A Randomised Controlled Trial on the Networks Integrating Anxiety and Metabolism in Anorexia Nervosa

Methods

Design The study is designed as a single-center, parallel-group RCT with two study arms: (1) calory intake in meal-1 and (2) no calory intake in meal-1. Participants and healthy controls are individually randomized to either one of the two arms, both delivered at the eating disorder clinic of the Ege University Hospital, Faculty of Medicine.

Primary outcome measure is brain patterns observed with magnetic resonance imaging and secondary outcomes include endocrine markers in blood, cardiovascular responses, self-reported symptom severity, and subsequent food consumption.

The study is planned in accordance with the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) and will be analysed and reported in accordance with the recommendations in consolidated standards of reporting trials (CONSORT), with special care taken to meet extended recommendations for both trials with patient reported outcomes and non-pharmacological trials.

Protocol Design and Conduct To ensure transparency and integrity throughout the study, all changes or deviations from the protocol will be thoroughly documented and reported. Any modifications to the protocol, including changes to study objectives, design, participant population, sample size, procedures, or significant administrative aspects, will necessitate a formal protocol amendment. Similarly, any alterations that could potentially impact participant safety, benefits, or risks will be carefully evaluated and documented. Proposed amendments will be reviewed and approved by the study team and submitted for approval to the national ethical review board before implementation. Additionally, all relevant study personnel will be promptly informed of these changes to ensure compliance and consistency across the study.

Recruitment The study will begin to recruit participants in February 2025 and the last measurements for the primary manuscript are expected to be collected by the end of 2026.

Recruitment, interventions and data collection will be performed in the specialized eating disorder clinic of the Faculty of Medicine at Ege University Hospital in Izmir (population approximately 4.5 million), Turkey. Before participants are recruited, we will ensure the implementation of Good Clinical Practice guidelines, the establishment of a functioning clinical research infrastructure and the training of study staff.

Participants will be recruited through four possible pathways: (1.) AN patients will be recruited from Ege University Faculty of Medicine Hospital, by physicians meeting these patients. Healthy controls will be recruited through (2.) announcements on Ege university campuses, (3.) local hospitals, and (4.) online platforms such as Instagram.

Individuals who agree to participate will be contacted by study personnel for further information about the study over the telephone. At that time an initial assessment for eligibility will be performed. For further eligibility assessments, please see the next heading. For adolescents that cannot be reached over the telephone, parents or legal guardians will be contacted. Potentially eligible participants will be scheduled to a study physician for further eligibility assessment and written informed consent.

Eligibility Female individuals of 13-18 years of age, who are currently patients at the eating disorder clinic and both newly diagnosed with AN according to the Diagnostic and Statistical Manual of mental disorders 5th edition (DSM-5) and treatment naïve will be eligible. Healthy control participants will be matched on age-, sex-, and education level with the AN-participants.

The clinical diagnosis/diagnoses for participants will be validated/ruled out by the Structured Clinical Interview for DSM-5 (SCID-5). Please see "Baseline measures for eligibility" for instrument details. The presence of other exclusion criteria will also be further explored at the clinical visit.

The exclusion criteria are: (1.) Having one or several chronic medical conditions that may interfere with or hinder participation, or affect study outcomes, (2.) Use of medications that affect hormone levels or eating behaviors, (3.) Having one or several severe psychiatric disorders other than AN (e.g., schizophrenia, major depressive disorder, bipolar disorder), (4.) Current drug abuse, pregnancy or potential pregnancy (for AN patients these conditions are excluded as a part of routine care. For healthy controls no objective testing will be performed), (5.) High risk of refeeding syndrome or severe complications related to eating disorders, (6.) Inability to comply with the nutritional program of the study, (7.) Allergies to foods included in the provided meal content, (8.) Cognitive impairments preventing comprehension of the study or the informed consent process, (9.) Contraindications for MRI (e.g., pacemaker, prosthesis, claustrophobia), and (10), fasting blood sugar above 6.0 mmol/l on the morning of the study, as this indicates either non compliance with overnight fasting or pre-diabetes.

Additional exclusion criterion for healthy controls: (1.) Having one or several eating disorders, current or past.

Unexpected findings of biochemical anomalies at T0 (please see "Assessments" below) will not be considered exclusion criteria and medical conditions will be referred for appropriate treatment.

Participant inclusion in the study will be determined by a study clinician based on the outlined eligibility criteria. In cases of uncertainty, the principal investigator will make the final decision. Importantly, to maintain impartiality and avoid selective recruitment bias, enrolling clinicians will be masked to future treatment allocations, as randomization will occur at a later point in time. Participants will receive both oral and written information about the study and will provide written informed consent before inclusion. For participants below 18 years of age, written informed consent will also be obtained from parents or legal guardians. Participation in the study is voluntary, and participants may withdraw at any time without any negative consequences or affected subsequent care. Participants may also choose to withdraw any personal data already collected.

Randomization When an eligible participant has been recruited and consented, they will be scheduled for randomization at a later timepoint. AN Participants will be individually randomized to one of the two study arms with a 1:1 allocation ratio, by a computer-generated allocation sequence using permuted blocks. The block sizes will vary randomly between two and four.

Randomization lists will have been pre-prepared by a separate researcher who is not involved in participant recruitment. Sealed envelopes will contain the study-ID for each participant and the meal assigned to that individual (caloric or non-caloric). When a participant is ready to enter the study, a researcher will draw an envelope and prepare the assigned meal for that participant in the evening, for consumption in the next morning.

Participants with AN will be admitted to the clinic's open inpatient unit on the evening prior to study participation to ensure a 12-hour overnight fast. On the following day, they will receive the specified interventions and undergo the outlined assessments. Once an AN participant has been randomized and completed the study, a matched healthy control participant will be recruited. Recruitment of healthy control participants will be conducted by study personnel who are masked to the randomization outcomes of the corresponding AN participant. This approach ensures that the allocation of both AN and healthy control participants remains concealed from recruiting researchers and those conducting assessments. Healthy control participants will undergo the same eligibility assessments as AN participants, be assigned their own study ID, and share the same randomization outcome as their corresponding AN participant. However, unlike AN participants, healthy control participants will not be admitted overnight.

Codes will be used to increase information confidentiality and participant anonymity, and participant allocation will be performed on the basis of these codes. Randomization will continue until n = 30 AN-patients and n = 30 healthy controls have completed the study as per-protocol participants.

The randomization results will not be revealed to anyone other than the individual preparing the assigned meal. The randomization step will thus be separated from the patient enrolment process and all the other processes of the study. Through that participants, study personnel and clinicians delivering any co-interventions will be masked to participants' allocation, and so will outcome-assessors and statistical analysts.

Intervention protocol Depending on their randomization result participants will be asked to consume one of two jellies, both composed by a dietitian and prepared by study personnel: one containing 400 kilocalories and the other calorie-free. Both participants and the person handing out the jelly will be unaware of its caloric content. Participants will be given 15 minutes to consume the jelly, without forcing them to do so, and the amount consumed will be recorded.

The caloric food used in the study will be prepared using jelly powder, water, powdered sugar, cinnamon, ginger, turmeric, and lemon zest. To make the mixture, 100 ml of hot water will be poured over the plain jelly powder and stirred until fully dissolved. Eighty grams of powdered sugar will then be added and stirred until completely integrated. For flavor enhancement, 1 teaspoon each of cinnamon, ginger, and turmeric, along with the zest of half a lemon, will be incorporated. Afterward, 100 ml of cold water will be added, and the mixture will be stirred thoroughly. The prepared mixture will be poured into single-serving containers, allowed to cool at room temperature, and then refrigerated for 2-3 hours to solidify.

The calorie-free mixture will be made with 200 ml water, agar-agar, a natural sweetener, cinnamon, ginger, turmeric, lemon zest, and lemon juice. These ingredients will be combined and brought to a boil on the stove. Once boiling, the mixture will be stirred continuously for 5 minutes until it begins to thicken. The heat will then be turned off, and the mixture will cool for 15 minutes. It will be transferred into containers and refrigerated for 3 hours to set.

The ingredients and preparation methods were carefully chosen to ensure that both mixtures have a similar appearance and taste.

Three hours after consuming the first mixture (meal-1), participants will be offered a second meal (meal-2). This meal will always contain calories and consist of a nutritionally balanced, standard oatmeal dish with a distinct taste from the first meal. Participants will be allowed to eat freely, up to a maximum of 500 kcal, to minimize the risk of medical complications such as refeeding syndrome in participants with AN. Participants will have 45 minutes to complete the meal, and the total amount consumed will be recorded.

Apart from meal-1 and meal-2, any oral intake apart from water will be prohibited during the time of the study.

Adherence to study protocol Protocol non-adherence is defined as: (1.) A participant withdraws their consent, choosing to discontinue the study from the time of randomization to the end of T2 data collection, (2.) A participant is discovered to have received the wrong type of meal in meal-1, (3.) A participant consumes ≤ 30 % of the food provided in meal-1, (4.) A participant eats or drinks anything other than the provided meals and water between T0 and T2 data collection, (5.) A participant refuses or is unable to undergo MRI-scanning at T2, (6.) A participant is unable to adhere to the study schedule, and (7.) An adverse event occurs that renders continued participation inappropriate, please see "Adverse events and security plan" below for details.

• Meal-1 food consumption [Срок оценки: 15 minutes post meal with/witout calories]
• Meal-2 food consumption [Срок оценки: Three hours post meal with/witout calories]
• Self-rated psychometrics [Срок оценки: 15 minutes, one hour and three hours post meal with/witout calories]
• Blood sample [Срок оценки: One hour post meal with/witout calories]
• Blood sample [Срок оценки: One hour post meal with/witout calories]
• Blood sample [Срок оценки: One hour post meal with/witout calories]
• Blood sample [Срок оценки: One hour post meal with/witout calories]
• Blood sample [Срок оценки: One hour post meal with/witout calories]
• Blood sample [Срок оценки: One hour post meal with/witout calories]
• Blood sample [Срок оценки: One hour post meal with/witout calories]
• Blood pressure [Срок оценки: One hour post meal with/witout calories]
• pulse [Срок оценки: One hour post meal with/witout calories]