Administrative information
Title {1} | Efficacy and tolerability of a specific blend of amino acids in patients with anorexia nervosa treated in a hospital setting. Study protocol for a randomized controlled trial |
Trial registration {2a and 2b} | ClinicalTrials.gov Identifier: NCT05290285 |
Protocol version {3} | 08/1/1/2022, V1 |
Funding {4} | Professional Dietetics S.p.A (Milan, Italy) will provide the treatment product used in this trial and help cover the costs of some of the analyses |
Author details {5a} | 1Department of Eating and Weight Disorders, Villa Garda Hospital, Via Monte Baldo, 89, 37016 Garda (Verona), Italy 2Center for Study and Research on Obesity, Department of Biomedical Technology and Translational Medicine (BIOMETRA), University of Milan, Via Vanvitelli, 22, 20129, Milan, Italy 3Department of Molecular and Translational Medicine (DMMT), Viale Europa, 11, Brescia University, Brescia, Italy |
Name and contact information for the trial sponsor {5b} | Riccardo Dalle Grave (rdalleg@gmail.com) is the Principal Investigator and contact person of the trial sponsor (Villa Garda Hospital) |
Role of sponsor {5c} | The present study is investigator-initiated. Professional Dietetics S.p.A had no role in study design and will not be involved in data collection, analysis, interpretation, or publication. The authors will not receive financial or non-financial support for the research or article authorship |
Introduction
Background and rationale {6a}
Objectives {7}
Trial design {8}
Methods: participants, interventions, and outcomes
Study setting {9}
Eligibility criteria {10}
Who will take informed consent? {26a}
Additional consent provisions for collection and use of participant data and biological specimens {26b}
Interventions
Explanation for the choice of comparators {6b}
Intervention description {11a}
PD-E07/AminoTher-PROTM | |||
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1 sachet (g) | 2 sachets (g) | g/100 | |
l-Leucine | 1.2 | 2.4 | 23.5 |
l-Lysine (chlorhydrate) | 0.9 | 1.8 | 17.6 |
l-Isoleucine | 0.6 | 1.2 | 11.7 |
l-Valine | 0.6 | 1.2 | 11.7 |
l-Threonine | 0.7 | 1.4 | 13.7 |
l-Cysteine | 0.2 | 0.3 | 2.9 |
l-Histidine | 0.2 | 0.3 | 2.9 |
l-Phenylalanine | 0.1 | 0.2 | 2–0 |
l-Methionine | 0.1 | 0.1 | 1.0 |
l-Tryptophan | 0–1 | 0.1 | 1.0 |
Vitamin B1 (thiamine chlorhydrate) | 0.0007 | 0.0001 | 0.0 |
Vitamin B6 (pyridoxine chlorhydrate) | 0.0009 | 0.0002 | 0.0 |
Citric acid | 0.409 | 0.82 | 8.0 |
Malic acid | 1.92 | ||
Succinic acid | 0.103 | 0.21 | 2.0 |
Total EAA (g) | 4.5 | 9.0 | 100.0 |
Total mixture (g) | 5.1 | 10.2 |
Intensive treatment of eating disorders
Core procedures |
• Assisted eating (three meals and one snack a day) |
• Weekly review meeting (with the clinical psychologist, dietician, nurse, and physician) |
• Collaborative weighing one a week |
• Individual CBT-E sessions |
• Group treatment sessions |
Nutritional rehabilitation protocol |
• Weight goal: BMI ≥ 19 |
• Expected speed of weight gain: 1 to 1.5 kg per week |
• Daily calorie content of the meal plan is established collaboratively with the patient according to the following guidelines: |
—Week 1: Menu A (1500 kcal) |
—Week 2: Menu B (2000 kcal) |
—Afterward: |
○ If the weight increases between 1 and 1.5 kg per week, the meal plan is maintained with the same calorie content as the previous week |
○ If the weight increases to less than 1 kg per week, the meal plan is increased by 500 kcal per day. For example, from Menu B to Menu C (2500 kcal) or from Menu C to Menu D (3000 kcal) |
○ If the weight increases more than 1.5 kg per week, the meal plan is reduced by 250 kcal per day. For example, from Menu C to Menu B / C (2250 kcal) |
○ When the patient reaches a BMI of 19, the program’s goal is to identify a weight range of 3 kg that can be achieved without adopting a calorie restriction. In most patients, weight maintenance occurs with a diet between 2000 (Menu B) and 2500 kcal (Menu C) |
Physical rehabilitation protocol |
• All patients above a BMI of 15 participate in two 30-min sessions per week of calisthenics with a physiotherapist |
Criteria for discontinuing or modifying allocated interventions {11b}
Strategies to improve adherence to interventions {11c}
Relevant concomitant care permitted or prohibited during the trial {11d}
Provisions for post-trial care {30}
Outcomes {12}
Primary outcome
Secondary outcomes
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Physical fitness measured with the Eurofit Physical Fitness Test Battery (EPFTB) [34].
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Physical activity measured with the Actiheart system, CamNtech Ltd., Cambridge, UK
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Weight regain measured with the Seca Digital Wheelchair Scale Model 664 (Seca, Hamburg, Germany).
Participant timeline {13}
Sample size {14}
Recruitment {15}
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Study procedures and objectives.
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Purposes, methods, inconveniences, risks, and benefits that the study may entail.
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Freedom to participate and withdraw from the study without affecting subsequent treatment.
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Compensation procedures and treatment of any damages deriving from participation in the study.
Assignment of interventions: allocation
Sequence generation {16a}
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Group 1: BMI ≤ 15.2 & age ≤20
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Group 2: BMI > 15.2 & age ≤ 20
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Group 3: BMI ≤15.2 & age > 20
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Group 4: BMI > 15.2 & age > 20
Concealment mechanism {16b}
Implementation {16c}
Assignment of interventions: blinding
Who will be blinded {17a}
Procedure for unblinding if needed {17b}
Data collection and management
Plans for assessment and collection of outcomes {18a}
Data collection form
Treatment progress evaluation form
Dropout form
Randomization form
Blood chemistry tests
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Fasting blood glucose, triglyceridemia, HDL, total VLDL and LDL cholesterol, C reactive protein, fasting insulin, ALT, AST, ALP; GGT, GFR, NA, K, Ca, P, and Vitamin D-25OH.
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Metabolomic analysis (n = 15–20 patients/group) [45]. Plasma and urine samples will be collected at T0 and T1, immediately frozen on dry ice, and stored at − 80° C. The samples will then be manipulated for “targeted” metabolomic analysis (glycolysis, TCA cycle, amino acid metabolism) by high-resolution LC–MS liquid chromatography-mass spectrometry (LC–MS), in addition to the analysis of amino acid levels with Biochrom30+ (amino acid analyzer).
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miRNome analysis (n = 15–20 patients/group) [45]. Plasma and urine samples will be collected at T0 and T1, immediately frozen on dry ice, and stored at − 80 °C. The samples will then be manipulated to analyze miRNome (Next Generation Sequencing, NGS system).
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Mitochondrial mass and bioenergetics (n = 15–20 patients/group). Peripheral blood samples will be collected at T0 and T1. The circulating PBMCs will be isolated within 2 h of collection, pelleted, and immediately frozen on dry ice, then stored in biobank. Samples will be manipulated to analyze the following parameters: (i) mtDNA content, (ii) enzymatic functions of the respiratory chain complexes, (iii) production of ATP, (iv) transcriptome (RNA-seq method), and (v) methylome (whole-genome DNA methylation analysis) [20].
Medical devices
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Dual-energy X-ray absorptiometry (DXA) (Prodigy Primo Lunar, A223040501, General Electric Company, Madison, WI 53,707–7550, USA-EnCORE TM 2009 (v13.31) software) will be used to measure LBM, and also total and regional fat mass and bone mass. Scans will be performed in the morning, and no special preparations will be made except to instruct participants to wear only underwear and no metal accessories.
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Bioelectrical Impedance (BIA) (Tanita HealthWare® Native Software, Tokyo, Japan) will be used to evaluate total body water on the same day the DXA scans are obtained. Participants are asked to take off their clothes, wear a hospital gown, and remain upright before stepping on the tool. They will be then instructed to stand on the scale barefoot in a wide, comfortable position with their arms relaxed and to the side, looking forward and remaining as relaxed as possible. Body composition data will be estimated using software provided by the manufacturer of the bioelectrical impedance analyzer.
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Seca Digital Wheelchair Scale Model 664 (Seca, Hamburg, Germany) will measure the weight and the height from which the BMI will be calculated.
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Vmax Encore 229 Sensormedics system (indirect calorimetry) to measure the Resting Energy Expenditure (REE). After a 12-h fast, participants lie supine in a quiet, dark room for 30 min before measuring VO2 and VCO2. The last 15 min of which will be also used to determine REE.
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Actiheart system, CamNtech Ltd., Cambridge, UK. It is a heart rate monitor combined with a uniaxial accelerometer. The monitor is applied to the left side of the chest and worn day and night for four consecutive days. Participants will be instructed to continue their usual activities, and the time spent in moderate to intense physical activity (≥ 3 MET) will be analyzed.
Physical fitness tests
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Eurofit Physical Fitness Test Battery (EPFTB) [34]. The EPFTB is a set of fitness tests that include (i) aerobic fitness (6-min walking test); (ii) musculoskeletal fitness (standing broad jump test, handgrip test, sit-up test); (iii) flexibility (sit-and-reach flexibility test); and (iv) motor fitness (flamingo balance test).
Eating disorder and general psychopathology
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EDE.17 interview [35, 36]. It is an interview validated in Italian that evaluates the severity of the ED psychopathology of the eating disorder in the last 28 days and allows you to make an accurate diagnosis of anorexia nervosa and other eating disorders according to the diagnostic criteria of the DSM-5.
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Eating Problem Check List (EPCL) [39]. It is a self-reported questionnaire validated in Italian that assesses ED attitudes and behaviors in the last 7 days.
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Starvation Symptom Inventory (SSI) [44]. It is a self-reported questionnaire validated in Italian that assesses the main symptoms secondary to starvation in the last 28 days.
Plans to promote participant retention and complete follow-up {18b}
Data management {19}
Data forms and data entry
Data transmission and editing
Data discrepancy inquiries and reports to core coordinating centers
Security and back-up of data
Description of hardware
Confidentiality {27}
Plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular analysis in this trial/future use {33}
Blood samples
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Peripheral blood mononuclear cell (PBMC) preparation. Blood will be collected in EDTA-coated tubes and then diluted with an equal volume of PBS + 2% FBS (Immunological Science, Bolney, Sussex) and divided into two identical aliquots. Diluted blood will be slowly layered on top of the Lymphoprep™ gradient (V = 1.077 g/ml; Stemcell Technologies, Vancouver, Canada) and centrifuged (400 × g, 35 min at room temperature, with the break off). PBMC layer will be collected in a new tube, washed in PBS + 2% FBS (100 × g for 10 min, the break off) to remove platelets, and then washed a second time (400 × g for 10 min, the break on). The PBMC pellet will be immediately frozen. The mitochondrial functions will be evaluated by measuring ATP production and enzyme functionality of respiratory chain complexes. The mitochondrial mass measurement [i.e., the mtDNA amount], the gene expression profile (i.e., RNA-seq), and the epigenomic characterization (i.e., methylome analysis) will be conducted to elucidate the mitochondrial biology, energy metabolism, and immunological phenotyping.
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Plasma samples will be used to measure the free amino acid profile as a biomarker of metabolic, cardiovascular, immune, and inflammatory diseases. Further, targeted metabolomics, miRNome, and circulating mtDNA will be evaluated.