Acute Nutritional Ketosis in VLCAD Deficiency

Study Overview
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Brief Summary
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To test if a ketone-ester based drink can boost muscle mitochondrial function in vivo in patients with VLCADD in order to establish a rational basis for therapeutic use in this disorder.

Detailed Description
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Exertional rhabdomyolysis is a common symptom in very long-chain acylCoA dehydrogenase deficient (VLCADD) patients. Failing muscle ATP homeostasis, due to impaired fatty acid oxidation, is the most likely cause. Therefore, supplementation with an alternative energy substrate to boost ATP homeostasis, such as an exogenous ketone ester (KE) drink, could be a therapeutic option. Previous results suggest that KE is preferentially oxidized in the tricyclic acid (TCA) cycle and improves physical endurance in athletes. Our primary objective is to test if KE boosts muscular ATP homeostasis in VLCADD patients to establish a rational basis for therapeutic use. VLCADD patients will be included in a randomized, blinded, placebo controlled, 2-way cross-over trial. Prior to each test, patients receive a KE drink or an isocaloric carbohydrate equivalent, and completed a 35 min cycling test on an upright bicycle, followed by 10 minutes of supine cycling inside a MR scanner. The protocol will be repeated after at least one week with the opposite drink.

Official Title
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Acute Nutritional Ketosis in VLCAD Deficiency: Testing the Metabolic Base for Therapeutic Use

Conditions
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VLCAD Deficiency, Fatty Acid Oxidation Defects

Intervention / Treatment
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* Dietary Supplement: ketone ester drink
* Behavioral: exercise
* Procedure: muscle biopsy
* Diagnostic Test: Magnetic Resonance Imaging


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: - Confirmed VLCADD by genetic profiling 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.

Ages Eligible for Study
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Minimum Age: 16 Years
Maximum Age: 65 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Other
Allocation: Randomized
Intervention Model: Crossover Assignment
Interventional Model Description: randomized, blinded, placebo controlled, 2-way cross-over trial
Masking: Double


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: ketone ester drink<br>Oral intake of ketone ester drink muscle biopsy exercise muscle biopsy Magnetic Resonance imaging | Dietary Supplement: ketone ester drink<br>* 395 mg of ketone ester/kg<br>* Other names: deltaG (R);Behavioral: exercise<br>* 35 min cycling test on an upright bicycle, followed by 10 minutes of supine cycling inside a MR scanner.<br>Procedure: muscle biopsy<br>* biopsy from the quadriceps muscle prior to and immediately after upright bicycling<br>Diagnostic Test: Magnetic Resonance Imaging<br>* 1H MR images and 31P MR spectra were acquired from the upper leg prior to-, during and after exercise<br>* Other names: Magnetic Resonance Spectroscopy;|
| Placebo Comparator: carbohydrate drink<br>Oral intake of isocaloric carbohydrate drinkmuscle biopsy exercise muscle biopsy Magnetic Resonance imaging | Behavioral: exercise<br>* 35 min cycling test on an upright bicycle, followed by 10 minutes of supine cycling inside a MR scanner.<br>Procedure: muscle biopsy<br>* biopsy from the quadriceps muscle prior to and immediately after upright bicycling<br>Diagnostic Test: Magnetic Resonance Imaging<br>* 1H MR images and 31P MR spectra were acquired from the upper leg prior to-, during and after exercise<br>* Other names: Magnetic Resonance Spectroscopy;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change of ATP concentration in millimolar | steady-state in vivo intramuscular concentration of ATP metabolites during rest and exercise. | During session 2 and 3: continuous measurements from t=75 minutes until t=85 minutes | 
| Change of PCr concentration in millimolar | steady-state in vivo intramuscular concentration of ATP metabolites during rest and exercise. | During session 2 and 3: continuous measurements from t=75 minutes until t=85 minutes | 
| Change of Pi concentration in millimolar | steady-state in vivo intramuscular concentration of ATP metabolites during rest and exercise. | During session 2 and 3: continuous measurements from t=75 minutes until t=85 minutes | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| kinetic rate constant of ATP synthesis in Hertz | rate constant of Pi and PCr recovery post-exercise | session 2 and 3, 10 minutes each time | 
| intramuscular concentration of H+ in millimolar | steady-state in vivo intramuscular concentration of H+ during rest and exercise | session 2 and 3, 10 minutes each time | 
| completion of 35 minute upright bicycling bout at FATMAX | (yes/no; if no, #minutes) | Session 2 and 3, 35 minutes | 
| completion of 10 minute supine bicycling bout at FATMAX in scanner | (yes/no; if no, #minutes) | Session 2 and 3, 10 minutes | 
| HR in beats per minute | heart rate, VO2 and VCO2 dynamics. During session 2+3 breath sampling will be done for 2 minutes per timepoint, simultaneously with blood sampling. | During session 1, 15 minutes During Session 2 + 3: 35 minutes | 
| VO2 in milliliter per minute per kilogram | heart rate, VO2 and VCO2 dynamics. During session 2+3 breath sampling will be done for 2 minutes per timepoint, simultaneously with blood sampling. | During session 1, 15 minutes During Session 2 + 3: 35 minutes | 
| VCO2 in milliliter per minute per kilogram | VCO2 dynamics during session 2+3 breath sampling for 2 minutes per timepoint, simultaneously with blood sampling. | During session 1, 15 minutes During Session 2 + 3: 35 minutes | 
| Changes in blood metabolites: D-betahydroxybutyrate in millimol per liter | Samples are taken at baseline, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75 minutes, 85 minutes and 265 minutes after ingestion of the testdrink | Session 2 and 3, 265 minutes per session | 
| Changes in blood metabolites: glucose in millimol per liter | Samples are taken at baseline, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75 minutes, 85 minutes and 265 min after ingestion of the testdrink | Session 2 and 3, 265 minutes per session | 
| Changes in blood metabolites: lactate in millimol per liter | Samples are taken at baseline, 30 minutes, 60 minutes, 85 minutes and 265 min after ingestion of the testdrink | Session 2 and 3, 265 minutes per session | 
| Changes in blood metabolites: insulin in picomol per liter | Samples are taken at baseline, 30 minutes, 60 minutes, 85 minutes and 265 min after ingestion of the testdrink | Session 2 and 3, 265 minutes per session | 
| Changes in blood metabolites: creatine kinase in units per liter | Samples are taken at baseline, 30 minutes, 60 minutes, 85 minutes and 265 min after ingestion of the testdrink | Session 2 and 3, 265 minutes per session | 
| Changes in blood metabolites: triglycerides in millimol per liter | Samples are taken at baseline, 30 minutes, 60 minutes, 85 minutes and 265 min after ingestion of the testdrink | Session 2 and 3, 265 minutes per session | 
| Changes in blood metabolites: LDL cholesterol in millimol per liter | Samples are taken at baseline, 30 minutes, 60 minutes, 85 minutes and 265 min after ingestion of the testdrink | Session 2 and 3, 265 minutes per session | 
| Changes in blood metabolites: free fatty acids in millimol per liter | Samples are taken at baseline, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75 minutes, 85 minutes and 265 min after ingestion of the test drink | Session 2 and 3, 265 minutes per session | 
| Changes in blood metabolites: total cholesterol in millimol per liter | Samples are taken at baseline, 30 minutes, 60 minutes, 85 minutes and 265 min after ingestion of the testdrink | Session 2 and 3, 265 minutes per session | 
| Changes in blood metabolites: HDL cholesterol in millimol per liter | Samples are taken at baseline, 30 minutes, 60 minutes, 85 minutes and 265 min after ingestion of the testdrink | Session 2 and 3, 265 minutes per session | 
| Changes in blood metabolites: acylcarnitines in micromol per liter | Samples are taken at baseline, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 75 minutes, 85 minutes and 265 min after ingestion of the test drink | Session 2 and 3, 265 minutes per session | 
| Subjective exertion | Measured with Borg score (range from 6 (rest) to 20 (extreme exertion)). | During Session 2 + 3, assessed during blood sampling, 265 minutes per session | 
| height in meters | height of patient | 1 minute during screening visit | 
| weight in kilogram | weight of patient to dose intervention and normalize outcome parameters | 1 minute during screening visit | 
| BMI in kg/m^2 | weight and height will be combined to report BMI in kg/m^2 | 1 minute during screening visit | 
| optional: TCA intermediates in muscle tissue (units is ratio of metabolite peak/ internal standard) and will be expressed as fold change from baseline | metabolomics (mass spectrometry) of muscle tissue on a voluntary basis | Session 2+3: before and after exercise, 20 minutes per session | 
| optional: glycolysis intermediates in muscle tissue (units is ratio of metabolite peak/ internal standard) and will be expressed as fold change from baseline | metabolomics (mass spectrometry) of muscle tissue on a voluntary basis | Session 2+3: before and after exercise, 20 minutes per session | 
| optional: acylcarnitines in muscle tissue (units is ratio of metabolite peak/ internal standard) and will be expressed as fold change from baseline | metabolomics (mass spectrometry) of muscle tissue on a voluntary basis | Session 2+3: before and after exercise, 20 minutes per session | 
| optional: D-betahydroxybutyrate in muscle tissue (units is ratio of metabolite peak/ internal standard) and will be expressed as fold change from baseline | metabolomics (mass spectrometry) of muscle tissue on a voluntary basis | Session 2+3: before and after exercise, 20 minutes per session | 
| optional: capillary density in muscle tissue based on CD31 staining (capillaries per millimeter^2) | individual phenotypic muscle properties on a voluntary basis. Immunohistochemistry. | Session 2+3: before and after exercise, 20 minutes per session | 
| optional: mitochondrial density based on ATPase, COX-SDH, SDH and NADH staining (intensity per microgram per minute). | individual phenotypic muscle properties on a voluntary basis. Immunohistochemistry. | Session 2+3: before and after exercise, 20 minutes per session | 
| optional: mitochondrial density based on as citrate synthase activity expressed as absorbance/s/mg. | individual phenotypic muscle properties on a voluntary basis. | Session 2+3: before and after exercise, 20 minutes per session | 
| optional: parameters for metabolism and mitochondrial function in muscle (AMPK, PPAR gamma, PGC1a, and GLUT4). All expressed as protein content as % of control. | individual phenotypic muscle properties on a voluntary basis. Westernblots. | Session 2+3: before and after exercise, 20 minutes per session | 
| optional: lipid accumulation based on Oil-Red-O staining (intensity of staining, and percentage positive-stained cells). | individual phenotypic muscle properties on a voluntary basis. Immunohistochemistry. | Session 2+3: before and after exercise, 20 minutes per session | 
| optional: muscle fiber type composition based on myosin heavy chain profiling. Type I, IIa, IIx fibres will be expressed as % of total fibres. | individual phenotypic muscle properties on a voluntary basis. | Session 2+3: before and after exercise, 20 minutes per session | 
| optional: muscle fiber type composition based on ATPase staining (intensity/ug/min). Type I, IIa, IIx fibres will be expressed as % of total fibres. | individual phenotypic muscle properties on a voluntary basis. Immunohistochemistry. | Session 2+3: before and after exercise, 20 minutes per session | 
| optional: glycogen content of muscle based on Periodic acid-Schiff (PAS) staining (intensity per millimeter^2) | individual phenotypic muscle properties on a voluntary basis. Immunohistochemistry. | Session 2+3: before and after exercise, 20 minutes per session | 
| optional: glycogen content of muscle measured as glucose released after enzymatic digestion with amyloglucosidase expressed as micromol per gram wet muscle weight. | individual phenotypic muscle properties on a voluntary basis. | Session 2+3: before and after exercise, 20 minutes per session | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Fatty acid Oxidation Disorders, ketone ester, VLCAD deficiency