Modulation of Circulating Levels of the Ketone Body 3-hydroxybutyrate in Patients With Chronic Heart Failure: Cardiovascular Effects
Phase: PHASE2
Conditions: Heart Failure, Ketosis, Ketonemia
Sponsor: University of Aarhus

 
 
KETO-CHF 
Project protocol 
Official Title: 
Modulation of circulating levels of the ketone body 3-hydroxybutyrate in patients 
with chronic heart failure: Cardiovascular and metabolic effects 
 
NCT Number: N/A 
 
Date: 15-06-2020 
 
 
 

Protocol 
Confidential 
 
Original protocol title: Modulation of circulating levels of the ketone body 3-hydroxybutyrate in patients with chronic heart failure: Cardiovascular 
and metabolic effects 
Kristian Hylleberg Christensen, Læge, PhD-studerende,  Afd. for Hjertesygdomme, Aarhus Universitetshospital Skejby, Aarhus, Denmark 
Scientific Committee ref.:  1-10-72-362-18 
 
 
2 
 
Study sites: 
Department of Cardiology 
Aarhus University Hospital, Aarhus  
Palle-Juul Jensens Boulevard 99 
DK-8200 Aarhus N, Denmark 
 
Medical Research Laboratory 
Aarhus University Hospital, Aarhus  
Nørrebrogade 44 
DK-8000 Aarhus N, Denmark 
Investigators: 
MD, Kristian Hylleberg Christensen (principal investigator) 
MD, PhD, Roni Nielsen  
MD, PhD, Lars Gormsen 
Ass. Professor, MD, PhD, Rikke Nørregard 
Professor, MD, DMSc, Steen Hvidtfeldt Poulsen 
Professor, MD, PhD, DMSc Niels Møller 
Professor, MD, PhD, DMSc Hans Erik Bøtker 
 
 
Sponsor investigator 
Professor, MD, PhD, DMSc Henrik Wiggers 
Department of Cardiology, Aarhus University Hospital, Palle-Juul Jensens Boulevard 99, DK-8200 Aarhus N, 
Denmark, phone: 78452026, email: henrikwiggers@dadlnet.dk 
 
Contact information: 
MD, Kristian Hylleberg Christensen 
Department of Cardiology  
Aarhus University Hospital  
Palle-Juul Jensens Boulevard 99 
DK-8200 Aarhus N, Denmark 
Phone: +45 2660 3577 

Protocol 
Confidential 
 
Original protocol title: Modulation of circulating levels of the ketone body 3-hydroxybutyrate in patients with chronic heart failure: Cardiovascular 
and metabolic effects 
Kristian Hylleberg Christensen, Læge, PhD-studerende,  Afd. for Hjertesygdomme, Aarhus Universitetshospital Skejby, Aarhus, Denmark 
Scientific Committee ref.:  1-10-72-362-18 
 
 
3 
Content 
1 Background .................................................................................................................................... 4 
2 Hypotheses .................................................................................................................................... 4 
3 Objectives ...................................................................................................................................... 4 
4 Design and endpoints .................................................................................................................... 4 
5 Methods ........................................................................................................................................ 7 
5.1.1 Swan-Ganz Monitoring: ....................................................................................................... 7 
5.1.2 Rest/Peak Exercise Echocardiography: ................................................................................ 7 
5.1.3 Whole-body metabolism: .................................................................................................... 7 
5.1.5 Cardiopulmonary Exercise Test: .......................................................................................... 8 
5.1.6 Blood samples: ..................................................................................................................... 8 
6 Power calculation and statistics .................................................................................................... 8 
7 Patient recruitment ....................................................................................................................... 8 
Inclusion criteria: .......................................................................................................................... 8 
Exclusion Criteria: ......................................................................................................................... 8 
Recruitment and informed consent ............................................................................................. 9 
8 Data collection and processing ..................................................................................................... 9 
Biobanks ........................................................................................................................................ 9 
9 Patient discomfort and risks ........................................................................................................ 10 
Blood samples (all studies) ......................................................................................................... 10 
Radiation ..................................................................................................................................... 10 
Right heart catherization ............................................................................................................ 10 
Echocardiography ....................................................................................................................... 10 
Muscle biopsies .......................................................................................................................... 11 
Adverse Effects of study medication .......................................................................................... 11 
Potential benefits for the participants ....................................................................................... 11 
10 Ethical considerations ............................................................................................................... 11 
11 Collaborators and feasibility...................................................................................................... 11 
12 Budget and Economy................................................................................................................. 12 
13 Publications ............................................................................................................................... 13 
15 References ................................................................................................................................. 13 
 

Protocol 
Confidential 
 
Original protocol title: Modulation of circulating levels of the ketone body 3-hydroxybutyrate in patients with chronic heart failure: Cardiovascular 
and metabolic effects 
Kristian Hylleberg Christensen, Læge, PhD-studerende,  Afd. for Hjertesygdomme, Aarhus Universitetshospital Skejby, Aarhus, Denmark 
Scientific Committee ref.:  1-10-72-362-18 
 
 
4 
1 Background 
Heart Failure (HF) is a major public health issue because the disease  affects 1-2% of the Western 
population[1] and the lifetime risk of HF is 20% [1]. HF is responsible for 1-2% of all healthcare 
expenditures and 5 % of all hospital admissions [2]. The cornerstone in the medical treatment of 
chronic HF is a combination of ACE-inhibitors/ATII-receptor antagonists, beta-blockers and 
mineralocorticoid receptor antagonists [3]. Despite major improvements in the management and 
care of patients with HF, the 1-year mortality in patients with HF is 13 % [4] and >50% of HF-
patients is admitted during a 2.5 year period [5]. Furthermore patients with HF have markedly 
decreased physical capacity and quality of life. Thus, there is a need for new treatment modalities 
in this group of patients. 
 
Ketone bodies are produced in the liver and are of vital importance in the human 
body for energy generation in the heart and brain during fasting, exercise and severe illness [9, 
10]. However, ketosis can be safely obtained using dietary supplements [10-12] and can increase 
exercise capacity in athletes [6]. The most important ketone bodies are 3-OHB and acetoacetate. 
Recently, it was demonstrated that patients with severe HF have increased myocardial utilization 
of the ketone body 3-hydroxybutyrate (3-OHB) [7]. It has been hypothesized that ketone bodies 
may act as “superfuel” for the failing heart [8]. In support of this, the glucose-lowering SGLT-2 
inhibitor empagliflozin reduces the risk of hospitalizations and cardiovascular death in diabetic 
patients with HF [9] and also increases circulating levels of 3-OHB [10].  
 
We have shown, using positron emission tomography, that ketone body infusion 
reduces myocardial glucose uptake and increases myocardial blood flow in healthy subjects [11]. 
Data from another study conducted by our group show a 40% increase in cardiac output during 
infusion of 3-OHB [12]. The mechanisms behind these marked hemodynamic effects are presently 
unknown, but could involve prostaglandin-release. 3-OHB is the endogenous ligand for the G 
protein-coupled receptor hydroxy-carboxylic acid 2 (HCA2) receptor. This receptor has 
downstream effects on cAMP and systemic effects via release of prostaglandins [13]. 
Presently there are no data on the clinical cardiovascular and metabolic effects of long - or short 
term oral ketone-supplementation in patients with chronic HF.  
2 Hypotheses 
Exercise capacity and cardiac function can be improved in patients with chronic HF by increasing 
circulation ketone bodies. This can be achieved through a dietary supplement of 3-OHB. 
3 Objectives 
1: To determine uptake kinetics of oral 3-OHB supplements in patients with chronic HF. 
2. Furthermore to investigate acute pharmacodynamic effects (i.e. hemodynamic effects) of oral 3-
OHB intake by two different ketone-supplements. 
3: To investigate the effect of 4 weeks modulation of circulating ketone body levels on cardiac 
function and exercise capacity in chronic HF patients. 
4 Design and endpoints 
The study consists of one pilot study (KETO-KINETICS) and one main study (KETO-CHF) with a 
hemodynamic substudy and a metabolic substudy. . The purpose of the pilot study is 1) to 
establish uptake kinetics and 2) acute hemodynamics of two oral ketone dietary supplements, and 
3) differentiate acute and chronic effects of oral ketone supplementation. There will be separate 
participant informations and consent forms for the substudies. Thus participants can opt only to 
participate in one substudy if they prefer. The study days are outlined in table 1-3.     

Protocol 
Confidential 
 
Original protocol title: Modulation of circulating levels of the ketone body 3-hydroxybutyrate in patients with chronic heart failure: Cardiovascular 
and metabolic effects 
Kristian Hylleberg Christensen, Læge, PhD-studerende,  Afd. for Hjertesygdomme, Aarhus Universitetshospital Skejby, Aarhus, Denmark 
Scientific Committee ref.:  1-10-72-362-18 
 
 
5 
KETO-KINETICS: KETO-CHF pilot study:  
Design: 10 patients are studied after an overnight fast, in random order on 3 visits, during a) oral 
intake of 36 g Perfect Keto Oral 3-OHB supplement, b) oral intake of 25 g HVMN Ketone Ester, and 
c) during a placebo isocaloric drink (similar energy content as the 36 g 3-OHB dose, containing 
1:1:2 mixtures of dextrose, fructose, and maltodextrin) [6]. 
Endpoints: Cardiac output, venous 3-OHB levels, LVEF, LV global strain. 
Methods: Swan-Ganz Monitoring, Echocardiography, ECG and blood pressure will be recorded 
hourly. 3-OHB levels are measured by venous blood samples every 60 minutes. Sampling will be 
performed hourly from 1 hour before till 5 hours after oral intake.  
KETO-CHF: Randomized, double blind crossover study with 20 stable chronic HF patients. 
Design: Patients are studied after 4 weeks of oral 3-OHB three times daily(36 g “Perfect Keto Base”  
or 25g HVMN Ketone Ester depending on the outcome of KETO-KINETICS three times) and 4 weeks 
isocaloric placebo-drink. Both “Perfect Keto Base” (Perfect Keto, Glendale Heights, IL, USA) and 
HVMN Ketone Ester (HVMN, San Francisco, CA, USA) are a commercially available ketone diet 
supplements. 
Endpoints: 1) Cardiac output (primary), mixed venous saturation (SVO2), pulmonary wedge 
pressure 2) Rest/Peak Exercise Echocardiography (LVEF, Global Strain), 3) Cardiopulmonary 
exercise test, 4) Whole-body substrate metabolism, insulin signalling and lipolysis. 
Methods: Swan-Ganz monitoring, echocardiography, exercise capacity, whole-body glucose, FFA 
and protein metabolism will be examined using isotope tracer techniques. Furthermore, muscle 
biopsies will be drawn for studies of insulin signalling and lipolysis (chapter 5).  
 
 
 
 
Table 1: Study Visits Overview 
Visit 
 
V0 
Screening 
Teleph. 
Contact  
 
 
 
V1 
Randomizat
ion 
 
 
V2 
Hemodynamic 
examination 1 
V3 
Metabolic 
Examinations 1 
V4 
Hemodynamic 
examinations 2 
V5 
Metabolic 
Examinations 2 
Time (Days, Weeks, 
Months) 
- 4W to 0 
-3½ W-0W 
0 
3-5W 
3-5W 
10-12W 
10-12W 
General 
 
 
 
 
Screening  
Randomization 
Placebo 
3-OHB 
Placebo 
3-OHB 
Figure 1: Patient Inclusion flowchart 

Protocol 
Confidential 
 
Original protocol title: Modulation of circulating levels of the ketone body 3-hydroxybutyrate in patients with chronic heart failure: Cardiovascular 
and metabolic effects 
Kristian Hylleberg Christensen, Læge, PhD-studerende,  Afd. for Hjertesygdomme, Aarhus Universitetshospital Skejby, Aarhus, Denmark 
Scientific Committee ref.:  1-10-72-362-18 
 
 
6 
Informed consent 
X 
 
 
 
In-and exclusion 
criteria 
X 
 
X 
 
Relevant medical/ 
surgical history 
 
 
X 
 
Relevant 
concomitant 
medication (1) 
 
 
X 
 
Randomize patient 
 
 
X 
 
Study medication 
 
 
 
 
Delivery 
(Trial 
period) 
 
X 
 
Crossover 
 
 
 
X 
 
Clinical assessment 
 
 
 
 
NYHA class 
 
 
X 
X 
X 
X 
X 
Height 
 
 
X 
 
Weight 
 
 
X 
X 
X 
X 
X 
Echocardiography 
 
 
 
X 
X 
 
Swan-Ganz Monit. 
 
 
 
X 
X 
 
CPX-test 
 
 
 
X 
X 
 
Whole-Body 
Metabolism 
 
 
 
X 
X 
Blood Samples 
 
 
 
X 
X 
X 
X 
Muscle Biopsies 
 
 
 
X 
X 
ECG 
 
 
X 
X 
X 
 
Blood pressure, 
heart rate 
X 
 
X 
X 
X 
X 
X 
Safety 
 
 
 
 
Reporting of: 
-Endpoints  
-SUSARs 
-selected AEs  
 
X 
X 
X 
X 
X 
X 
 
Table 2: Hemodynamic study day overview 
Time 
07.30 08.30 09.15 09.30 10.30 10.50 11.10 11.30 12.30 
Arrival 
x 
 
 
 
 
 
 
 
 
Swan Ganz 
 
x 
 
 
 
 
 
 
 
Exercisetest 
 
 
 
 
x 
 
 
 
 
Blood Samples 
 
 
x 
 
x 
x 
x 
x 
 
Echocardiography 
 
 
x 
 
x 
x 
x 
x 
 
Invasive measurements 
 
 
x 
 
x 
x 
x 
x 
 

Protocol 
Confidential 
 
Original protocol title: Modulation of circulating levels of the ketone body 3-hydroxybutyrate in patients with chronic heart failure: Cardiovascular 
and metabolic effects 
Kristian Hylleberg Christensen, Læge, PhD-studerende,  Afd. for Hjertesygdomme, Aarhus Universitetshospital Skejby, Aarhus, Denmark 
Scientific Committee ref.:  1-10-72-362-18 
 
 
7 
3-OHB/placebo ingestion 
 
 
 
x 
 
 
 
 
 
End of study day 
 
 
 
 
 
 
 
 
x 
 
Table 3: Metabolic study day overview 
 
Time 
07.30 
08.30 
09.30 
10.30 
11.30 
12.30 
13.30 
14.30 
Arrival 
x 
 
 
 
 
 
 
 
Basal period 
 
 
 
 
 
 
 
 
3-OHB sipping 
 
 
 
 
 
 
 
 
3-OHB sipping + Clamp 
 
 
 
 
 
 
 
 
[3-3H]Glucose-Infusion 
 
 
 
 
 
 
 
 
[9,10-3H]Palmitate-infusion 
 
 
 
 
 
 
 
 
Muscle Biopsy 
 
 
x 
 
 
 
x 
 
Blood samples  
x 
 
x 
x 
x 
x 
x 
 
5 Methods 
5.1.1 Swan-Ganz Monitoring:  
This is a standard procedure at the clinical research site and will be performed in a dedicated 
cardiac catheter lab with experienced physicians.  Standard triple-lumen Swan-Ganz thermister 
and balloon-tipped catheters will be used. Catheters will be introduced using the internal jugular 
vein, using ultrasound guidance, and advanced using pressure waveform and fluoroscopy into the 
pulmonary artery. Cardiac output will be measured using the thermodilution method.  
Measurements will be made at rest, during peak exercise and post-exercise.  
5.1.2 Rest/Peak Exercise Echocardiography: 
We intend to use a GE VIVID 7 or 9E system (GE Medical System, Horten, Norway) with a 2.5-MHz 
transducer for transthoracic echocardiography. For image analysis EchoPAC (GE-Vingmed 
Ultrasound, Horten, Norway) will be used. All measurements will be the averaged over 3 
consecutive heart cycles in end-expiration. 2D echocardiography will be performed according to 
national guidelines [14]. In case of poor image quality, a contrast agent (SonoVue/Optison) will be 
used to opacify the left ventricle (LV) [15, 16]. Regarding LV measurements, the following 
parameters will be recorded: 2D [16], regional wall motion score (16 segment model), LVEDV and 
LVESV, left atrial volume, tissue Doppler study of the mitral annulus and LV strain and strain-
rate[17]. 
5.1.3 Whole-body metabolism: 
Glucose metabolism is determined by isotope dilution technique with constant infusion of [3-3H] 
glucose (12 μCi / min) from two hours before and 60 minutes after intake of ketones or isocaloric 
placebo.  
FFA Turnover is determined by isotope dilution technique with 2 times 1-hour constant infusion of 
[9.10-3H] palmitate (0.3 μCi / min, totalling 36 μCi) before and 60 minutes after oral intake. Blood 
samples to determine FFA concentration and specific activity will be taken at baseline and after 
30, 40, 50 and 60 minute infusion. Plasma FFA (palmitate) concentration and specific activity are 
determined by HPLC[18]. 

Protocol 
Confidential 
 
Original protocol title: Modulation of circulating levels of the ketone body 3-hydroxybutyrate in patients with chronic heart failure: Cardiovascular 
and metabolic effects 
Kristian Hylleberg Christensen, Læge, PhD-studerende,  Afd. for Hjertesygdomme, Aarhus Universitetshospital Skejby, Aarhus, Denmark 
Scientific Committee ref.:  1-10-72-362-18 
 
 
8 
5.1.4 Muscle biopsies 
Samples are taken from the vastus lateralis by sterile technique. Proteins involved in insulin-
signalling and lipolysis will be determined by Western Blot technique. 
5.1.5 Cardiopulmonary Exercise Test: 
Cardiopulmonary exercise capacity will be performed using a staged bicycle exercise test with 
measurements of oxygen consumption and carbon dioxide excretion. After resting measurements 
are obtained patients will start exercising at 0 watts and using 3 minute intervals increase to 20 
watts and then with 10 watt increments every 3 minutes until peak exercise (Borg > 18). At each 
level of exercise hemodynamic and echocardiographic measurements will be recorded. A 
preliminary exercise test will be performed prior to the baseline study to make the patient familiar 
with the procedure. 
5.1.6 Blood samples: 
Venous blood samples for  measurements of plasma insulin, free fatty acids, glucose, ketone 
bodies, lactate, haemoglobin, P-Na+, P-K+. Will be measured three times during each visit 
P-LDL, P-HDL and P-Cholesterol (total), prostaglandin E2, I2, F2α, thromboxane A, erythropoietin, 
thrombopoietin and granulocyte stimulating factor will be measured twice during the pilot study 
and the hemodynamic study days. 
6 Power calculation and statistics 
Power calculations 
In the pilot study we plan to include 8 patients. However, if necessary because of drop-outs or 
missing values up to 10 patients will be included. 
Coefficiency of variation (CV) regarding Cardiac output by pulmonary artery catherization is 
determined to be 4 % [19]. By enrolling 8 patients in the KETO-KINETIC pilot study, we will be able 
to detect a relative difference of 6% with a power of 90% and a two-sided significance level of 5% 
In the KETO-CHF study by enrolling 20 patients in the hemodynamic examinations, and allowing up 
to 20 % missing values, we will be able to determine a relative difference of 4 %, and thus detect 
changes related to clinical outcome [20], with a power of 90% and a two-sided significance level of 
5%. 
In the metabolic studies, previous studies have shown an SD of the difference of 8% and an effect 
size of 10%. With power and significance level as described above a sample size of 8 patients are 
needed. To allow 20% missing values 10 patients will be included in the metabolic studies. 
Reporting of endpoints: 
For these studies normally distributed variables will be presented as mean±SD. Non-parametric 
statistics and appropriate log-transformation will be performed if assumption of normality is not 
met. A two-tailed p value of 0.05 or less will be considered statistically significant.  
Differences will be compared by appropriate paired statistical tests. Regression analysis will be 
performed and correlation coefficients will be presented when appropriate. 
7 Patient recruitment 
Patients with stable HF will be recruited from the outpatient HF clinic at Aarhus University 
Hospital. 
Inclusion criteria:  Chronic HF: NYHA class II-III, left ventricular ejection fraction (LVEF) <40% 
Exclusion Criteria: Diabetes or HbA1c >48 mmol/mol, significant cardiac valve disease, severe 
stable angina pectoris, severe comorbidity as judged by the investigator, inability to give informed 
consent  

Protocol 
Confidential 
 
Original protocol title: Modulation of circulating levels of the ketone body 3-hydroxybutyrate in patients with chronic heart failure: Cardiovascular 
and metabolic effects 
Kristian Hylleberg Christensen, Læge, PhD-studerende,  Afd. for Hjertesygdomme, Aarhus Universitetshospital Skejby, Aarhus, Denmark 
Scientific Committee ref.:  1-10-72-362-18 
 
 
9 
Recruitment and informed consent  
Study participants will be recruited from the participating centres. Participants will receive oral 
and written information regarding the study together with the brochure: " Volunteer’s rights in a 
health science research project” (“Forsøgspersoners rettigheder i et sundhedsvidenskabeligt 
forskningsprojekt“) and an agreement form requesting accept to be contacted by the investigators 
for further information. Interested subjects will have to sign the enclosed agreement form and 
return it before being contacted. Subjects agreeing to receive further information will be 
contacted by phone. The subjects are informed about the project's background, objectives, 
methods, risks, side effects, disadvantages, benefits and potential usefulness, and that there may 
be unforeseen risks and impacts of participation in the project. The subjects are informed that 
participation is voluntary and that he / she at any time without reason can withdraw their consent 
and drop out of the project without any consequences for their future treatment. The information 
is repeated orally upon arrival for the screening visit. The subjects will have the opportunity to ask 
questions and to bring a companion at this visit. The information conversation will take place in 
privacy in an investigation room at Department of Cardiology, Aarhus University Hospital and 
carried out by any of the investigators. Before signing the consent form, the participant will have 
the opportunity to re-consider for at least 24 hours. Copies of participant information and consent 
form will be given to the subjects. No study-related examinations will be conducted until after the 
informed consent form has been signed. The investigator will ensure that the participant is 
adequately informed both orally and written about the study, background, design, risks, side 
effects, disadvantages, benefits and usefulness. The investigators are responsible for ensuring 
obtained written informed consent from all subjects before enrolment in the study. 
Information on individual medical data will be given by a medical doctor affiliated with the study. 
However, the individual subject’s desire not to know his or her own data will be respected.  
By participating in the study, subjects agree that relevant information from medical records that 
potentially can influence on the study may be passed to the responsible investigators. This may be 
information relating to medicine intake and previous and current diseases and treatments. 
8 Data collection and processing  
Data collection and processing will be carried out in accordance with current legislation on 
processing of personal data, and the general data protection regulation. 
Source data will be recorded in the patient's electronic patient record or on specific worksheets. 
Invasive hemodynamics and echocardiograpic data will be stored at the Department of Cardiology 
Aarhus University Hospital. A centralized electronic Case Report Form (CRF) will be constructed for 
data capture. Data will be stored in coded form in 15 years according to recommendations from 
the Data Monitoring Board. After which it will be transmitted to the Danish Data Archives.  
Biobanks 
A research biobank will be established at Department of Endocrinology and Metabolism, Aarhus 
University Hospital. This research biobank will store blood samples in coded form for later analysis 
(>7 days). Blood samples will be stored at -80°C. After completion of the project, excess biological 
material (blood plasma and muscle biopsies) will be transferred to a biobank for future research. 
This biobank will be registered separately with the Central Denmark Region.   

Protocol 
Confidential 
 
Original protocol title: Modulation of circulating levels of the ketone body 3-hydroxybutyrate in patients with chronic heart failure: Cardiovascular 
and metabolic effects 
Kristian Hylleberg Christensen, Læge, PhD-studerende,  Afd. for Hjertesygdomme, Aarhus Universitetshospital Skejby, Aarhus, Denmark 
Scientific Committee ref.:  1-10-72-362-18 
 
 
10 
9 Patient discomfort and risks  
The enrolled volunteers should expect to spend approx. 1 hour at the screening visit and 6-8 hours 
at each study visits. Participants will have their travel expenses from participating in the project 
reimbursed. In case of injury, patients are covered under The Patient Compensation Association. 
Blood samples (all studies) 
The studies will require venous puncture which may inflict a short pain and a risk of a small 
hematoma. There is a minimal risk for infection by the point of insertion. A total of 340 mL of 
blood will be drawn for the KETO-KINETICS study distributed over the three study days. In the 
KETO-CHF 110 mL of blood will be drawn at each visit. This should cause no side effects. 
Radiation 
The risk for adverse health effects from cancer is proportional to the amount of radiation dose 
absorbed and strongly dependent on age. When reviewing the numbers below, the natural 
incidence of fatal cancer in Denmark of about 25%  and the 5-year mortality of 43% in Danish 
heart failure patients [2] should be taken into account. Radiation doses for the KETO-KINETICS  and 
Keto-CHF study is 6 mSv and 4 mSv each respectively, corresponding to IRCP category IIb. Thus, 
participation in one study increases cancer risk from 25% to 25.02% in the KETO-CHF study and 
25.03% in the KETO-KINETICS study. 
Right heart catherization 
This method has been implemented at our centre for decades both in daily practice and for 
research use. The applied local anaesthetic, may cause a short stinging pain (few seconds) and in 
some cases cause temporally hoarseness (1-2 hours). Catheter placement through the internal 
jugular vein will be performed under sterile conditions. The catheter will be advanced to the 
pulmonary artery for measurements of pressure and cardiac output. Accidental puncture of the 
carotid artery will be avoided by transcutaneous ultrasound guidance. A single dose of antibiotics 
will be given when appropriate (pacemakers, artificial valves, etc). At the puncture site of the neck, 
a small hematoma can appear which does not require any further treatment. The procedure will 
be performed by experienced physicians, and according to the guidelines from the Danish Society 
of Cardiology the complication rate is described as particularly low. Brief periods of atrial 
fibrillation or self-limiting ventricular arrhythmias are the most common. There are reports of right 
atrial or right ventricle perforation using stiff catheters, but no such episodes were reported using 
balloon tipped catheters. Such balloon tipped catheters will be applied in the present project. In a 
study of 7218 patients undergoing right heart catherization, a fatal episode related to the 
procedure was described in one patient due to rupture of a pulmonary artery [21]. This patient 
had severe pulmonary hypertension but no HFrEF. Thus, a patient category with a completely 
different disease located in the pulmonary vessels. No such patients will be enrolled in the present 
study. Finally, it should be mentioned that the method is applied in an ongoing trial (TEMPO) at 
Dept. of Cardiology Aarhus University, and in a recent published study in which healthy Danish 
volunteers were investigated, and no adverse events were reported [22]. 
Echocardiography 
Echocardiography uses standard ultrasound techniques to image the heart. It is performed with 
the patient in the left lateral position and is without risk or discomfort for the patient.  

Protocol 
Confidential 
 
Original protocol title: Modulation of circulating levels of the ketone body 3-hydroxybutyrate in patients with chronic heart failure: Cardiovascular 
and metabolic effects 
Kristian Hylleberg Christensen, Læge, PhD-studerende,  Afd. for Hjertesygdomme, Aarhus Universitetshospital Skejby, Aarhus, Denmark 
Scientific Committee ref.:  1-10-72-362-18 
 
 
11 
Muscle biopsies 
During the metabolic examination days two muscle biopsies will be drawn. A local anaesthetic will 
be used to minimize patient discomfort. After the biopsy, a hematoma will develop as well as 
soreness of the muscle. In rare cases a local infection may occur. No patients on oral anticoagulant 
treatment will be subjected to this procedure. 
Adverse Effects of study medication 
Ketone supplements may cause mild gastrointestinal discomfort and mild headaches in the first 
days entering a regimen. Patients are therefore subjected to a four day run-in period (2 days of 
KETO-base supplement and 2 days of placebo) before randomization. Only those able to tolerate 
side-effects will be included in the study. In case of unacceptable side-effects during the study 
period, doses will be reduced step-wise until reaching no – or accepted level of side-effects. 
Blood samples for determination of K+, Na+, Ca2+ and Mg2+ will be drawn after two- four days of 
treatment and 1-2 weeks later during the study period in the first 6 patients in the KETO-CHF 
study. If deemed necessary by the investigators this procedure will be carried out in the remaining 
14 patients as well. 
Potential benefits for the participants 
Participants will potentially benefit from the 3-OHB supplement applied in this study. Participants 
with HFrEF will be evaluated for their present cardiac status. The right sided heart catherization is 
the gold standard to obtain measures of cardiac output and importantly cardiac filling pressures 
(preload) conditions. During daily practice, HFrEF patients often have their heart failure 
medication adjusted after right sided heart catherization as non-invasive assessment of preload 
conditions are often very difficult or impossible to obtain. Thus, each HFrEF participant will benefit 
by having their heart failure and diuretic treatment re-evaluated and eventually adjusted or 
intensified. Furthermore, the study could contribute with pivotal information on a potential future 
treatment of patients suffering from HFrEF, and determine whether a clinical trial of long-term 
hyperketonaemia should be initiated.  
10 Ethical considerations 
The project will be carried out in accordance with the principles of the Helsinki Declaration II. The 
protocol, including the written participant information and consent forms, must be finally 
approved by the Research Ethics Committee of the Central Denmark Region and the Danish Data 
Protection Agency. Data obtained from the subjects are protected under the Act concerning the 
processing of personal data and health law. The study will be conducted according to the Good 
Clinical Practice guidelines and will be reported to clinicaltrials.gov before initiation. The study 
does not need approval from the Danish Medicines Agency since the patients do not receive a 
drug but a dietary intervention (LMST journal number: 2018110888). 
Based on the previously mentioned literature, the overall risk of side effects when participating in 
these studies is estimated to be relatively low. The results of these studies will contribute with 
new and essential knowledge of HFrEF and potentially new treatment approaches. Therefore, the 
investigators are convinced that the possible risks and side effects are outweighed by the 
expected benefits from the conduct of this study. 
11 Collaborators and feasibility 
Dr. Kristian Hylleberg Christensen (KHC) is already trained in echocardiography and management 
of acute and chronic HF. KHC will be trained in imaging techniques needed for the study by the 

Protocol 
Confidential 
 
Original protocol title: Modulation of circulating levels of the ketone body 3-hydroxybutyrate in patients with chronic heart failure: Cardiovascular 
and metabolic effects 
Kristian Hylleberg Christensen, Læge, PhD-studerende,  Afd. for Hjertesygdomme, Aarhus Universitetshospital Skejby, Aarhus, Denmark 
Scientific Committee ref.:  1-10-72-362-18 
 
 
12 
research group. KHC will be the principal investigator in this study. Dr Roni Nielsen (RN) defended 
his PhD thesis on myocardial metabolism in heart failure in 2013. He has continuously been 
working with heart failure, cardiac PET imaging and metabolism and has experience with all 
applied methods. Associate professor Henrik Wiggers (HW) and Professor Hans Erik Bøtker (HEB), 
Dept. of Cardiology are specialists in heart failure; they both have bridged the gap between 
cardiology and metabolism and have considerable experience in the field of myocardial PET. 
Professor Niels Møller (NM), Dept. of Endocrinology and Medicine, is an expert in human in vivo 
metabolism and clinical nutrition and has considerable administrative experience. He has directed 
similar projects and has a formal education in management and administration. Consultant 
physician Lars Gormsen (LG), Dept. of Nuclear Medicine, has major experience in scientific and 
clinical interpretation of myocardial PET imaging. 
This project is initiated by the multidisciplinary research group, and the proposal could not be 
conducted without the added synergistic contributions from all fields. The present studies will 
cement and further develop this fruitful scientific milieu in the cross-fields between clinical 
metabolism, cardiology, nuclear medicine and experimental clinical research. All methods 
described are well established, validated, used in many publications and known well known to the 
investigators. Similar study populations have been enrolled in previous trials; hence, inclusion of 
participants is feasible. 
12 Budget and Economy 
Operating costs – raw expenses: 
Utensils 
: 
40 x 1.250 DKK  
= 50.000 DKK 
Right heart catheters 
: 
40 x 1800 DKK  
= 72.000 DKK  
Electronic centralized CRF   
: 
 
= 25.000 DKK 
Transportation of participants  
: 
80 x 50 x 3,75 DKK = 15.000 DKK 
Analysis of hormones and metabolites (40 analyses) : 
      
= 150.000 DKK 
Purchase, purification and package of 3-OHB 
: 
 
= 90.000 DKK 
Metabolism Studies 
: 
 
= 200.000 DKK 
Subtotal 
:  
 
 602.000 DKK 
 
VIP salaries 
PhD salary 12 months  
:  
12 x 34.852DKK 
= 418.228 DKK 
PhD study fee 3 years 
 
 
 180.000 DKK 
 
TAP salaries 
Technician 3 months 
: 
 
= 105.000 DKK 
 
Grand total 
: 
 
= 1.305.228 DKK 
 
Present funding 
Det Fri Forskningsråd 
: 
 
= 1.800.000 DKK 
Novo Nordisk Fonden 
: 
 
= 1.600.000 DKK 
 

Protocol 
Confidential 
 
Original protocol title: Modulation of circulating levels of the ketone body 3-hydroxybutyrate in patients with chronic heart failure: Cardiovascular 
and metabolic effects 
Kristian Hylleberg Christensen, Læge, PhD-studerende,  Afd. for Hjertesygdomme, Aarhus Universitetshospital Skejby, Aarhus, Denmark 
Scientific Committee ref.:  1-10-72-362-18 
 
 
13 
The funds are administered by a research account that is subjected to public revision. No 
investigators have direct access research accounts. Professor Niels Møller is a member of the 
Independent Research Council on Medical Sciences. The investigators have no personal financial 
affiliations to any of the listed funds or companies. 
13 Publications 
Based on the proposed study we aim to publish 2 scientific papers. Positive, negative and 
inconclusive results will be published. Prior to publication, results will be presented at renowned 
international cardiovascular congresses. The papers will be submitted to international peer-
reviewed journals with highest possible journal impact factor. 
15 References 
1. 
Mosterd, A. and A.W. Hoes, Clinical epidemiology of heart failure. Heart, 2007. 93(9): p. 1137-46. 
2. 
Guha, K. and T. McDonagh, Heart Failure Epidemiology: European Perspective. Curr Cardiol Rev, 
2013. 9(2): p. 123-7. 
3. 
Ponikowski, P., et al., 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic 
heart failureThe Task Force for the diagnosis and treatment of acute and chronic heart failure of the 
European Society of Cardiology (ESC)Developed with the special contribution of the Heart Failure 
Association (HFA) of the ESC. European Heart Journal, 2016. 37(27): p. 2129-2200. 
4. 
Nakano, A., et al., Trends in quality of care among patients with incident heart failure in Denmark 
2003–2010: a nationwide cohort study. BMC Health Serv Res, 2013. 13: p. 391. 
5. 
Schou, M., et al., Extended heart failure clinic follow-up in low-risk patients: a randomized clinical 
trial (NorthStar). Eur Heart J, 2013. 34(6): p. 432-42. 
6. 
Cox, P.J., et al., Nutritional Ketosis Alters Fuel Preference and Thereby Endurance Performance in 
Athletes. Cell Metab, 2016. 24(2): p. 256-68. 
7. 
Bedi, K.C., Jr., et al., Evidence for Intramyocardial Disruption of Lipid Metabolism and Increased 
Myocardial Ketone Utilization in Advanced Human Heart Failure. Circulation, 2016. 133(8): p. 706-
16. 
8. 
Lopaschuk, G.D. and J.R. Ussher, Evolving Concepts of Myocardial Energy Metabolism: More Than 
Just Fats and Carbohydrates. Circ Res, 2016. 119(11): p. 1173-1176. 
9. 
Fitchett, D., et al., Heart failure outcomes with empagliflozin in patients with type 2 diabetes at high 
cardiovascular risk: results of the EMPA-REG OUTCOME(R) trial. Eur Heart J, 2016. 37(19): p. 1526-
34. 
10. 
Ferrannini, E., et al., Shift to Fatty Substrate Utilization in Response to Sodium-Glucose 
Cotransporter 2 Inhibition in Subjects Without Diabetes and Patients With Type 2 Diabetes. 
Diabetes, 2016. 65(5): p. 1190-5. 
11. 
Gormsen, L.C., et al., Ketone Body Infusion With 3-Hydroxybutyrate Reduces Myocardial Glucose 
Uptake and Increases Blood Flow in Humans: A Positron Emission Tomography Study. J Am Heart 
Assoc, 2017. 6(3). 
12. 
Nielsen, R., et al., The ketone body 3-hydroxybutyrate increases cardiac output and left ventricular 
contractile function in heart failure patients with reduced ejection fraction. European Journal of 
Heart Failure, 2018. 20(S1): p. 381. 
13. 
Offermanns, S., Hydroxy-Carboxylic Acid Receptor Actions in Metabolism. Trends Endocrinol Metab, 
2017. 28(3): p. 227-236. 
14. 
Møller, J., et al., Ekkokardiografi. Dansk Cardiologisk Selskab NBV, 2017. 
15. 
Mulvagh, S.L., et al., American Society of Echocardiography Consensus Statement on the Clinical 
Applications of Ultrasonic Contrast Agents in Echocardiography. J Am Soc Echocardiogr, 2008. 
21(11): p. 1179-201; quiz 1281. 

Protocol 
Confidential 
 
Original protocol title: Modulation of circulating levels of the ketone body 3-hydroxybutyrate in patients with chronic heart failure: Cardiovascular 
and metabolic effects 
Kristian Hylleberg Christensen, Læge, PhD-studerende,  Afd. for Hjertesygdomme, Aarhus Universitetshospital Skejby, Aarhus, Denmark 
Scientific Committee ref.:  1-10-72-362-18 
 
 
14 
16. 
Malm, S., et al., Real-time simultaneous triplane contrast echocardiography gives rapid, accurate, 
and reproducible assessment of left ventricular volumes and ejection fraction: a comparison with 
magnetic resonance imaging. J Am Soc Echocardiogr, 2006. 19(12): p. 1494-501. 
17. 
Munk, K., et al., Remote Ischemic Conditioning in Patients With Myocardial Infarction Treated With 
Primary Angioplasty: Impact on Left Ventricular Function Assessed by Comprehensive 
Echocardiography and Gated Single-Photon Emission CT. Circ Cardiovasc Imaging, 2010. 3(6): p. 
656-62. 
18. 
Miles, J.M., et al., Validation of a new method for determination of free fatty acid turnover. Am J 
Physiol, 1987. 252(3 Pt 1): p. E431-8. 
19. 
Kroon, M., A.B. Groeneveld, and Y.M. Smulders, Cardiac output measurement by pulse dye 
densitometry: comparison with pulmonary artery thermodilution in post-cardiac surgery patients. J 
Clin Monit Comput, 2005. 19(6): p. 395-9. 
20. 
Doughty, R.N., et al., Effects of carvedilol on left ventricular remodeling after acute myocardial 
infarction: the CAPRICORN Echo Substudy. Circulation, 2004. 109(2): p. 201-6. 
21. 
Hoeper, M.M., et al., Complications of right heart catheterization procedures in patients with 
pulmonary hypertension in experienced centers. J Am Coll Cardiol, 2006. 48(12): p. 2546-52. 
22. 
Wolsk, E., et al., The Influence of Age on Hemodynamic Parameters During Rest and Exercise in 
Healthy Individuals. JACC Heart Fail, 2017. 5(5): p. 337-346.