Albumin Catabolic Rate Measured by a Stable Isotope
Phase: NA
Conditions: Liver Disease Chronic
Sponsor: Ake Norberg

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Albumin catabolic rate measured by 
a stable isotope  
 
 
 
Primary Investigator 
Åke Norberg  
Karolinska University Hospital, Huddinge 
Perioperative Medicine and Intensive Care 
 
Study Design 
An open physiologic study of healthy subjects and 
patients with liver cirrhosis 
 
Karolinska Institutet  
Study number 
4-2885/2022 
 
 
Karolinska University Hospital 
Study number 
K2022-10941 
 
Clinicaltrials.gov number  
NCT xxxxxxxx  
 
Version 
1.01 
 
Date 
2023-07-11 
 
 
 
 

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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 
SPONSOR AND PRIMARY INVESTIGATOR .............................................................................. 6 
4.2 
CO-INVESTIGATORS AND OTHER PARTICIPATING RESEARCHERS .................................... 6 
4.3 
MONITORING ............................................................................................................................ 6 
5 
BACKGROUND ............................................................................................................................... 7 
5.1 
ALBUMIN AND PHENYLALANINE - BACKGROUND .............................................................. 7 
5.2 
ALBUMIN – MEASUREMENT OF CATABOLIC RATE .............................................................. 7 
5.3 
ALBUMIN –MEASUREMENT OF SYNTHESIS .......................................................................... 8 
5.4 
OXIDATION OF ALBUMIN ........................................................................................................ 8 
6 
STUDY AIMS .................................................................................................................................... 8 
7 
RESEARCH QUESTION .................................................................................................................. 9 
8 
ENDPOINTS ..................................................................................................................................... 9 
9 
STUDY DESIGN ............................................................................................................................... 9 
10 
STUDY PROCEDURES ................................................................................................................... 9 
10.1 
INFORMED CONSENT ............................................................................................................... 9 
10.2 
DAY 0. MEASUREMENT OF FRACTIONAL SYNTHESIS RATE .............................................. 10 
10.3 
DAY 7, 14, 21, 28, 42, 56, 70, AND 84. MEASUREMENT OF FRACTIONAL CATABOLIC RATE.
 10 
11 
METHODS AND ANALYSIS .......................................................................................................... 10 
11.1 
MEASUREMENT METHODS ................................................................................................... 10 
11.2 
SAMPLE HANDLING ............................................................................................................... 10 
12 
SUBJECTS .................................................................................................................................... 11 
12.1 
INCLUSION CRITERIA, HEALTHY VOLUNTEERS ................................................................. 11 
12.2 
INCLUSION CRITERIA, PATIENTS WITH LIVER CIRRHOSIS ................................................ 11 
12.3 
EXCLUSION CRITERIA ........................................................................................................... 11 
13 
CRITERIA FOR DISCONTINUATION ............................................................................................ 11 
13.1 
PATIENT RELATED CRITERIA ............................................................................................... 11 
13.2 
INVESTIGATOR’S CRITERIA AND EVALUABILITY .............................................................. 11 
13.3 
SUBJECT LOG .......................................................................................................................... 11 
14 
ASSESSMENT OF SAFETY AND EFFICACY .............................................................................. 12 
15 
STATISTICS AND DATA MAMAGEMENT ................................................................................... 12 
15.1 
DATA MANAGEMENT ............................................................................................................ 12 
15.2 
STATISTICAL ANALYSIS ........................................................................................................ 12 
15.3 
DETERMINATION OF THE NUMBER OF SUBJECTS .............................................................. 12 
16 
ACCESS TO SOURCE DATA ....................................................................................................... 13 
17 
QUALITY CONTROL ..................................................................................................................... 13 
17.1 
MONITORERING ..................................................................................................................... 13 
18 
ETHICS .......................................................................................................................................... 13 

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19 
DATA MAMAGEMENT AND ARCHIVE ........................................................................................ 13 
19.1 
CASE REPORT FORMS ............................................................................................................ 13 
19.2 
ARCHIVE ................................................................................................................................. 13 
20 
FINANCIAL SUPPORT AND INSURANCE ................................................................................... 14 
21 
PUBLICATION OF RESULTS ....................................................................................................... 14 
22 
REFERENCES ............................................................................................................................... 14 
23 
SIGNATURE ................................................................................................................................... 14 
 
 
 
 

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2 SUMMARY 
PROTOCOL IDENTITY AND AIM 
Protocol title 
Albumin catabolic rate measured by a stable isotope. 
Study aim 
1. Measurement of albumin catabolic rate by an oral non-radioactive 
tracer, D5-Phe in a group of research volunteers and a group of 
patients with known liver cirrhosis. Comparison of the catabolic rate 
between the groups, and with historical controls measured by 
radioiodine-labeled albumin. 
 
2a. Evaluate how few and which measurement points are sufficient 
for use in clinical practice for the D5-Phe method while maintaining 
precision in the estimation of albumin half-life. 
 
2b. Measurement of the fractional synthesis rate of albumin with the 
oral non-radioactive tracer, D5-Phe. 
 
2c. Modeling of albumin degradation rate using oxidized albumin, i.e. 
two isoforms of mercaptoalbumin HNA-1 and HNA-2 and their 
change over time expressed as the fraction labeled with D5-Phe 
 
2d. Correlate the amount of oxidized albumin with the albumin 
catabolic rate. 
 
METHODOLOGY 
Study design 
Open explorative physiologic study 
Primary research question 
Does the albumin catabolic rate measured by the D5-Phe method 
differ between volunteers and patients with liver cirrhosis, and how 
does the catabolic rate measured by the new method compare to 
historical measurements with radioiodine-labeled albumin? 
Effect parameters 
Albumin fractional catabolic rate, albumin fractional synthesis rate, 
plasma albumin, changes in oxidized d5-phe-albumin over time. 
Safety parameters 
Vital parameters 
 
 
INVESTIGATED POPULATION 
Subjects 
Healthy volunteers, n=12 
 
Patients with known cirrhosis of the liver, n = 12 
 
TIME PLAN 
First subject included 
September 2023 
Last patient included 
September 2028 
Last patient completed 
December 2028 
 
 
 

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3 ABBREVIATIONS 
  
Abbreviation 
Unit 
Explanation 
D5-Phe 
 
Deuterium labeled phenylalanine 
ACR 
mg/kg/d 
Absolute catabolic rate 
ASR 
mg/kg/d 
Absolute synthesis rate 
AUC 
 
Area under the curve of the time-concentration plot 
CRF 
 
Case Report Form 
FCR 
% per day 
Fractional catabolic rate 
FSR 
% per day 
Fractional synthesis rate 
GCP 
 
Good Clinical Practice 
GDPR 
 
General Data Protection Regulation 
HMA 
 
Human merkaptoalbumin (normal form, unharmed 
molecule) 
HNA-1 
 
Human non-merkaptoalbumin 1 (oxidized form, 
reversible) 
HNA-2 
 
Human non-merkaptoalbumin 2 (oxidized irreversible) 
kDa 
u 
kilo Dalton indicated the size of a molecule  
MPE 
% 
Mol percent excess 
PV 
L 
Plasma volume 
SEK 
 
Swedish currency 
TC 
 
Take Care, the hospital record system 
 
 
 
 
 

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4 ADMINISTRATIVE INFORMATION 
  
4.1 SPONSOR AND PRIMARY INVESTIGATOR 
Åke Norberg, M.D., Ph.D., Ass Professor 
Karolinska Institutet, CLINTEC 
Perioperative Medicine and Intensive Care 
Karolinska University Hospital, Huddinge B31 
141 86 Stockholm 
Tel: 08-123 800 00 
Fax: 08-779 54 24 
E-mail: ake.norberg@regionstockholm.se  
 
4.2 CO-INVESTIGATORS AND OTHER PARTICIPATING RESEARCHERS 
 
Olav Rooyackers, Ph.D., Professor 
Karolinska Institutet, CLINTEC 
Perioperative Medicine and Intensive Care 
Karolinska University Hospital, Huddinge B31 
141 86 Stockholm 
 
Andreas Komáromi, Senior Consultant 
Karolinska Institutet, CLINTEC 
Perioperative Medicine and Intensive Care 
Karolinska University Hospital, Huddinge B31 
141 86 Stockholm 
 
Staffan Wahlin, M.D. Ph.D. Ass Professor 
Karolinska Institutet, Department of Medicine 
ME Upper Abdomen 
Karolinska University Hospital, Huddinge 
141 86 Stockholm 
 
4.3 MONITORING 
Clinical Research Unit, KFE, Solna 
Contact: Johan Mårtensson 
Perioperative Medicine and Intensive Care 
Karolinska University Hospital, Solna  
171 76 Stockholm 
 
 

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5 BACKGROUND 
5.1 ALBUMIN AND PHENYLALANINE - BACKGROUND  
Albumin is a medium-sized molecule, 69 kDa, and constitutes the dominant protein in plasma (40 g/liter 
out of a total of 70 g/liter). According to the literature, albumin is catabolized with a half-life of 14–20 
days, and it is synthesized in the liver. The synthesis takes place as prealbumin whose tertiary structure 
is configured for export from the liver. The process from start of synthesis to export takes about 30 
minutes. Albumin has several physiological functions, constitutes 70% of the oncotic pressure in 
plasma, functions as a transport protein and as an endogenous scavenger (binds and neutralizes free 
acid radicals). Lack of albumin can be acute or chronic. In acute deficiency, edema often occurs. 
Low plasma albumin appears in liver disease, kidney disease, sepsis, malnutrition, and other situations. 
It is a predictor of shorter life expectancy and of complications associated with major surgery. Low 
levels can be an expression of low synthesis, as in liver disease, but in some cases, synthesis is 
increased despite low values in plasma, as in nephrosis or critical illness in intensive care. Measuring 
the turnover rate of albumin and other proteins can be of great value to better understand various 
diseases and their mechanisms. 
Degradation of albumin is controlled to some extent from the liver, which means that patients with 
severe liver disease can compensate for their reduced synthesis ability by saving on the existing 
albumin molecules [Levitt 2016]. However, the literature is quite limited due to the difficulties associated 
with radioactive isotope studies, and there is a great need for new methods that can be less invasive or 
avoid radiation. 
Phenylalanine is an essential amino acid which, due to its low free concentration in plasma and 
intracellularly, is particularly suitable for stable isotope studies of protein turnover. It can be labeled by 
deuterium, often five atoms or more, and thus different from naturally occurring phenylalanine. 
Phenylalanine is suitable as a tracer for measuring the degradation rate of proteins as it is found in 
large quantities in various proteins, can be detected with gas chromatography and mass spectrometry 
and has many exchangeable hydrogen atoms, which enables high analytical sensitivity. We know of 
only one report in the literature that used stable isotopes for endogenous labeling of proteins, but no 
individual proteins were assessed in the way we intend to do [Holm 2013]. 
5.2 ALBUMIN – MEASUREMENT OF CATABOLIC RATE  
The gold standard for measurement of albumin catabolic rate is by radioiodine labeled human serum 
albumin. 
The new idea to be evaluated in this study is to use phenylalanine (D5-Phe) orally, to measure the 
breakdown rate of albumin. The amino acid is incorporated into the albumin molecules during their new 
synthesis, and one should then achieve 4 advantages compared to the method that uses radioiodine-
labeled albumin: 
1) Avoiding radiation, and the costs and extensive bureaucracy that surrounds even the use of very low 
doses of radioactive tracers. 
2) The tracer cannot detach from the albumin molecule as it forms an integral part of the amino acids in 
the albumin molecule. 
3) It is difficult to see any influence of the labeling itself which could shorten the lifespan of the albumin 
molecule. 
4) If this works well for albumin, then one could measure other proteins in the body with slow turnover in 
the same way, which would be a valuable new method. 
To be able to transfer the study results to the clinic, it would be good if the number of sampling 
occasions could be reduced. This will be studied based on the data we receive from the group of 
healthy volunteers. In a second step, we will examine patients with known liver cirrhosis, but then with a 
much sparser sampling. Apart from day 0 when the stable isotope is ingested, we expect that 2-4 more 
sampling occasions may be sufficient. 
 
 

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5.3 ALBUMIN –MEASUREMENT OF SYNTHESIS  
The synthesis rate of albumin can be calculated after intravenous administration of isotopically labeled 
amino acid, i.e. phenylalanine labeled with 5 deuterium atoms (D5-Phe). The appearance over time of 
labeled amino acid in newly synthesized albumin is measured [Ballmer 1990]. Estimation of the 
precursor pool in hepatocytes is necessary for calculation of synthesis rate. Liver biopsy in a research 
context is usually unacceptable because invasiveness, so therefore the "flooding dose" technique has 
been developed. The method is based on the idea that all pools in the body are flooded by a relatively 
high dose of intravenous phenylalanine so that the plasma levels thus give a reasonable figure of the 
precursor pool, i.e. the levels inside the hepatocytes. 
A second idea of this study is to give an oral "flooding dose" and measure synthesis rate. The 
assumption that plasma levels of phenylalanine reflect the precursor pool in the hepatocytes with oral 
tracer has not been validated, but in this first step we want to see if we get the same steep rise of D5-
Phe-albumin as with intravenous tracer and if the plasma levels of D5-Phe harmonize with a reasonable 
precursor pool. This part of the study is exploratory, but if the results are promising, we can proceed 
with validation. 
At steady state, the rate of synthesis must be equal to the rate of degradation, but there are sources of 
error and variations over time that deserve to be elucidated and may be of great value in the planning of 
future studies of albumin turnover.  
5.4 OXIDATION OF ALBUMIN  
The albumin molecule binds to free acid radicals (scavenger function) with its sulfhydryl group in the 
Cys-34 position (human mercaptoalbumin HMA). During oxidative stress, the sulfhydryl group is 
oxidized to sulfinic acid (human non-mercaptoalbumin 1, HNA 1). This is a reversible process. Upon 
continued oxidation, sulfonic acid is formed, which appears to be irreversible (human non-
mercaptoalbumin 2, HNA2). The result is a damaged albumin molecule that has lost its scavenger 
function. Oxidative stress with high levels of HNA2 may have a decisive role in the pathogenesis of 
severe liver failure [Oettl 2013]. 
There is no data in the literature regarding changes in oxidized albumin over time. We want to try to 
model albumin turnover by looking at how these fractions and the proportions between them change 
over time. We also want to see if we can reproduce previous results suggesting that the amount of 
oxidized albumin is higher in patients with liver cirrhosis [Oettl 2013]. Finally, we will investigate the 
association between albumin oxidation and albumin catabolic rate. 
6 STUDY AIMS 
The primary aim of the study is to compare the albumin degradation rate in healthy volunteers and in a 
group of patients with liver cirrhosis. We will also compare the obtained values with historical controls 
measured by radioiodine-labeled albumin, both in our own data from previous studies and in the 
literature. 
The goal is partly to get a more accurate measurement, partly to get rid of the use of radioactive tracers 
in future metabolic studies. 
There are 3 secondary objectives: 
a) For the method to be clinically useful, the number of measurement points must be reduced. We will 
determine which measuring points could optimally be used in clinical practice to still be able to estimate 
the albumin degradation rate with reasonable accuracy with the peroral D5-Phe-albumin method. Of 
special interest is to determine the earliest time point when the tracer in the precursor pool is negligible 
and all albumin compartments are well stirred, i.e. when you can take the first sample after dosing and 
be sure that it is on the terminal slope. This can be examined by looking at residuals in the terminal 
slope of the graph that presents enrichment of D5-Phe over time. After intravenous injection of 
radioiodine-labeled albumin, we have previously seen that one should wait 2 weeks, but it is possible 
that an even longer time is needed when using D5-phenylalanine orally. 
b) As a spin-off pilot study of the above, we want to see if an oral flooding dose of D5-Phe can be used 
to measure albumin synthesis, compared to our previous measurements with intravenous tracer. That 
would be a great methodological gain. 

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c) As a second spin-off, we will measure D5-Phe oxidized albumin and see if the rate of albumin 
degradation can be modeled from these early metabolites. We will also investigate the association 
between albumin catabolic rate and the oxidized forms of albumin. 
Collected information can contribute to the optimization of future isotope studies regarding the turnover 
rate of albumin, so these can be conducted without the use of radioactive tracer and hopefully also give 
a more accurate value. 
 
7 RESEARCH QUESTION 
PRIMARY QUESTION  
Is there a difference between the rate of albumin degradation as measured by D5-Phe-albumin in 
healthy research subjects compared to patients with liver cirrhosis? Does albumin degradation rate 
measured with D5-Phe albumin differ from historical controls measured with radioiodine-labeled 
albumin? 
SECONDARY QUESTIONS 
a) How few and which measurement points could be used clinically with the D5-Phe method to still 
obtain meaningful measurements of the albumin degradation rate? 
b) Can orally administered D5-Phe be used to obtain reasonable values of albumin fractional synthesis 
rate compared to historical controls as measured by intravenous administration of the same tracer in 
our previous studies? 
c) Can measurement of different forms of oxidized d5-Phe-albumin over time be used to model the rate 
of albumin degradation? 
d) Does the rate of albumin degradation correlate with the amount of oxidized albumin? 
8 ENDPOINTS 
FCR measured by D5-Phe-albumin, FSR measured by D5-Phe-albumin, P-albumin, HNA-1, HNA-2. 
9 STUDY DESIGN 
This study is an open, exploratory pilot study in two stages. In step 1, healthy volunteers are examined. 
On day 0, an oral dose of phenylalanine 45 mg per kilogram of body weight 50% MPE D5-
phenylalanine is given, and repeated blood samples are taken over 120 minutes to measure FSR. 
Plasma volume will be calculated anthropometrically. 
Afterwards, the research subjects return for single venipunctures around days 7, 14, 21, 28, 42, 56, 70 
and 84 to measure D5-Phe-albumin, P-albumin, and labeled and unlabeled oxidized forms of albumin 
(human non-mercaptoalbumin), HNA 1 and HNA- 2, respectively. 
After analysis of the collected data, phase 2 of the study begins with patients who have cirrhosis of the 
liver. The procedure is identical to the above except that the sampling after day 0 takes place on fewer 
occasions, tentatively at 3, 8 and 12 weeks, but the protocol may be slightly adjusted based on the 
results from the healthy research subjects. 
 
10 STUDY PROCEDURES 
10.1 INFORMED CONSENT 
 
The research subjects are informed orally and in writing about the study and the study-specific 
procedures by the principal researcher, another researcher or research nurse. After that, written 
consent is obtained. They then undergo a simple health examination and routine blood samples are 
taken. 

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A researcher evaluates inclusion and exclusion criteria, so that there is no contraindication to 
participating in the study. Here, record is kept in the existing record system (Take Care) that the 
research person participates in a clinical study in accordance with the Patient Data Act and the 
regulations of the National Board of Health and Welfare. 
 
10.2 DAY 0. MEASUREMENT OF FRACTIONAL SYNTHESIS RATE 
In the morning, the research subject arrives fasting at the research department at Perioperative 
Medicine and Intensive Care, Huddinge. Pregnancy tests are taken on female research subjects of 
childbearing age, 40–55 years. A peripheral venous line is placed for repeated blood sampling. Two 
grams of D5-Phe is taken per os as a suspension. Blood samples are taken at 0, 10, 20, 30, 50, 70, 90, 
and 120 minutes for measurement of FSR, that is D5-Phe and D5-Phe-albumin. Actual sampling times 
are documented in the CRF. 
 
The oxidized forms of albumin will also be measured in some of these samples. 
 
Blood samples for determination of P-albumin, B-Hb and B-hematocrit are taken for determination by 
routine lab. Actual sampling times are documented in the CRF. 
 
10.3 DAY 7, 14, 21, 28, 42, 56, 70, AND 84. MEASUREMENT OF FRACTIONAL 
CATABOLIC RATE. 
The healthy volunteers come back on 8 occasions for venous sampling of P-albumin, D5-Phe-albumin, 
and the oxidized forms of albumin as absolute values and the ratio between labeled and unlabeled 
fractions. Sampling occasions are documented in the CRF. Exact dates and times are documented but 
may differ from those in the headline. 
Patients with liver cirrhosis only come back on preliminary 3 occasions, after approximately 21, 56 and 
84 days. 
 
11 METHODS AND ANALYSIS 
11.1 MEASUREMENT METHODS 
Quantitative measurement of enrichment of isotopically labeled phenylalanine is done using gas 
chromatography-mass spectrometry at the Karolinska Stable Isotope Core where Olav Rooyackers is 
the laboratory director. Quantification of total phenylalanine (the precursor) is performed by mass 
spectrometry against an internal standard. Oxidized forms of albumin will be measured by high 
performance liquid chromatography. 
11.2 SAMPLE HANDLING 
The samples for the measurement of stable isotopes will be labeled in a de-identified way and stored in 
the existing biobank at the Anesthesia and Intensive Care Clinic Karolinska University Hospital 
Huddinge at minus 80ºC while waiting for the material collection to be completed and then analyzed in 
the same analysis round to obtain the best possible precision in accordance with local routines. Åke 
Norberg is the responsible part for the sample collection. All sample material will be destroyed once the 
study is published. The code key is kept in a locked room in the anesthesia department, internal 
address K32, at Karolinska University Hospital, Huddinge. 

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12 SUBJECTS  
Due to the many return visits, the healthy research subjects will be recruited through advertising within 
the staff at Karolinska University Hospital Huddinge. The short visits for blood sampling can then take 
place in connection with the employees' work shifts. 
Liver cirrhosis patients will be recruited from the Clinic of Hepatology, ME Upper Abdomen, Karolinska 
University Hospital, Huddinge. 
12.1 INCLUSION CRITERIA, HEALTHY VOLUNTEERS 
Ø Healthy volunteers, females and males, ≥ 40 years old 
Ø Good peripheral blood vessels 
Ø Signed written consent. 
12.2 INCLUSION CRITERIA, PATIENTS WITH LIVER CIRRHOSIS 
Ø Adult patients with known compensated liver cirrhosis and radiological or endoscopic signs of 
portal hypertension, such as varices, splenomegaly, or shunts. 
Ø Signed written consent. 
12.3 EXCLUSION CRITERIA 
Ø Planned surgical procedure within 3 months (due to possible blood loss i.e. loss of tracer) 
Ø Pregnancy at dosing 
Ø Phenylketonuria 
Ø Participating in other study with stable isotopes within 60 days. 
Ø Circumstance that causes the responsible researcher to assess the research person's 
participation as inappropriate. 
13 CRITERIA FOR DISCONTINUATION 
13.1 PATIENT RELATED CRITERIA 
The research subject can at any time cancel his participation in the study without giving a reason. If 
possible, the researcher/nurse can write the reason for the end of the study in the CRF. 
Ø 
Acute serious illness in research subjects 
Ø 
Decompensated liver failure with bleeding or ascites drainage 
Ø 
The research person chooses to cancel his participation. 
13.2 INVESTIGATOR’S CRITERIA AND EVALUABILITY 
The principal investigator can decide that the research subject cannot continue in the study. This can be 
caused by lack of evaluability or other reasons detailed below: 
Ø Technical failure of equipment 
Ø Personnel reasons that make safe and standardized method analysis impossible 
This must be stated in the CRF with the date and time. 
13.3 SUBJECT LOG 
The investigator will keep a record of all subjects who have been asked about participation and 
research subjects who have been on screening visits in a log list. The log list is kept in the investigator's 

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folder which is available at the department of Perioperative Medicine and Intensive Care. It shows all 
screened subjects, included and not included. 
14 ASSESSMENT OF SAFETY AND EFFICACY 
Phenylalanine is an essential amino acid found naturally in various proteins in food and in all 
commercial enteral nutrition solutions. Deuterium is a stable isotope of hydrogen that occurs naturally in 
0.015% of all water molecules: The risk with oral intake of phenylalanine 45 mg per kilogram of body 
weight 50% MPE D5-phenylalanine is considered negligible. 
The research subjects are monitored at the Research Department at the Unit for Perioperative Medicine 
and Intensive Care, Karolinska University Hospital Huddinge, by staff who are well acquainted with 
emergency events. Monitoring of the research subject's heart rate, ECG, respiratory rate, saturation 
(oximetry), blood pressure and temperature is done before and during the first 120 min after oral 
administration of stable isotope on day 0 in accordance with the research unit's routines. 
Laboratory samples are analyzed at the ISO-certified Karolinska University Laboratory, Huddinge. 
 
15 STATISTICS AND DATA MAMAGEMENT 
15.1 DATA MANAGEMENT 
For the research subjects, the form from the health check will be de-identified and attached to the CRF. 
Monitoring curve day 0 is attached to the CRF. Otherwise, data is retrieved from the hospital electronic 
records (Take Care, TC) and placed decoded in the CRF.  
All data concerning the endpoints will be put into a database that will be locked when the data collection 
is complete. De-identified and coded demographic and study data will be compiled and entered in an 
electronic database. 
15.2 STATISTICAL ANALYSIS 
Demographic data such as age, height, weight, BMI, gender, and routine lab, will be presented as 
number, mean and standard deviation, or median and range depending on the nature of the data. The 
liver cirrhosis patients will furthermore be presented with diagnosis and existing measures of liver 
disease severity in TC, such as the Child-Pugh score and the Model for End-stage Liver Disease. 
Plasma half-lives of D5-Phe-albumin will be calculated from the assumed log-linear decline over time, 
the terminal slope λz, and analyze of residuals will be performed to determine when the terminal slope 
is reached. The groups will be compared by t-test or Mann-Whitney U-test depending on the 
appearance of the data. We will also do non-compartmental analysis of the data. 
All values of FCR and FSR will be included in a planned joint pharmacokinetic analysis later in the study 
program. 
15.3 DETERMINATION OF THE NUMBER OF SUBJECTS 
No previous measurements of albumin degradation rate measured with D5-Phe are available in the 
literature. 
With radioiodine-labeled albumin there are measurements showing that the FCR for volunteers is 9.6 ± 
1.5% per day, against 6.9 ± 1.9 for cirrhotic patients [Hasch 1967]. With these numbers corresponding 
to an effect size of 1.56, we would reach 80% power with 8 people in each group, two-sided test and a 
significance level of 5%. However, these measurements were made on inpatients, and it is possible that 
outpatient cirrhosis patients at Huddinge, who are expected to be stable in their liver failure for 3 
months, are somewhat less impaired in their liver function, i.e. that the difference between the groups is 
somewhat smaller. 

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If we look at our own data on synthesis measurements with the flooding-dose technique for volunteers 
[Dumitrescu 2018] and liver disease patients [Amouzandeh 2023 submitted manuscript], we have FSR 
values of 12.8 ± 2.3% per day respectively 6.1 ± 3 ,2. At steady state, fractional synthesis rate is equal 
to fractional degradation rate and then 5 people in each group would be enough to reach 80% power to 
detect this difference with a two-tailed test and a significance level of 5%. 
Overall, we think that with 12 people in each group, we can withstand 2 dropouts per group and still be 
reasonably confident of having a good power. The risk of dropouts is affected by the long observation 
period, 12 weeks. With 10 observations in each group, you reach 80% power to find an effect size of 
1.35 with a two-sided test and a significance level of 5%. 
16 ACCESS TO SOURCE DATA 
The principal investigator is responsible for ensuring that the patient record manager at the clinic 
establishes a confidentiality relationship with the monitor and other independent reviewer (i.e. auditor) 
who is given permission to verify the data in the patient record. For this, written consent is required from 
the research subject. 
17 QUALITY CONTROL 
17.1 MONITORERING 
The investigator and staff must set aside time for monitoring. The principal investigator is responsible 
for monitoring being done by an independent monitor with knowledge of GCP, to ensure that the study 
is conducted according to GCP, GDPR and according to the study protocol. The sponsor has 
contracted the Clinical Research Unit KFE at Perioperative Medicine and Intensive Care, Karolinska 
Solna for monitoring this study. 
18 ETHICS 
The principal investigator is responsible for the application for approval from the ethics review authority. 
The study will be conducted according to the study protocol, GCP, GDPR and the Declaration of 
Helsinki. 
19 DATA MAMAGEMENT AND ARCHIVE 
19.1 CASE REPORT FORMS 
The data collected in the study will be registered in so-called Case Report Forms (CRF) where the 
research person or patient is only identified with a code number and initials. The code key is stored in 
such a way that unauthorized persons cannot access it. De-identified and coded copies of lab lists, 
patient record entries and diagnoses (Take Care) and monitoring records will also be included in the 
CRF. Data collected in the study in the CRF will be transferred to a database that the principal 
investigator is responsible for. 
19.2 ARCHIVE 
Study documents will be filed for at least 10 years. The documents shall be filed in readable stated for 
future audit or inspection by authorities. 

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20 FINANCIAL SUPPORT AND INSURANCE 
The study is funded by research grants. The research subjects are insured by the patient injury 
insurance.  
Healthy volunteers will receive grants for loss of income, in total 2000 SEK, for only the first day 1000 
SEK. Patients with liver cirrhosis will get 400 SEK per visit for travel expenses.  
21 PUBLICATION OF RESULTS 
The intention is to publish the results in a scientific journal, together with previously unpublished data 
from one of our previous radioiodine studies of albumin kinetics. 
22 REFERENCES 
 
Ballmer PE, McNurlan MA, Milne E, et al. Measurement of albumin synthesis in humans: a new 
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23 SIGNATURE 
Huddinge 2023-07-04 
 
 
 
 
 
Åke Norberg, Associate Professor, Sponsor, and Principal Investigator