hers further 
participation in the study, without giving any cause.  
If possible, the reason for discontinuation shall be stated in the CRF with date and time. 
13.2 INVESTIGATORS CRITERIA AND EVALUABILITY  
The primary investigator can determine that the patient cannot stay in the study. This can be 
founded on lack of evaluability or the reasons stated below:  
• 
Errors in technical equipment 
• 
Staff shortage that makes safe and standardized methods impossible.  
The reason for discontinuation shall be stated in the CRF with date and time.   
13.3 SUBJECT LOG 
The investigator will keep a record of all eligible patients on a log list. The log list is stored in 
the investigator brochure available Perioperative Medicine and Intensive Care at Karolinska 
University Hospital Huddinge, in a locked room. It will show all screened subjects, included 
and not included.  
14 TREATMENT 
There is no study specific treatment. Study tracers are only used to monitor body physiology, 
not to treat the patient. 
15 ASSESSMENT OF SAFETY AND EFFICACY 
The patients are monitored according to local routines at Karolinska University Hospital 
Huddinge. No external monitoring related to this study is planned.  
15.1 ASSESSMENT OF ANALYSIS RESULTS 

        
 
 
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Laboratory will be analyzed at the ISO certified Karolinska University Laboratory, Huddinge. 
Blood gases are analyzed at the Department of Anesthesiology. P-albumin will be analyzed by 
immunochemical method through the Studiecenter at the Karolinska University Laboratory, 
Solna. X-albumin and ELISAs will be performed at Karolinska Stable Isotope Core Facility at 
KFC Novum. 
15.2 ASSESSMENT OF PATIENT SAFETY 
Patients will be monitored in the operating room, at the intensive care unit and at the 
transplant surgical ward, respectively, for the mass balance sampling. Stable isotopes will be 
administered in the ICU or in the transplant surgical ward by a staff that is familiar with 
emergency incidents. Monitoring of the patient's heart rate, ECG, respiratory rate, oxygen 
saturation (oximetry), and blood pressure are taken before and during the first 90 min after 
administration. 
15.3 ASSESSMENT OF ANALYSIS RESULTS 
Laboratory samples, taken during and after surgery, are analyzed at the ISO certified 
Karolinska University Laboratory, Huddinge. Blood gases are analyzed by analyzers at the 
Department of Anesthesiology and Intensive Care Unit, respectively. P-albumin will be 
analyzed by immunochemical method through the Studiecenter at the Karolinska University 
Laboratory, Solna. ELISAs and X-albumin (blood products, drains etc) will be analyzed at 
Karolinska Stable Isotope Core Facility at KFC Novum. Other samples are analyzed as 
described in paragraph 11. 
16 MANAGEMENT AND REPORTING OF ADVERSE EVENTS 
All patients undergoing a liver transplantation will in the perioperative course experience 
incidents that would under other circumstances be described as AE/SAE. Major surgery in 
itself is associated with hospitalization. Therefore expected effects of anesthesia and surgery 
will not be reported, even if they may be considered undesirable. Such effects do not relate to 
the planned measurement procedures and they are not controlled by the investigator. Given 
the observational character of the study However, events that may influence the results of the 
study or whether subjects are evaluable or not will be noted in the CRF. 
17 STATISTICS AND DATA MANAGEMENT 
17.1 DATA MANAGEMENT 
All laboratory values will be printed from the patient record (Take Care, TC) de-identified and 
attached to the CRF. All values of vital parameters during in connection with tracer 
administration will be documented directly on a coded anesthesia paper record by the 
research nurse or the investigator. This journal is a part of the CRF. Other documents that are 
stored in the CRF for coding and de-identification are: medical record from the Department of 
Transplant Surgery, medication list from TC, medication list from CliniSoft (the medical record 
system at the Intensive Care Unit).  
Data from the mass spectrometer measurements of phenylalanine will be exported as a data 
sheets via a USB stick. 

        
 
 
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All the data regarding end-points will be added in an Access database that is locked when 
data collection is complete. 
De-identified and coded patient demographics will be prepared and included in the Access 
database. 
17.2 STATISTICAL ANALYSIS 
In a previous study on selected patients undergoing liver transplantation at our department, we 
found a net leakage of albumin of 37 ± 17 g at end of surgery and of 48 ± 33 g at the third 
postoperative day. In our hospital, post-operative albumin infusions to keep plasma albumin at 
25 g/l are very common, suggestive of ongoing losses in this patient group.  
The effect and time course of ischemic liver injury on albumin synthesis has not been 
investigated, but in the previous pilot study albumin synthesis was higher than normal values 
on post-operative day 3. All data will be presented as mean with standard deviation (SD) or as 
median and range of data if not normally distributed. 
Furthermore, the mutual correlations between the mass balance albumin parameters and 
albumin synthesis and P-albumin will be calculated and presented with correlation coefficients. 
Patients will be dichotomized to “leaky” and “not so leaky”, and possible predictors analyzed 
by univariate and multivariate logistic regression. 
With 103 subjects there is a power of 80% to detect an anticipated effect size of 0.11 with 3 
predictors and a 2-sided significance level of 5%. This equals the situation where the 3rd 
predictor gives an increase in the squared correlation coefficient r2 from 0.30 to 0.37 or from 
0.50 to 0.54. Although this is rather hypothetical it seems likely that such inferences might 
occur, and we will find them if 2 years production of liver transplants are investigated. If only 2 
predictors are used an effect size of 0.095 can be found with 80% power. 
Correlations will be reported as Pearson’s correlation coefficient r or Spearman's rank 
correlation rs depending on the data's nature. 
Demographic preoperative data such as age, height, weight, BMI, gender, routine lab, history 
of weight loss, diagnoses, and current medications will be presented as numbers, averages 
and standard deviation, or as median range as applicable.  
17.3 DETERMINATION OF THE NUMBER OF SUBJECTS 
On page 4 we state that the number of subjects is 240 in the main study. This refers to an 
overestimation of the total number of liver transplantations during a approximatively 2 year 
period at our hospital and the primary outcome of albumin mass balance.  
The primary endpoint of the study (cumulative perioperative albumin shift at POD7) has never 
been investigated before. On POD3 48 ± 33 g, p=0.0017 versus baseline was found in our 
previous study on selected patients undergoing liver transplantation [Amouzandeh 2017]. This 
corresponds to a effect size of 1.4. Although data in consecutive patients are likely to be even 
more scattered a power of 90% is reached with n=100 for an effect size of 0.33 with a one 
sample 2-sided t-test and a significance level of 5%.  
We plan to do linear regression with the thus detected “leakiness” as dependent variable and 
look at possible predictors. We will also dichotomize the patients in “leaky” and “not so leaky” 
(cut off to be estimated by ROC-curve analysis) and explore possible predictors by logistic 
regression and multiple logistic regression. The size of the data base becomes important to 
the quality of the multiple regression procedure, but data quantity will be governed by our 
patient flow and research resources. Data collection over much more than 2 years is likely to 
be confounded by changes in routines over time. 

        
 
 
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The large number of correlations mentioned above must be seen against the background that 
this is an exploratory pilot study of albumin mass balance and albumin kinetic parameters in 
liver transplantation. Because this is an unselected patient group for this kind of 
measurements, there is an intention that the study also should be hypothesis generating. 
Sub study A (children, n=20) is included in the above mentioned number.  
Sub study B (prolonged ICU stay, n=20) is a cohort included in the main study above.  
The expected numbers in substudies A and B are the main reason to extend the whole study 
to approximately 2 years, and are based on historical records. Although data are likely to be 
scattered, with n=10-20 there is a reasonable chance to perform statistical inference analysis. 
The groups will also be compared with the complete data set.  
 
 
18 ACCESS TO SOURCE DATA 
The primary investigator is responsible that there is a confidentiality agreement between a 
monitor or any other independent reviewer (i.e. auditor) given permission to verify the data in 
the patient record and the patient record responsible party at Perioperative Medicine and 
Intensive Care, Karolinska Huddinge. This requires the patient's written consent. 
19 ETHICS 
19.1 ETHICAL REVIEW BOARD 
The investigator is responsible for the application for approval from the Ethics Review Board.  
. 
19.2 ETHICAL CONDUCT OF THE STUDY 
The study will be conducted according to the study protocol, GCP, regulatory requirements 
and the Helsinki Declaration. 
Our practice is to not suggest a blood sampling protocol to patients that exceeds 100 
mLunless the sampling period is prolonged. 
 
20 DATA MANAGEMENT AND ARCHIVE 
20.1 CASE REPORT FORMS 
Data collected in the study will be recorded in Case Report Forms (CRF) in which the patient 
is only identified with a code number and initials. The code key kept by the investigator in such 
a way that unauthorized persons cannot take part of it. Anonymous and coded copies of 
medical records such as anesthesia records, monitoring sheet for post-operative care, journal 
text (Take Care) and laboratory lists (Take Care) will also be included in the CRF. 

        
 
 
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Data collected in the study and registered in the CRF will be transferred to a database that the 
sponsor is responsible of. 
20.2 ARCHIVE 
Study documents will be filed at least 10 years after the study report is submitted to the 
Swedish MPA. The documents should be archived in a legible condition for eventual future 
audit or inspection by authorities. 
21 FINANCIAL SUPPORT AND INSURANCE 
The study is funded by research grants. The patients are insured through medical insurance 
and patient injury insurance. 
22 PUBLICATION OF RESULTS 
The intention is to publish the results in a scientific journal. The main study and the two 
planned sub studies will be published separately. 
 
 
 

        
 
 
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23 REFERENCES 
Annane D, Sebille V, Charpentier C, et al. (2013) Effects of fluid resuscitation with colloids vs 
crystalloids on mortality in critically ill patients presenting with hypovolemic shock: the CRISTAL 
randomized trial. JAMA, 310: 1809-17 
Ballmer PE, McNurlan MA, Milne E, et al. (1990). Measurement of albumin synthesis in humans: a new 
approach employing stable isotopes. Am J Physiol E797-803 
Ballmer PE, Reichen J, McNurlan MA, et al. (1996). Albumin but not fibrinogen synthesis correlates with 
galactose elimination capacity in patients with cirrhosis of the liver. Hepatology 24: 53-9 
Barle H, Januszkiewicz A, Hållström L, et al. (2002). Albumin synthesis in humans increases 
immediately following the administration of endotoxin. Clin Sci 103: 525-31 
Caironi P, Tognoni G, Masson S, et al (2014). Albumin replacement in severe sepsis or septic shock. N 
Engl J Med, 370: 1412-21 
Caso G, Feiner J, Mileva I, et al. (2007) Response of albumin synthesis to oral nutrients in young and 
elderly subjects. Am J Clin Nutr 85: 446-51 
Delaney AP, Dan A, McCaffrey J, et al. (2011) The role of albumin as a resuscitation fluid for patients 
with sepsis: a systematic review and meta-analysis. Crit Care Med 39: 386-91 
Dellinger RP, Levy MM, Rhodes A, et al. (2013) Surviving Sepsis Campaign: international guidelines for 
management of severe sepsis and septic shock, 2012. Intensive Care Med 39: 165-228 
Finfer S, Bellomo R, Boyce N, et al. (2004) A comparison of albumin and saline for fluid resuscitation in 
the intensive care unit. N Engl J Med 350: 2247-56 
Groeneveld AB, Navickis RJ, Wilkes MM. (2011) Update on the comparative safety of colloids: a 
systematic review of clinical studies. Ann Surg 253: 470-83 
Kaysen GA. (1998) Albumin turnover in renal disease. Mineral Electrolyte Metab 24: 55-63 
Margarson MP, Soni NC. (2002) Effects of albumin supplementation on microvascular permeability in 
septic patients. J Appl Physiol 92: 2139-45 
Myburgh JA, Finfer S, Bellomo R, et al. (2012) Hydroxyethyl starch or saline for fluid resuscitation in 
intensive care. N Engl J Med 367: 1901-11 
Norberg Å, Rooyackers O, Segersvärd R, et al. (2015) Albumin kinetics in patients undergoing major 
abdominal surgery. PLoS One 10: e0136371 
Norberg Å, Segersvärd R, Rooyackers O, et al (2016). Leakage of albumin in major abdominal surgery.  
Crit Care 20: 113  
 Parving HH, Ranek L, Lassen NA. (1977). Increased transcapillary escape rate of albumin in patients 
with cirrhosis of the liver. Scand J Clin Lab Invest 37: 643-8 
Perel P, Roberts I, Ker K. (2013) Colloids versus crystalloids for fluid resuscitation in critically ill 
patients. Cochrane Database Syst Rev, 2: CD000567 
Perner A, Haase N, Guttormsen AB, et al. (2012) Hydroxyethyl starch 130/0.42 versus Ringer's acetate 
in severe sepsis. N Engl J Med 367: 124-34 
Rehm M, Haller M, Brechtelsbauer H, et al. (1998) Extra protein loss not caused by surgical bleeding in 
patients with ovarian cancer. Acta Anaesthesiol Scand 42: 39-46 
Rhodes A, Evans LE, Alhazzani W, et al (2017) Surviving Sepsis Campaign: International Guidelines for 
Management of Sepsis and Septic Shock: 2016. Crit Care Med 45: 486-552 

        
 
 
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Salo J, Gines A, Gines P, et al. (1997). Effect of therapeutic paracentesis on plasma volume and 
transvascular escape rate of albumin in patients with cirrhosis. J Hepatol 27: 645-53 
 
 
 

        
 
 
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24 SIGNATURE 
This page should be signed by the sponsor and the primary investigator. 
 
Primary Investigator and sponsor 
Åke Norberg, MD, PhD, Associate Professor 
Dept. of Perioperative Medicine and Intensive Care  
Karolinska University Hospital Huddinge 
 
 
 
 
Signature 
 
Date 
 
 
 
 
 
 
 

        
 
 
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25 ATTACHMENTS 
25.1 FLOW CHART 
 
 
 
 
RECRUITMENT INFO
RECRUITMENT ORAL CONSENT
DAY OF SURGERY
POD 1-21
POD 4-21 in ICU
Written patient information
X
 
 
Inclusion/exclusion criteria
X
X
Oral study information
X
 
 
Written consent
X
Diseaseses, medication, demographic data
X
Blood sampling: P-Albumin and mass balance
X
X
Blood sampling ROUTINE
X
X
Measurement of albumin synthesis
X
Sampling for ELISA
 
X
X