Acute Nutritional Ketosis in VLCAD Deficiency: Testing the Metabolic Base for Therapeutic Use
Phase: NA
Conditions: VLCAD Deficiency, Fatty Acid Oxidation Defects
Sponsor: University Medical Center Groningen

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PROTOCOL TITLE: 
‘Acute Nutritional Ketosis in VLCAD Deficiency: testing the metabolic basis for therapeutic use’ 
 
 
Protocol ID 
‘Acute Nutritional Ketosis in Patients with VLCAD 
Deficiency: testing the metabolic basis for 
therapeutic use’ 
Short title 
Nutritional Ketosis therapy in VLCAD deficiency 
Version 
2.1 
Date 
15.2.2017 
Coordinating investigator/project 
leader 
Dr. J.A.L. Jeneson, PhD 
Division of Neuroscience/ BCN-Neuroimaging MR 
Center 
University Medical Center Groningen 
Hanzeplein 2 
9700 RB Groningen 
Tel: 050-363 8989 
Email: j.a.l.jeneson@umcg.nl 
 
Principal investigator(s) 
(inDutch: uitvoerder) 
Multicenter research: per site 
Site 1: UMCG 
J.A.L. Jeneson, PhD 
Dept of Neuroscience/ Neuroimaging Center 
Hanzeplein 2, 9700 RB Groningen 
Tel: 050-363 8989 
Email: j.a.l.jeneson@umcg.nl 
Site 2: University of Oxford 
Prof. dr. Kieran Clarke 
Department of Anatomy, Physiology and Genetics 
Cardiac Metabolic Research Group 
University of Oxford, Oxford, UK 
Tel +44 
Email: kieran.clarke@dpag.ox.ac.uk 
Pete Cox, MD PhD 
Cardiac Metabolic Research Center 
Department of Physiology 
University of Oxford, Oxford, UK 
Tel: +44 
Email: pete.cox123@hotmail.com 
Site 3: University Medical Center Utrecht 
Dr. G. Visser, paediatrician metabolic diseases 
UMC Utrecht - department of Paediatric metabolic 
diseases 
Lundlaan 6, 3584 EA Utrecht, the Netherlands 
Telnr. 088-7554003 

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E-mail: g.visser-4@umcutrecht.nl 
Drs. JC Bleeker, MD 
UMC Utrecht - department of Paediatric metabolic 
diseases, Lundlaan 6, 3584 EA Utrecht, the 
Netherlands 
Telnr. 088-7554003 
E-mail: j.c.bleeker-2@umcutrecht.nl 
Subsidizing party: 
Nvt 
Sponsor 
(in 
Dutch: 
verrichter/opdrachtgever) 
University Medical Centre Groningen 
Hanzeplein 1 
9700 RB Groningen 
Independent physician(s) 
Dr.  T.  Dercks 
Metabolic paediatrician 
Hanzeplein 1 
9700 RB Groningen 
Tel: 050-3611080 
Email: t.j.dercks@umcg.nl 
Laboratory sites 
Site 1: MRS data collection 
NeuroImaging Center, University Medical Center 
Groningen, Anton Deusinglaan 1, Groningen 
Site 2:  blood and tissue samples 
Cardiac Metabolic Reseach Group, Dept of Genetics, 
Anatomy and Physiology, University of Oxford, South 
Parks Road 15, Oxford, UK. 
Site 3: blood and urines samples 
Laboratory for Metabolic Disease, Academic Medical 
Center University of Amsterdam, Meibergdreef 2, 
Amsterdam 

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PROTOCOL SIGNATURE SHEET 
 
 
 
Name 
Signature 
Date 
Sponsor or legal representative: 
Prof dr Gert ter Horst, 
managing Director 
- Neuroimaging Center 
Anton Deusinglaan 1, Groningen 
 
For non-commercial research, 
Head of Department: 
<include name and function> 
Prof dr Gert ter Horst, 
managing Director 
- Neuroimaging Center 
Anton Deusinglaan 1, Groningen 
 
 
Coordinating 
Investigator/Project 
leader/Principal Investigator: 
J.A.L. Jeneson, PhD 
Dept of Neuroscience 
BCN-Neuroimaging Center 
Hanzeplein 2 
9700 RB Groningen 
 
 
 

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TABLE OF CONTENTS 
1. 
INTRODUCTION AND RATIONALE ............................................................................. 14 
2. 
OBJECTIVES ................................................................................................................ 19 
3. STUDY DESIGN .............................................................................................................. 19 
4. STUDY POPULATION ..................................................................................................... 21 
4.1 Population (base) ........................................................................................................ 21 
4.2 Inclusion criteria .......................................................................................................... 21 
4.3 Exclusion criteria ......................................................................................................... 21 
4.4 Sample size calculation ............................................................................................... 22 
5. TREATMENT OF SUBJECTS .......................................................................................... 22 
5.1. Investigational product/treatment ............................................................................... 22 
5.2. Use of co-intervention (if applicable) .......................................................................... 22 
5.3. Escape medication (if applicable) ............................................................................... 22 
6. INVESTIGATIONAL MEDICINAL PRODUCT ................................................................... 22 
7. NON-INVESTIGATIONAL PRODUCT .............................................................................. 23 
8. METHODS ....................................................................................................................... 23 
8.1 Study parameters/endpoints ....................................................................................... 23 
8.1.1 Main study parameter/endpoint ............................................................................ 23 
8.1.2 Secondary study parameters/endpoints (if applicable) .......................................... 23 
8.2 Randomisation, blinding and treatment allocation ....................................................... 23 
8.3 Study procedures ........................................................................................................ 23 
8.4 Withdrawal of individual subjects ................................................................................. 25 
8.5 Replacement of individual subjects after withdrawal ................................................... 25 
8.6 Follow-up of subjects withdrawn from treatment .......................................................... 26 
8.7 Premature termination of the study ............................................................................. 26 
9. SAFETY REPORTING ..................................................................................................... 26 
9.1 Section 10 WMO event ............................................................................................... 26 
9.2 Adverse and serious adverse events .......................................................................... 26 
9.2.1 Suspected unexpected serious adverse reactions (SUSAR) ................................. 27 
9.3 Annual safety report .................................................................................................... 28 
9.4 Follow-up of adverse events ....................................................................................... 28 
9.5 Data Safety Monitoring Board (DSMB) ........................................................................ 28 
10 STATISTICAL ANALYSIS ............................................................................................... 28 
10.1. Descriptive statistics ................................................................................................. 28 
10.2 Univariate analysis .................................................................................................... 28 
11. ETHICAL CONSIDERATIONS ....................................................................................... 29 
11.1 Regulation statement ................................................................................................ 29 
11.2 Recruitment and consent .......................................................................................... 29 
11.3 Objection by minors or incapacitated subjects ...........................................................29 
11.4 Benefits and risks assessment, group relatedness .................................................... 29 
11.5 Compensation for injury ............................................................................................ 30 

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12. ADMINISTRATIVE ASPECTS AND PUBLICATION ....................................................... 31 
12.1. Handling and storage of data and documents .......................................................... 31 
12.2 Monitoring and Quality assurance ............................................................................. 31 
12.3 Amendments ............................................................................................................. 32 
12.4 Annual progress report (Not applicable) .................................................................... 32 
12.5 End of study report .................................................................................................... 32 
12.6 Public disclosure and publication policy .................................................................... 32 
14. REFERENCES ............................................................................................................... 34 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS 
 
 
31 P MRS 
31Phosphorus magnetic resonance spectroscopy 
τHHbrecovery 
Time constant of HHb during recovery from exercise 
ABR 
ABR form, General Assessment and Registration form, is the application 
form that is required for submission to the accredited Ethics Committee 
(In Dutch, ABR = Algemene Beoordeling en Registratie) 
AE 
Adverse Event 
AR 
Adverse Reaction 
ATP 
Adenosine triphosphate 
ATT 
Adipose tissue thickness 
CA 
Competent Authority 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: 
Centrale Commissie Mensgebonden Onderzoek 
CK 
Creatinine Kinase 
CV 
Curriculum Vitae 
DPF 
Differential pathlength factor 
DSMB 
Data Safety Monitoring Board 
EU 
European Union 
EudraCT 
European drug regulatory affairs Clinical Trials 
FAO 
Fatty Acid Oxidation 
FEV1 
Forced expiratory volume in one second 
GCP 
Good Clinical Practice 
HAES 
Habitual Activity Estimation Scale 
HMP 
Hexose monophosphate 
IB 
Investigator’s Brochure 
IC 
Informed Consent 
IMCL 
Intra myocellular lipids 
IMP 
Investigational Medicinal Product 
IMPD 
Investigational Medicinal Product Dossier 
KO 
Knock Out 
METC 
Medical research ethics committee (MREC); in Dutch: medisch   ethische 
toetsing commissie (METC) 

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MRS 
Magnetic Resonance Spectroscopy 
NK 
Nm 
Nutritional Ketosis 
Nanometer 
O2 
Oxygen 
PCr 
Phosphocreatine 
Pi 
Inorganic phosphorus 
PPE 
(S)AE 
Physical performance enhancement 
(Serious) Adverse Event 
SPC 
Summary of Product Characteristics (in Dutch: officiële productinfomatie 
IB1-tekst) 
Sponsor 
The sponsor is the party that commissions the organisation or 
performance of the research, for example a pharmaceutical 
company, academic hospital, scientific organisation or investigator. A 
party that provides funding for a study but does not commission it is not 
regarded as the sponsor, but referred to as a subsidising party. 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
VLCADD 
Very Long Chain Acyl-CoA Dehydrogenase Deficiency 
Wbp 
Personal 
Data 
Protection 
Act 
(in 
Dutch: 
Wet 
Bescherming 
Persoonsgevens) 
WMO 
Medical Research Involving Human Subjects Act (in Dutch: Wet Medisch- 
wetenschappelijk Onderzoek met Mensen 
WOB 
Work of breathing 

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SUMMARY 
Rationale: Very Long-Chain Acyl-CoA Dehydrogenase deficiency (VLCADD) is a rare  
inborn error of fatty acid metabolism with a broad clinical presentation ranging from infant 
fatality to exercise intolerance and elevated risk of exertional rhabdomyolysis with onset in 
childhood or adolescence. These episodes of rhabdomyolysis can result in permanent 
muscle damage and deterioration of activity level of adult patients. We recently obtained 
evidence that these symptoms are in part due to a lower energetic efficiency of upper leg 
muscle fibers that aggravates reliance on carbohydrate stores in this disease, rendering the 
organ vulnerable to an exertional energy crisis (Diekman et al, PloS One 2016). While no 
effective treatment has been available for VLCADD, it has long been proposed that 
nutritional ketosis (NK) could be highly beneficial to patients. Amongst others, ketone bodies 
could take on the role of primary energy source in exercising muscle. The problem has been 
that, until now, no vehicle for establishing NK in humans without undesired side effects has 
been available. A breakthrough has finally been achieved by collaborator Kieran Clarke in 
Oxford whose team has recently produced an edible ketone ester that can achieve acute NK 
in human subjects via oral ingestion without any undesired side-effects. It was found that the 
ketone ester produced significant physical performance enhancement in rodents and human 
athletes (Cox et al., Science (in review)). 
The effect has been attributed to enhanced muscle mitochondrial function in addition to 
glycogen sparing. Here, we will investigate if acute NK in adolescent and adult symptomatic 
VLCAD deficient patients can boost muscle mitochondrial function in vivo. If so, a rational 
basis will have been established for therapeutic use in this metabolic myopathy. This study 
will include patients from the age of 16 years that could benefit from NK as well. Since 
rhabdomyolysis often has an onset in adolescence, confirmation of our hypothesis would 
lead to therapeutic use in that age group as well. Moreover, older patients often suffer from 
severe exercise intolerance and loss of muscle function due to earlier episodes of 
rhabdomyolysis, leaving most of them unable to complete our study protocol. Due to the 
rarity of the disease and earlier argumentation, it’s vital to include adolescent subjects to 
reach sufficient power in this study. As such, this study constitutes a vital first step towards 
possible validation of an effective treatment for patients with VLCADD that may improve the 
quality of life including an active lifestyle with overall health benefits.  
 
Objective: To test if acute NK boosts muscle mitochondrial function in vivo in patients with 
VLCADD in order to establish a rational basis for therapeutic use in this disorder. A 
secondary objective is to gather data to test the working hypothesis that the positive effect of 
acute NK on muscle mitochondrial function results from enhanced substrate supply to the 
tricyclic acid (TCA) cycle. 
Study design: randomised, blinded, placebo controlled, 2-way cross over trial. 

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Study population: Six Patients with VLCADD between 16 and 65 years of age, seen   
for evaluation in the VLCADD expertise centre of the UMC Utrecht. 
 
Intervention (if applicable): Oral intake of nutritional drinks; prolonged moderate-intensity exercise on 
bicycle ergometer; in vivo Magnetic Resonance Spectroscopy; muscle microbiopsy; venipuncture. 
Main study parameters/endpoints: 
o Whole body: 
Physical performance during a 45 bicycle exercise test at individual 
FATMAX workload (typically of the order of 40%MVO2). Specifically: 
- 
completion of 40 min upright exercise bout at FATMAX workload 
(yes/no; if no, #minutes) 
- 
completion of 5 min in-magnet exercise bout at FATMAX workload 
(yes/no; if no, #minutes) 
- 
subjective fatigue and muscle ache score after each exercise bout 
(scale 0-10) and 24 hours after session (telephone consultation) 
- 
HAES score questionnaire 
o Muscle: 
- 
resting quadriceps phosphocreatine (PCr) and inorganic phosphate 
concentrations; pH 
- 
exercise quadriceps phosphocreatine (PCr) and inorganic phosphate 
concentrations; pH 
- 
post-exercise rates of recovery of quadriceps PCr, Pi and pH 
- 
optional: resting and post-exercise muscle levels of glycogen, lactate, 3- 
OH butyrate, and (very) long (C18-C14) to medium (C12-C8) to short 
chain (C6-C2) acetyl-carnitines (on a patient-voluntary basis) 
o Blood: 
levels of 3-OH butyrate, glucose, bloodgas, lactate, acylcarnitines and 
creatine kinase at rest 
and post-exercise 
o Urine: myoglobine 
 
Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness: In clinical practice, many patients with VLCADD  wonder to which  
extent they are able to exercise safely. Any means of boosting this capacity will greatly 
improve the quality of life and long-term health in this patient population. Acute nutritional 
ketosis after ingestion of a novel edible ketone ester is a very promising novel therapy 
towards this aim and has already been tested with positive effects in human athletes. This 
study will gain insight whether a sound metabolic basis exists for its therapeutic use in this 
patient population. 

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Patients will be asked to participate in three study sessions: 
session I (site: nearest Academic Medical Center, Pediatric test ward). 
- standard maximal cardiopulmonary exercise test (CPET; bicycle ergometry with 
indirect calorimetry) 
objective: determination of VO2max in order to set the desired exercise workload of maximal 
individual rate of fat oxidation (FATMAX; typically ~40% MVO2) in sessions 2 and 3. 
- venipuncture 
objective: measure basal metabolic profile and rise in blood lactate and CK levels in 
response to CPET. 
 
sessions II and III (site: Neuroimaging Magnetic Resonance Center UMC Groningen). 
- venipuncture + cannulation 
objective: prepare subject for serial (every 10 min) blood sampling for off-site metabolic 
profiling 
- oral intake of nutritional drink A or B (ketone and placebo drinks, respectively) 
Each subject will receive both drinks over the course of the trial in a blinded fashion 
– i.e., if drink A in session II, then drink B in session III, and vice versa. 
- microbiopsy from the quadriceps muscle (optional) 
1st biopsy: 20 min after oral drink; 2nd biopsy: immediately following end of exercise bout 1. 
objective: metabolomic profiling of the state of the energy metabolic network in muscle and 
mapping of individual phenotypic muscle properties (fiber type, mitochondrial density, 
capillary density).Biopsies will be obtained from the m. vastus lateralis of the left leg. 
Hereto, a preparatory superficial incision of the skin and fascii will be made under local 
sedation and bandaged, allowing fast and reproducible biopsy sampling prior to and 
immediately following exercise bout 1 below. 
 
- 40 min upright bicycling exercise at FATMAX outside MR scanner 
(exercise bout 1) 
Blood samples are collected every 10 minutes. 
 
- microbiopsy from the quadriceps muscle (optional) 
Biopsy will be obtained from the m. vastus lateralis of the left leg (see above). 
 
- 5 min supine bicycling exercise at 50%MVO2 inside MR scanner (exercise bout 2) 
serial in vivo 31P MR spectra will be recorded non-invasively from the m. vastus lateralis of 
the right leg as previously (protocols 12-211/K (VLCAD; UMC Utrecht); NL41313.042.12 
(MCAD; UMG) to determine energy and proton balance in the muscle during exercise and 
the kinetics of metabolic recovery following exercise. A blood sample will be taken after the 
subject has exited the scanner room. Final blood samples including on-site analysis of 
CK levels are taken up to 3 hr post-session III to monitor any rhabdomyolytic event. 
 
- 24 hours after session II and III, co-investigator Bleeker will call each subject to evaluate 
subjective fatigue and muscle complaints after the subjet left the test facility. 
 
Time between study sessions I and II will be at least 7 days; time between study sessions II 
and III will be 7 days. For session I, patients will be asked to abstain from food 3 hrs prior to 
the test. For sessions II and III, participants will be asked to keep a ‘food diary’ for the week 
prior to the test in order to monitor the caloric intake and macronutrient content of each  
participant. For sessions II and III, patients will travel to Groningen the evening prior to the 
study and will be housed in a hotel across the street from UMCG. Patients are able to take a 

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late evening snack or meal of their choice in correspondence with their individual record of 
maximal fasting duration, given a start time of the study at 08:00 am the following day. 
Patients will be allowed the same light breakfast according their normal diet before they will 
receive the caloric drive before the test. The breakfast will be the same for session II and III. 
 
No adverse effects of this study are expected. Of the listed interventions that are proposed 
here, all but one – i.e., ingestion of the ketone caloric drink - have previously been included 
and approved in clinical investigations of metabolic myopathy patients by members of the 
investigative team in Amsterdam, Utrecht and/or Groningen: venipuncture and microbiopsy 
of the quadriceps muscle prior to and after exercise (protocol 04/1984 (MCADD; AMC 
Amsterdam); protocol NL41313.042.12 (MCADD; UMCG)); bouts of prolonged bicycle 
exercise outside and inside a MR scanner including in vivo MR spectroscopic data collection 
(protocol 12-211/K (VLCADD; UMC Utrecht; protocol NL41313.042.12 (MCADD; UMCG)). 
 
Of the interventions, only the burden of collecting tissue samples from the quadriceps 
muscle by microbiopsy is rated as moderate (as opposed to ratings of nil to minor burden for 
all other interventions1). As such, this intervention will only be performed on a voluntary 
basis; it will not affect enrollment in the study. Muscle tissue sampling is, however, needed – 
and therefore desirable - to test the secondary hypothesis that NK boosts muscular 
mitochondrial function specifically at the level of substrate supply to the TCA cycle. Without 
the biopsy data, this secondary hypothesis cannot be validated. It would be highly desirable 
to know the outcome of that test since the information is needed to develop a proper 
mechanistic understanding of any therapeutic value of ketone administration in human 
metabolic myopathies such as fatty acid oxidation defects. On these grounds, the burden of 
microbiopsy is considered justifiable. 
 
The safety of the nutritional drinks that will be administered and the expected acute, 
transient mild ketosis (concentrations of ketone bodies < 3 mM) has been thoroughly tested 
and documented in healthy subjects (see references 9,10). The drink has been designated 
as ‘GRAS’ (= Generally Recognized as Safe) by the Federal Drug Administration of the 
 
1 the short, maximum exercise test in session I will use anaerobic derived energy. As patients with 
VLCADD do not have a problem in glycolysis, the burden for this test will be nihil. The endurance test 
at submaximal level might induce muscle pain temporarily. the burden for patients is classified as 
minimal and patients will be monitored accordingly during the study period. Blood will be drawn 
intravenously from patients; this implies a minimal burden. 

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United States allowing it to be used as a foodstuff in the USA. No particular risk for VLCADD 
patients is expected with respect to oral ingestion of the ketone ester since its metabolites 
are natural, organic compounds released without any caution or proton load that are readily 
oxidizable by cellular mitochondria irrespective of enzymatic defects in fatty acid oxidation. 
 
The benefit to VLCADD patients of participation in the study is potentially enormous. In our 
previous applications for exercise and MRS testing in metabolic myopathy (protocols 12- 
211/K (VLCAD; UMC Utrecht) and NL41313.042.12 (MCAD), respectively), we put forward 
that the benefit of those studies would be that it would help establish a platform to conduct 
studies on the effectiveness of medication. Here, we now exploit this validated platform to 
test the metabolic effects of a nutritional drink that may revolutionize therapy of human 
metabolic myopathies due to defects in carbohydrate or fat metabolism. Ultimately, an 
effective treatment for VLCADD may be available for the first time to improve the quality of 
life of patients, including the opportunity to engage in a more active lifestyle with generic 
(including cardiovascular and anti-diabetic) health benefits. 

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1. INTRODUCTION AND RATIONALE 
 
Disorders of long-chain fatty acid beta-oxidation (FAO) comprise genetic deficiencies of fatty 
acid transport from the cytosol to the mitochondrial matrix and mitochondrial beta-oxidation  
of acyl-CoA compounds with a chain length of 14-20 carbon atoms. One of the  most 
common long-chain fatty acid oxidation defects in humans is very long-chain acyl-CoA 
dehydrogenase deficiency VLCADD (OMIM 201475, EC 1.3.99.13; [1]). Impairment of  
energy production from exogenous or endogenous fatty acids is the most prominent feature 
of VLCADD [1]. Clinical symptoms, therefore, arise or are exacerbated during catabolic 
situations e.g. during exercise, illness or fasting when lipids are released from endogenous 
stores and have to be used as energy substrate. Organs most frequently involved are those 
using long-chain fatty acids as primary energy source, such as the heart and skeletal 
muscles. Age at onset, manifestation patterns and clinical severity differ between patients. 
The most severe presentations are Reye-like symptoms, cardiomyopathy and SIDS (sudden 
infant death syndrome) in the neonatal period. Milder clinical phenotypes manifest with 
hypoketotic hypoglycaemia during intercurrent illness and exercise-induced skeletal 
myopathy as well as episodic rhabdomyolysis. Patients may present with different symptoms 
at different ages. 
 
Rhabdomyolyis 
One of the most prominent symptoms of VLCADD patients is myopathy [1,2]. It most often 
involves the breakdown of muscle (rhabdomyolysis). Myopathy is provoked by fasting, fever, 
cold, medication and exercise from infancy to adulthood, but often has its onset in 
adolescence. In clinical practice, parents of pediatric patients and adult patients themselves 
wonder therefore to which extent they will be able to perform exercise. The pathophysiology 
of myopathy and rhabdomyolysis in general is not completely understood. The likely 
mechanisms behind the exercise-induced rhabdomyolysis and myopathy in VLCADD are a 
failing compensatory mechanism during requirement of extra energy (eg during exercise, 
fever, fasting). The symptoms are thought to result from one or more different 
pathophysiological mechanisms: (i) energy shortage in (exercising) muscle due to the FAO 
defect; (ii) toxicity caused by the accumulation of FAO intermediates. Reported muscle 
biopsies show a range from normal muscle fibre structure, to diffuse lipid accumulation and 
aspecific abnormalities. No ragged red fibres have been reported. Based on the recurrent 
episodes of rhabdomyolysis, myopathy in VLCADD might also be caused by meta-
flammation: inflammation caused by abnormal metabolites. The accumulation of long chain 
acylcarnitine esters has been implicated in muscle pathology in VLCADD, particularly in the 
heart. But, there is also evidence that ischemic changes in  heart 

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might be due to impaired energy production rather than from long chain acylcarnitines 
accumulation, emphasizing the importance of available free carnitine to recycle CoA. 
 
Therapy in VLCADD. 
Clinical management of VLCADD patients poses a major challenge. At present, there is no 
effective therapy to alleviate the debilitating condition of severe exercise intolerance and 
myalgia with risk of episodic rhabdomyolysis [3]. Patients are typically advised to engage 
only in light physical work in combination with timely and ample intake of carbohydrates 
and/or medium-chain triglycerides (MCT). It has, however, long been hypothesized that 
nutritional ketosis (NK) could be highly beneficial to patients with metabolic myopathies such 
as VLCADD [4]. Amongst others, ketone bodies could take on the role of primary energy 
source in exercising muscle. The problem has been, however, that, until now, no vehicle for 
establishing NK in humans without undesired side effects has been available [4]. 
 
Ketone bodies the evolutionary adaptation to power metabolism 
Ketone bodies, acetoacetate and D-β-hydroxybutyrate, are the metabolic response to energy 
crisis, representing a powerful evolutionary mechanism to sustain life by altering oxidative 
fuel selection [5]. The teleological advantages of ketone body metabolism are clear during 
conditions where survival of the organism depends upon optimal mitochondrial  efficiency, 
and conservation of fuel reserves Ketosis may also provide thermodynamic advantages over 
other carbon substrates by increasing the free energy conserved in ATP (ΔGATP) by the 
oxidation of ketones during mitochondrial oxidative phosphorylation [6]. The combination of 
improved energetic efficiency and fuel sparing is vitally important not only during famine, but 
could also provide clues to new methods of sustaining human performance. Recent work 
within the group of collaborator Kieran Clarke at University of Oxford on the effects of oral 
administration of ketone bodies during exercise has demonstrated a significant, 4% 
improvement in the power output of elite athletes over 30 minutes (seated rowing ergometer; 
Figure below; [7]). This degree of improvement in this population of athletes is of significant 
competitive advantage. 

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The effects of acute nutritional ketosis on physical performance in athletes. 
(A). Protocol and nutritional composition of ketone and control drinks. (B). blood concentrations of 
ketone body over time showing increased ketosis after ingestion of ketone drink and oxidation of 
ketone from blood stream during exercise. (C). Bland-Altman plot of performance. (D) Uniform 
erformance improvement over all weight and gender classes of athlete. (E). Power profiles over 30 
min of maximal exercise, with a 4% greater power output in the ketone arm at the end of exercise vs. 
control. (from [7]). 
 
Harnessing ketosis for the preservation of muscle performance 
In order to harness the physiological advantages of ketosis, a ketone ester was developed to 
deliver nutritional ketosis without elevations in fatty acids, or depletion of carbohydrate 
reserves (as is seen in starvation or endogenous ketosis) [8]. This exogenous form of ketosis 
enables the creation of a novel physiological condition with which to re-evaluate human 
performance, and metabolic regulation. Developed by TDeltaS Ltd. in association with the 
University of Oxford, this nutritional ketone body is not commercially available and to this 
point has been tested on over 200 people without adverse effects The safety, tolerability and 
toxicology profile of this ester has been published previously [9]. The drink has been 
designated as GRAS by the FDA allowing it to be used as a foodstuff in the USA. The ketone 
ester has previously been used in clinical research by Dr Pete Cox at the University of 
Oxford; LREC reference numbers O7/Q1605/37, 10/H0605/10, 11/SC/0223, 14/EE/0063, 
and 14/LO/0288 as well as in previous work conducted in partnership with the UK military 
MODREC 298/PPE/11. In accordance with University of Oxford policy, the Intellectual 
property rights to this ketone ester are linked to an investigator (prof dr Kieran Clarke) who 
has a declared interest. 

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Ketosis alters muscle fuel metabolism in working muscle 
Strong supportive evidence for the importance of ketosis in altering working muscle 
metabolism dates back in excess of 60 years. Most notably the work of Randle in 1963, who 
demonstrated that the presence of ketone bodies inhibits glycolytic flux and increases the 
quantity of glycogen in both rat cardiac muscle and diaphragm [10] . The thermodynamic 
potential of ketosis was shown by Kashiwaya and colleagues, who demonstrated a dramatic 
28% increase in hydraulic efficiency over glucose by the addition of ketones to the perfusate 
of a working heart model [11]. 
We have recently characterised the effects of nutritional ketosis on skeletal muscle 
metabolism, showing the clear evolutionary advantages of ketosis in sparing not only 
carbohydrate and fat metabolism, but in preserving muscle protein breakdown. The  
metabolic alterations induced by nutritional ketosis on the major energy pathways before and 
after exercise in humans are shown below. 
 
 
The effects of nutritional ketosis on muscle metabolism before (pre) and after (post) 1 hour of exercise 
at 75% of WMax. (A). Branched chain amino acid (BCAA) concentrations are significantly reduced 
following ketosis vs. other nutritional substrates. (B). Branched chain amino acid levels in muscle are 
related to pyruvate levels as the end products of glucose breakdown. (C). Conservation of muscle 
BCAA is proportional to the muscle ketone concentration, as a result of ketone bodies reducing  
muscle glucose use (D). (E). Schematic representation of the differences between fat and glucose 
metabolism in exercise are shown vs. ketosis (F). (From: [7]) 

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Nutritional intervention in metabolic myopathy 
Enzymatic deficiencies affecting the metabolism of fats are multiple, albeit rare disorders 
characterized by consistent clinical phenotypes. The primary symptoms of defective 
metabolism of fat vary in severity depending on the metabolic location of the defect, and the 
degree of expression. However common to almost all conditions are an intolerance of 
exercise, skeletal or cardiac muscle myopathy, and a reliance on exogenous dietary fuels to 
sustain muscular energy demands. 
 
Whilst the mainstay of therapy for these patients is the maintenance of blood borne 
substrates via adherence to various diets [12-14], these regimes are often inadequate to 
allow a normal physical lifestyle. In severe cases muscle damage, and other organ 
dysfunction reduces life expectancy and can render patients severely incapacitated. An 
alternative nutritional strategy to supply fuels in the form of ketone bodies may offer a new 
approach in the management of these conditions, without the unwanted effects of bolus 
glucose intake [3], or unpalatable dietary intervention. 
 
Ketosis in metabolic myopathy 
Previously, inducing a physiological ketosis required unpleasant Atkin’s style high fat, low 
carbohydrate diets [15], or infusions of dissolved ketones salts [16]. Neither of these methods 
are applicable in VLCAD deficiency for obvious reasons. Now it is possible to deliver 
nutritional ketone bodies in drink form, making the oral nutritional delivery of ketones possible 
for the first time in humans [7]. This proposal seeks approval for a first clinical investigation 
into the possible benefits of acute NK delivered via the ketone ester drink in human VLCADD 
patients, specifically the metabolic basis for any enhanced muscle function. 
 
Collaboration with the Dutch VLCADD expertise centre 
Since 2007 Very Long Chain Acyl-CoA Dehydrogenase Deficiency (VLCADD) is included in 
the Dutch neonatal screening program (NSP). VLCADD is a rare disease (1:80000) and has 
only been discovered recently in 1992. Knowledge about disease course and long term effect 
of therapy is scarce. Therefore, the UMC Utrecht in collaboration with the Laboratory Genetic 
Metabolic Diseases (Academic Medical Center Amsterdam) and RIVM started an expertise 
centre in which all patients with VLCADD in the Netherlands can be seen. The primary goal  
of this expertise center is to document the clinical and biochemical phenotype and disease 
course of all diagnosed VLCADD patients in the Netherlands (METC nr 10-430/C). The 
Center partakes in the present investigative team to investigate the clinical benefits of acute 
NK in human VLCADD (dr Visser). The PI of the present application (dr Jeneson) has 

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previously collaborated with the center in a clinical investigation into muscle ATP metabolism 
in VLCADD patients (protocol 12-211/K (UMC Utrecht)). 
 
Inclusion of adolescent patients 
Due to the rarity of the disease this study will include patients from the age of 16 years that 
could benefit from NK as well. Since rhabdomyolysis often has an onset in adolescence, 
confirmation of our hypothesis would lead to therapeutic use in that age group as well. 
Moreover, older patients often suffer from severe exercise intolerance and loss of muscle 
function due to earlier episodes of rhabdomyolysis, leaving most of them unable to complete 
our study protocol. Originally, all adult VLCADD patients known by the Dutch expertise 
center were provided with information on the study. Unfortunately, 1.5 years after the first 
inclusion only four patients participated. The main reason the other patients did not want to 
participate was the intensity of the protocol. Although the exercise test is at moderate 
intensity, the physical condition of these patients withholds them from riding a bike. 
Therefore an amendment was made to the original protocol to lower the minimum age to 16 
years to reach sufficient power for this study. Unfortunately not many adolescent patients are 
known in The Netherlands, lowering the minimum age to 16 years will make participation 
possible for one patient. Lowering the minimum age even further won’t make a difference, 
since the patients between 10-16 years are both rare and severely affected. 
 
In vivo 31P MRS read-out of muscle mitochondrial performance 
Phosphorus magnetic resonance spectroscopy (31P MRS) allows for non-invasive read-out of 
intramuscular pH and concentrations of Pi, PCr and ATP in human tissues (17). As such, 
dynamic 31P MRS recording of these metabolic variables in muscle offers a window onto the 
integrated in vivo performance of the cellular metabolic networks that produce ATP from the 
breakdown of fat and carbohydrates to maintain energy balance during exercise. 31P MRS- 
derived parameters such as the rate constant of PCr and Pi recovery post-exercise offer 
unsurpassed indices of in vivo mitochondrial function that have proven most useful in the 
clinical investigation of human metabolic myopathy (17). Recently, we applied this method to 
investigate energy balance in quadriceps muscle of VLCADD patients during an endurance 
exercise task (Diekman, manuscript in preparation). We found no evidence for impaired 
mitochondrial ATP synthetic function. However, changes in Pi and PCr during a standardized 
in-magnet bicycling exercise test were threefold larger than in healthy controls suggesting 
lower contractile efficiency of VLCADD muscle. Such a muscle phenotypic adaptation would 
aggravate the problem in VLCADD of exclusive dependence on turnover of the finite muscle 
carbohydrate store for ATP production, in turn explaining the known elevated risk for 
exertional rhabdomyolysis in VLCADD (Diekman, manuscript in preparation). Conversely, 
these results suggest that any boost of muscle mitochondrial function from acute NK should 

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be an effective therapy to improve exercise tolerance and lower the risk of exertional 
rhabdomyolisis in VLCADD. 
 
2. OBJECTIVES 
 
1. To investigate the potential of a novel dietary substrate preparation to  enhance 
muscle mitochondrial function in VLCADD via acute nutritional ketosis. 
 
2. To investigate if tri-citric acid cycle substrate supply in muscle of patients with 
VLCADDis boosted by acute nutritional ketosis. 
 
3. STUDY DESIGN 
 
This is a randomised, blinded, placebo controlled, two-way cross over trial. Patients with 
VLCADD will be recruited by the VLCADD Expertise Center at UMC Utrecht. Nutritional  
ketosis will be compared to a placebo, delivered as an isocaloric drink before exercise. An 
individualised, standardised bicycle exercise protocol will be completed by each participant 
with collection of blood metabolites, respired gases, and subjective ratings of fatigue and 
muscle complaints. In addition in vivo 31P MR Spectroscopic recordings will be made from  
the quadriceps muscle before (resting), during and post-exercise. An optional muscle biopsy 
will be obtained from the quadriceps before and after exercise. 
Patients will be asked to participate in three study sessions: 
 
session I (site: nearest Academic Medical Center, Pediatric test ward). 
- standard maximal cardiopulmonary exercise test (CPET; bicycle ergometry with 
indirect calorimetry) 
objective: determination of VO2max in order to set the desired exercise workload of maximal 
individual rate of fat oxidation (FATMAX; typically ~40% MVO2) in sessions II and III below. 
- venipuncture 
objective: measure rise in blood lactate and creatine kinase (CK) levels in response to CPET 
 
sessions II and III (site: Neuroimaging Magnetic Resonance Center UMC Groningen). 
- venipuncture + canulation 
objective: prepare subject for serial (every 10 min) blood sampling for off-site metabolic 
profiling 
- oral intake of nutritional drink A or B (ketone and placebo drinks, respectively) 
Each subject will receive both drinks over the course of the trial in a blinded fashion 
– i.e., if drink A in session II, then drink B in session III, and vice versa. 
- microbiopsy from the quadriceps muscle (optional) 
1st biopsy: 20 min after oral drink; 2nd biopsy: immediately following end of exercise bout. 
objective: metabolomic profiling of the state of the energy metabolic network in muscle and 
mapping of individual phenotypic muscle properties (fiber type, mitochondrial density, 
capillary density).Biopsies will be obtained from the m. vastus lateralis of the left leg. 
Hereto, a preparatory superficial incision of the skin and fascii will be made under local 
sedation and bandaged, allowing fast and reproducible biopsy sampling prior to and 
immediately following exercise bout 1 below. 
 

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- 40 min upright bicycling exercise at FATMAX outside MR scanner 
(exercise bout 1) 
Blood samples are collected every 10 minutes. 
Subjects state subjective fatigue and muscle ache following exercise bout. 
 
- microbiopsy from the quadriceps muscle (optional) 
Biopsies will be obtained from the m. vastus lateralis of the left leg (see above). 
 
- 5 min supine bicycling exercise at FATMAX inside MR scanner (exercise bout 2) 
serial in vivo 31P MR spectra will be recorded non-invasively from the m. vastus lateralis of 
the right leg as previously (protocols 12-211/K (VLCAD; UMC Utrecht); NL41313.042.12 
(MCAD; UMG) to determine energy and proton balance in the muscle during exercise and 
the kinetics of metabolic recovery following exercise. Subjective fatigue and muscle ache 
scores and a blood sample will be taken after the subject has exited the scanner room. Final 
blood samples including on-site analysis of CK levels are taken up to 3 hr post-session III to 
monitor any rhabdomyolytic event. 
 
- 
24 hours after session II and III, co-investigator Bleeker will call each subject to evaluate 
subjective fatigue and muscle complaints after the subjet left the test facility. 
 
Time interval between study sessions I and II will be at least 7 days; between study sessions 
II and III: 7 days. The night prior to sessions II and III, patients will be housed in close vicinity 
to the UMCG. Patients will be instructed to take a late evening snack or meal according to 
their individual record of maximal fasting duration, given a start time of the study at 08:00 am 
the following day. Patients will be allowed a light breakfast according their normal diet before 
they will receive the caloric drive before the test. The breakfast will be the same for session II 
and III. 
 
The total duration of the first session will be around 45 minutes, with 30 minutes of 
preparation and 8-12 minutes of cycling (exact time length dependent on the maximal 
endurance of the participant). The total duration of the second and third session will be 
around 5 hours including 45 minutes of preparation, 45-60 minutes of bicycling for the 
submaximal endurance exercise test and 3 hours of post-exercise resting where after the last 
blood and urine sample will be collected. During this 3 hour period, participants will be asked 
to fill out a HAES diary and can watch a DVD to pass time. 
 
 
 

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4. STUDY POPULATION 
4.1 Population (base) 
Six patients with VLCADD between 16 and 65 years of age, seen by dr. G. Visser          
for evaluation in the VLCADD expertise center of the UMC Utrecht. 
 
4.2 Inclusion criteria 
• 
Confirmed VLCADD by genetic profiling. 
• 
age 16-65. 
 
4.3 Exclusion criteria 
• 
contraindications for MRI studies (assessed by standardised questionnaire as 
previously used in METC 08-267/K; see UMCG section F METC documents) 
• 
inability to perform bicycle exercise. 
• 
recent episode of rhabdomyolysis, or treatment for acute renal failure in the past 2 
months. 
• 
intercurrent illness which may influence exercise tolerance (anaemia, musculoskeletal 
injury, or other undiagnosed illness under investigation). 
• 
known coronary artery disease, positive history for angina, or changes on ECG 
suggestive of previous ischaemia without a negative stress test. 
• 
insulin-dependent diabetes mellitus. 
• 
loss of, or an inability to give informed consent. 
• 
pregnancy or current breastfeeding, or females not taking the oral contraceptive pill 
(this is due to the variability in hormonal patterns and substrate levels with different 
parts of the menstrual cycle). 
• 
any other cause which in the opinion of the investigators, may affect the volunteers 
ability to participate in the study 
 
4.4 Sample size calculation 
Exact sample size calculation for this study is difficult for several reasons: 
 
1. VLCADD is a rare metabolic disorder. The UMCU Center currently has 6 patients that 
may qualify for this particular study. 
2. The proposed study is the very first of its kind. No equivalent means of imposing 
acute NK has previously been available. As such, there are no data available on the 
magnitude of any enhancement effect of the nutritional intervention on metabolic 
performance in this particular group of patients. 
 
As mentioned in Section 3, this study is a randomised, blinded, placebo controlled, two-way 
cross over trial. In previous case-control design studies, it has been well established that 
≥30% differences in the two categories of MRS-derived metabolic parameters – i.e., steady- 
state muscle pH, Pi and PCr levels, and kinetic rate constants of metabolic recovery post- 
exercise - can be reliably and reproducibly identified by the proposed investigational methods 
(e.g. [18]). We previously successfully detected statistically significant abnormalities in 
muscle MRS parameters (P<0.05; two-sided) in a case-control study of VLCADD featuring 5 
patients (Diekman et al, PloS One 2016). 

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5. TREATMENT OF SUBJECTS 
 
5.1. Investigational product/treatment 
Nutritional drink and placebo (“sports drinks”). 
 
5.2. Use of co-intervention 
Prolonged moderate-intensity exercise. 
 
5.3. Escape medication 
In case of physical complaints, the exercise will be terminated prematurely. Paracetamol is 
available if needed by the patient. Additionally, dextrose tablets will be available to administer 
when clinical symptoms of hypoglycaemia (e.g. headache, or light headedness) occur. 
 
6. INVESTIGATIONAL MEDICINAL PRODUCT 
This section is not applicable for this study. 
 
7. NON-INVESTIGATIONAL PRODUCT 
This section is not applicable for this study 
 
8. METHODS 
8.1 Study parameters/endpoints 
 
8.1.1 Main study parameter/endpoint 
 
(i) steady-state in vivo intramuscular levels of Pi, PCr, and pH during exercise versus rest. 
(ii) kinetic rate constants of metabolic recovery post-exercise. 
 
8.1.2 Secondary study parameters/endpoints 
- completion of 40 min upright bicycling bout at FATMAX (yes/no; if no, #minutes) 
- completion of 5 min supine bicycling bout at FATMAX in scanner (yes/no; if no, #minutes) 
- subjective fatigue and muscle ache score after each exercise bout (scale 0-10) and 24 
hours after exercise 
- blood levels of 3-OH butyrate, acetoacetate, acylcarnitines, glucose, lactate and pH prior to 
and post-exercise. 
- optional: muscle levels of glycogen, lactate, 3-OH butyrate, and (very) long (C18-C14) to 
medium (C12-C8) to short chain (C6-C2) acetyl-carnitines prior to and immediately post- 
exercise (on a patient-voluntary basis) 
 
8.2 Randomisation, blinding and treatment allocation 
Patients will be handed an unlabeled caloric drink (placebo and ketone ester drink) prior to 
the endurance exercise bout in each of the two study sessions at UMCG. All patients will 
receive both drinks over the course of the study. Co-investigator Bleeker, MD, who will 

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conduct the studies at UMCG together with PI Jeneson, will plan, randomize and record the 
order of placebo and ketone ester administration to the patients. Jeneson, who will analyze  
all MRS data, will not have, nor seek access to, these records thus ensuring blinded MRS 
data analysis. 
 
8.3 Study procedures 
 
The study is introduced to the patient by co-investigator Bleeker, MD, of the VLCADD 
expertise 
center 
in 
Utrecht 
in 
correspondence 
with 
their 
individual 
metabolic 
paediatrician/internist. The study will be announced during the ‘patient information day’ that’s 
annually held by the  the lcFAO expertise centre and by electronic newsletter to which   most 
VLCADD patients or their caretakers are subscribed. Eligible patients will receive an 
information letter about the study. After a two week consideration period co-investigator 
Bleeker will send a reminder letter to subjects who did not respond to the inquiry. This letter 
will repeat the invitation to participate in the study. If after one more week the subject fails to 
reply, it is assumed that the subject is not interested and he or she will not be contacted any 
further. If the subject is interested and has given informed consent, the patient is included in 
the study, and the subject will be checked for the inclusion- and exclusion criteria The 
investigator informs the subject about final inclusion, or possible exclusion of the study on 
basis of the criteria. 
 
Session I: Determination of the VO2max (CPET) and FATMAX. 
Participants are asked to cycle at maximal speed on an ergometric bicycle on a functional 
test ward at the nearest UMC location (AMC, UMC Utrecht or UMC Groningen). Prior to this 
session they will be asked to complete a HAES questionnaire containing questions on 
exercise frequency and the presence of physical complaints during or after exercise. 
 
Sessions II and III. 
In the week prior to testing, participants will be asked to keep a ‘food diary’ for the week prior 
to the test in order to monitor the caloric intake and macronutrient content of each participant. 
For sessions II and III, patients will travel to Groningen the evening prior to the study and will 
be housed in a hotel across the street from UMCG. Patients will be instructed to take a late 
evening snack or meal in correspondence with their individual record of maximal fasting 
duration, given a start time of the study at 08:00 am the following day. Patients will be 
allowed the same light breakfast according their normal diet before they will receive the 
caloric drive before the test. The breakfast will be the same for session II and III. 
 
Venipuncture and blood sampling: 
Blood samples of 1-2 ml will be drawn via a 3-way tap from a 22 G venous canula (for 
subject comfort) inserted into a forearm vein at the start of each study visit. Blood samples 

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for free fatty acids, glucose and other assays will be taken and stored on ice, subsequently 
centrifuged, and stored at -80 until further processing. Up to 7 samples will be taken from the 
canula (~15 ml of blood total): #1 and #2 at baseline (in duplo), #3-5 during exercise (every 
10 min), #6 after 40 minutes of moderate-intensity exercise, and #7 after the final MRS- 
analysis when the participant has recovered from exercise. 
 
Muscle biopsy: 
Participants are given the option at the start of the trial of undergoing muscle biopsy. If the 
patient has given informed consent to muscle biopsy, samples will be taken before and after 
exercise testing protocol during one or both study visits (2 samples/run; maximal of four 
biopsies for the complete study). 0.25 gm. of muscle tissue will be collected using 
percutaneous needle biopsies from the lower third of the Vastus Lateralis muscle with a 
biopsy gun (Bard Monopty, Bard Biopsy Systems, USA; a sterile standard biopsy technique 
(SOP).Muscle biopsy will be obtained from new incisions under local anaesthesia. Tissue 
samples will be frozen immediately in liquid nitrogen and stored at -80 oC until further 
analysis. Assays include glycogen and the metabolites of the glycolytic, TCA cycles. If 
sufficient tissue is available, enzymatic activity of mitochondrial and cytosol proteins, muscle 
fibre types, amino acids, and fat stores will also be determined. 
 
Storage of tissue samples 
All samples will be frozen immediately and stored at -80o C in the laboratory for Liver, 
Digestive and Metabolic diseases of the department of Paediatrics at UMCG. 
 
31P MRS 
MR data gathering will be performed using a 3.0 T whole-body MR-scanner (Intera; Philips 
Medical Systems, Best, The Netherlands). Subjects are introduced to the scanner and 
exercise apparatus and instructed. MRS data gathering will be performed with a surface coil 
wrapped over the quadriceps muscle. Conventional anatomical MRI scans will be acquired 
for positioning and scanner preparation. 31P MR spectra will be acquired serially to measure 
dynamics in intramuscular PCr, ATP, Pi, and pH using standard 31P-MRS protocols. 
 
8.4 Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without 
any consequences. Subjects can withdraw from the study by writing the coordinating 
investigator a letter, stating that they would like to withdraw from the study. The 
investigator can decide to withdraw a subject from the study for urgent medical 
reasons. 
 
8.4.1 Specific criteria for withdrawal (if applicable) 
Not applicable. 
 
8.5 Replacement of individual subjects after withdrawal 
In case a patient decides to withdraw from the study, a new patient will be recruited if 

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possible. If a patient decides to withdraw from the study prematurely because of clinical 
complaints during the prolonged moderate-intensity exercise, no new participant will be 
recruited. The data that have been obtained before termination will then be used as much as 
possible during data analysis. 
 
8.6 Follow-up of subjects withdrawn from treatment 
Patients who decide to withdraw from the study will be followed at the outpatient clinic at 
regular intervals. The follow-up scheme will remain similar to the scheme that was followed 
before participation in the study. Withdrawal from the study has no consequences for 
treatment and follow-up of the patients. 
 
8.7 Premature termination of the study 
 
Patients will have to terminate the study prematurely in the unlikely event of (symptoms of) 
hypoglycaemia during exercise. Dextrose tablets will be available to increase the blood 
glucose concentration in case of such a hypoglycaemia. 
 
9. SAFETY REPORTING 
9.1 Section 10 WMO event 
In accordance to section 10, subsection 1, of the WMO, the investigator will inform the 
subjects and the reviewing accredited METC if anything occurs, on the basis of which it 
appears that the disadvantages of participation may be significantly greater than was 
foreseen in the research proposal. The study will be suspended pending further review by 
the accredited METC, except insofar as suspension would jeopardise the subjects’ 
health. The investigator will take care that all subjects are kept informed. 
 
9.2 Adverse and serious adverse events 
Adverse events are defined as any undesirable experience occurring to a subject during 
the study, whether or not considered related to prolonged moderate-intensity exercise or 
MRS-analysis. All adverse events reported spontaneously by the subject or observed by 
the investigator or his staff will be recorded. 
A serious adverse event is any untoward medical occurrence or effect that at any dose: 
- results in death; 
- is life threatening (at the time of the event); 
- requires hospitalisation or prolongation of existing inpatients’ hospitalisation; 
- results in persistent or significant disability or incapacity; 
- is a congenital anomaly or birth defect; 
- is a new event of the trial likely to affect the safety of the subjects, such as an 
unexpected outcome of an adverse reaction, lack of efficacy of an IMP used for the 
treatment of a life threatening disease, major safety finding from a newly completed 
animal study, etc. 

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All SAEs will be reported through the web portal ToetsingOnline to the accredited METC 
that approved the protocol, within 15 days after the sponsor has first knowledge of the 
serious adverse reactions. 
SAEs that result in death or are life threatening should be reported expedited. The 
expedited reporting will occur not later than 7 days after the responsible investigator has 
first knowledge of the adverse reaction. This is for a preliminary report with another 8 
days for completion of the report. 
 
9.2.1 Suspected unexpected serious adverse reactions (SUSAR) 
Chapter 9.2.2 is not applicable for this study. 
Adverse reactions are all untoward and unintended responses to an investigational 
product related to any dose administered. 
Unexpected adverse reactions are adverse reactions, of which the nature, or 
severity, is not consistent with the applicable product information (e.g. Investigator’s 
Brochure for an unapproved IMP or Summary of Product Characteristics (SPC) for 
an authorised medicinal product). 
The sponsor will report expedited the following SUSARs through the web portal 
ToetsingOnline to the METC: 
− SUSARs that have arisen in the clinical trial that was assessed by the METC; 
− SUSARs that have arisen in other clinical trials of the same sponsor and with the 
same medicinal product, and that could have consequences for the safety of the 
subjects involved in the clinical trial that was assessed by the METC. 
The remaining SUSARs are recorded in an overview list (line-listing) that will be 
submitted once every half year to the METC. This line-listing provides an overview 
of all SUSARs from the study medicine, accompanied by a brief report highlighting 
the main points of concern. 
The expedited reporting of SUSARs through the web portal ToetsingOnline is 
sufficient as notification to the competent authority. 
The sponsor will report expedited all SUSARs to the competent authorities in other 
Member States, according to the requirements of the Member States. 
The expedited reporting will occur not later than 15 days after the sponsor has first 
knowledge of the adverse reactions. For fatal or life threatening cases the term will 
be maximal 7 days for a preliminary report with another 8 days for completion of the 
report. 

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9.3 Annual safety report 
In addition to the expedited reporting of SUSARs, the sponsor will submit, once a 
year throughout the clinical trial, a safety report to the accredited METC, competent 
authority, Medicine Evaluation Board and competent authorities of the concerned 
Member States. 
This safety report consists of: 
− a list of all suspected (unexpected or expected) serious adverse reactions, along 
with an aggregated summary table of all reported serious adverse reactions, 
ordered by organ system, per study; 
− a report concerning the safety of the subjects, consisting of a complete safety 
analysis and an evaluation of the balance between the efficacy and the 
harmfulness of the medicine under investigation. 
 
9.4 Follow-up of adverse events 
All adverse events will be followed until they have abated, or until a stable situation has 
been reached. Depending on the event, follow up may require additional tests or medical 
procedures as indicated, and/or referral to the general physician or a medical specialist. 
 
9.5 Data Safety Monitoring Board (DSMB) 
Due to the short duration of the study, no Data Safety Monitoring Board will be appointed. 
 
 
10 STATISTICAL ANALYSIS 
10.1. Descriptive statistics 
Data will be expressed both quantitatively (values of PCr, ATP, Pi, pH, rate constants 
tau_PCr and tau_Pi,  expressed as median and range), and qualitatively (MRS-spectra). 
 
10.2 Univariate analysis 
Results will be presented by means of confidence intervalsThe significance level will be set 
at p<0.05. Statistical analysis of the results will be performed using standard two-sided 
Mann-Whitney testing and will be conducted using Origin software (Caltech, USA, version 
6.1). 
 
10.3 Multivariate analysis 
not applicable 
 
10.4 Interim analysis 
Not applicable. 

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11. ETHICAL CONSIDERATIONS 
11.1 Regulation statement 
The study will be conducted according to the principles of the Declaration of Helsinki Brazil, 
October 2013, and in accordance with the Medical Research Involving Human Subjects Act 
(WMO). 
11.2 Recruitment and consent 
Patients will be informed about the study during follow-up visits to the outpatient clinic, or 
during the patient information day that is organized every year in the UMC Groningen. They 
will be recruited by means of information that is administered both orally, and written in a 
patient information letter. After information has been administered orally, potential 
participants will receive written patient information. The physician will inform the patients and 
supply the written patient information. Patients will be asked to reply to the request within 14 
days after oral and written information has been provided. This period can be extended 14 
days extra. If after 14 days patients didn’t reply after the provided written information, they 
will receive a recall letter. If subjects don’t respond after 14 days they wil not be contacted 
any further. In order to approve of participation in the study, participants have to return the 
signed informed consent form by mail to the coordinating investigator. After the informed 
consent form has been received, the coordinating investigator will contact the participant by 
phone to provide further details if needed. All participants will be asked to sign a written 
consent form. 
 
11.4 Benefits and risks assessment, group relatedness 
In clinical practice, many patients with VLCADD wonder to which extent they are able to 
exercise safely. Any means of boosting this capacity will greatly improve the quality of life  
and long-term health in this patient population. Acute nutritional ketosis after ingestion of a 
novel edible ketone ester is a very promising novel therapy towards this aim and has already 
been tested with positive effects in human athletes. This study will gain insight whether a 
sound metabolic basis exists for its therapeutic use in this patient population. 
 
No adverse effects of this study are expected. Of the listed interventions that are proposed 
here, all but one – i.e., ingestion of the ketone caloric drink - have previously been included 
and approved in clinical investigations of metabolic myopathy patients by members of the 
investigativce team in Amsterdam, Utrecht and/or Groningen: venipuncture and microbiopsy 
of the quadriceps muscle prior to and after exercise (protocol 04/1984 (MCADD; AMC 
Amsterdam); protocol NL41313.042.12 (MCADD; UMCG)); bouts of prolonged bicycle 
exercise outside and inside a MR scanner including in vivo MR spectroscopic data collection 
(protocol 12-211/K (VLCADD; UMC Utrecht; protocol NL41313.042.12 (MCADD; UMCG)). 
Of the interventions, only the burden of collecting tissue samples from the quadriceps 
muscle by microbiopsy is rated as moderate (as opposed to ratings of nil to minor burden for 

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all other interventions2). As such, this intervention will only be performed on a voluntary 
basis; it will not affect enrollment in the study. Muscle tissue sampling is needed to test the 
secondary hypothesis that NK boosts muscular mitochondrial function specifically at the level 
of substrate supply to the TCA cycle; therefore, the burden of microbiopsy is considered 
justifiable. 
 
The safety of the nutritional drinks that will be administered and the expected acute, 
transient mild ketosis (concentrations of ketone bodies < 3 mM) has been thoroughly tested 
and documented in healthy subjects (see references 9,10). The drink has been designated 
as ‘GRAS’ (= Generally Recognized as Safe) by the Federal Drug Administration of the 
United States allowing it to be used as a foodstuff in the USA. No particular risk for VLCADD 
patients is expected with respect to oral ingestion of the ketone ester since its metabolites 
are natural, organic compounds released without any cation or proton load that are readily 
oxidizable by cellular mitochondria irrespective of enzymatic defects in fatty acid oxidation. 
 
The benefit to VLCADD patients of participation in the study is potentially enormous. In our 
previous applications for exercise and MRS testing in metabolic myopathy (protocols 12- 
211/K (VLCAD; UMC Utrecht) and NL41313.042.12 (MCAD), respectively), we put forward 
that the benefit of those studies would be that it would help establish a platform to conduct 
studies on the effectiveness of medication. Here, we now exploit this validated platform to 
test the metabolic effects of a nutritional drink that may revolutionize therapy of human 
metabolic myopathies due to defects in carbohydrate or fat metabolism. Ultimately, an 
effective treatment for VLCADD may be available for the first time to improve the quality of 
life of patients, including the opportunity to engage in a more active lifestyle with generic 
(including cardiovascular and anti-diabetic) health benefits. 
11.5 Compensation for injury 
The sponsor/investigator has a liability insurance which is in accordance with article 7, 
subsection 6 of the WMO. 
The sponsor (also) has an insurance which is in accordance with the legal requirements in the 
Netherlands (Article 7 WMO and the Measure regarding Compulsory Insurance for Clinical Research 
in Humans of 23th June 2003). This insurance provides cover for damage to research subjects 
through injury or death caused by the study. 
 
 
 
 
2 the short, maximum exercise test in session I will use anaerobic derived energy. As patients with 
VLCADD do not have a problem in glycolysis, the burden for this test will be nihil. The endurance test 
at submaximal level might induce muscle pain temporarily. the burden for patients is classified as 
minimal and patients will be monitored accordingly during the study period. Blood will be drawn 
intravenously from patients; this implies a minimal burden. 

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1. € 450.000,-- (i.e. four hundred and fifty thousand Euro) for death or injury for each 
subject who participates in the Research; 
2. € 3.500.000,-- (i.e. three million five hundred thousand Euro) for death or injury for 
all subjects who participate in the Research; 
 
3. € 5.000.000,-- (i.e. five million Euro) for the total damage incurred by the organisation for 
all damage disclosed by scientific research for the Sponsor as ‘verrichter’ in the meaning of 
said Act in each year of insurance coverage. 
The insurance applies to the damage that becomes apparent during the study or within 4 
years after the end of the study. 
 
11.6 Incentives (if applicable) 
Not applicable. 
 
 
12. ADMINISTRATIVE ASPECTS AND PUBLICATION 
12.1. Handling and storage of data and documents 
Each participant will obtain a code, and data will be 
handled according to this code. The code cannot be directly traced back to the participant. 
The key to the code is stored in a file on the computer of the principal investigator, which is 
secured by a login code and password of which only the coordinating investigator knows. 
Only the principal investigator, the coordinating investigator and the Monitor Board have 
access to the source data. 
Signed informed consent forms will be stored in a separate research file of the patient. These 
files will be stored in a cabinet with a lock, together with the signed informed consents of the 
control participants. Only the coordinating investigator has the key to this lock. The data are 
kept until 20 years after termination of the study. 
 
12.2 Monitoring and Quality assurance 
Quality assurance will be performed by an independent monitor. The monitor will be a 
colleague who is independent of the current study. The members of the research team 
will meet with the monitor before advent of the study, and after the study has been 
completed (“close-out visit”). The meeting before the advent of the study is used to 
discuss how monitoring will take place, and to analyse what both the monitor and the 
members of the research team expect of the monitoring. The close-out visit will be 
combined with a monitoring visit. Furthermore, the monitor will monitor the study after 
analysis of the first 3 participants. 
Monitoring consists of: 
- Checking the presence and correctness of 100% of the informed consent forms. 
- 100% monitoring of in- and exclusion criteria of first 3 participants who are included 

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for the study, and monitoring of 10% of the other participants who are included. 
- Analysis of agreement between the data in the database, and the data in the source 
documents. 10% of all data will be monitored. 
- 100% verification of reported SAEs and SUSARs. Data from 10% of the participants 
will be checked for possible missed SAEs and SUSARs. 
The monitor will write a report about each visit, which will be discussed with the principal 
investigator and the coordinating investigator. The principal investigator will store the 
reports. 
 
12.3 Amendments 
Amendments are changes made to the research after a favourable opinion by the 
accredited METC has been given. All amendments will be notified to the METC that gave 
a favourable opinion. 
 
12.4 Annual progress report 
The investigator will submit a summary of the progress of the trial to the accredited METC 
once a year. Information will be provided on the date of inclusion of the first subject, numbers 
of subjects included and numbers of subjects that have completed the trial, serious adverse 
events/ serious adverse reactions, other problems, and amendments. 
 
12.5 End of study report 
The investigator will notify the accredited METC of the end of the study within a period of 
8 weeks. The end of the study is defined as the last patient’s last visit. 
In case the study is ended prematurely, the investigator will notify the accredited METC, 
including the reasons for the premature termination. 
Within one year after the end of the study, the investigator/sponsor will submit a final 
study report with the results of the study, including any publications/abstracts of the study, 
to the accredited METC. 
 
12.6 Public disclosure and publication policy 
Results from the study will be presented at the patient information day that is annually hosted 
by the lcFAO expertise center in the UMCU. Additionally, a manuscript will be written about 
the obtained results. This manuscript will subsequently be offered for publication in a peer- 
reviewed journal. 
 
13. STRUCTURED RISK ANALYSIS 
This chapter is not applicable for this study 

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