This is a non-blinded clinical trial that evaluated individuals with ART-associated lipodystrophy by clinical follow-ups of those who met the inclusion/exclusion criteria of this study. Evaluations were carried out in a Reference Hospital. The research took place during the pandemic period when we experiencing a great scarcity of resources and inputs, it was decided not to blind, maintaining the diet therapy guidelines for both study groups, we aim to promote positive impacts and homogenize as much as possible concomitant changes in eating behavior, which could be biases in the research, it is highlighted that the participants do not have links with each other or explicit relationships that could facilitate communication of the type of intervention that was carried out.

This study was carried out from January 2020 to June 2022 and was supported by Resolution 251 of 1957 of the Brazilian National Council of Ethics and Research as a phase IV Research. Good clinical research practices and due submission to the Brazilian registry platform of clinical trials-ReBEC were followed. The study period described comprised the time for standardization of collection instruments, aiming to favor quality in data collection and results obtained (January 2020 to June 2022), in addition to the time for effective research (July 2021 to June 2022), period necessary so that the benefits of omega-3 supplementation could be identified. Furthermore, it is worth highlighting that because it is a reference hospital related to the single health system, in which patients attend the endocrinology outpatient clinic once a week, on Fridays, in a controlled quantity, with return visits every three months, there was their inclusion in the study gradually, according to prior scheduling by the hospital. The study period included the COVID pandemic, therefore, in some interventions, it was necessary to redo the study with 6 patients, due to problems with returning to carry out the return consultation, as they lived in other interior municipalities, where travel was affected and replacement of 1 patient who died during the period.

Sample calculation was performed considering a 5% alpha level, 95% confidence intervals, and two groups (intervention/control) followed for three months. The group selection plan, to guarantee the randomization process, included the allocation of all patient names, which was obtained from the hospital database, in a Microsoft Excel spreadsheet, from which random numbering was assigned, generated by the program, we sequentially separated individuals from 1 - 50 as select for the supplementation groups and from 51 - 100 for the guidance groups. In this way, ensuring that the researcher does not interfere with the allocation of patients into the different groups.

Sample size was calculated for each treatment group based on a previous study [11] . The mean of the studied variable, a 6.45˚ mean phase angle, a 1.09 SD, a 0.645 magnitude of effect (10% × 6.45˚); a 0.645/1.09 = 0.59 standardized effect magnitude; a 0.05 alpha level; and a 0.20 Beta error were considered, totaling 32 individuals in the supplementation/guidance group and 32 individuals in the guidance group (sample calculation for T-test) [12] . In our assessment of body composition using the bioimpedance technique, BioDynamics 450 equipment was used with an electric current intensity of 800 µA and a fixed frequency of 50 kHz (kilohertz), which ran through patients’ entire body, using four electrodes, The equipment used is a certified by the National Health Surveillance Agency (ANVISA)/Ministry of Health and used at international scientific research level. The electrical current and frequency used by the device allow it to partially cross the cell membrane to measure resistance and reactance, in order to estimate the fat-free mass, in addition to accurately providing the phase angle, the main object of the research. A study by Back, 2020, which compared three devices that measure bioimpedance components, highlighted that all three can be used in the clinical management of patients, but highlighting the BioDynamics 450, due to its cost-benefit, size of the equipment and the fact that it is portable, making transport easier, compared to octopolar models, for example [13] .

The technique included comfortably positioning individuals on a hospital stretcher without shoes, socks, watches, bracelets, or the like in the dorsal decubitus position and affixing two electrodes on their feet (the distal electrode at the base of their middle finger and the proximal electrode a little above their ankle joint line between their medial and lateral malleoli) and two electrodes on their right hand (the distal electrode at the base of their middle finger and the proximal electrode a little above their wrist joint line, coinciding with their styloid process), which were connected to a monitor by a sensor cable. The portion of participants’ skin used to attach these electrodes showed no injuries and was previously cleaned with alcohol to remove their oiliness. Electrodes were placed at least five cm apart. After recording the necessary data regarding individuals’ age, gender, height, phase angle and body cell mass results were provided in less than one minute, recorded, and subsequently analyzed.

The variables extracted from the bioimpedance test for analysis were phase angle; muscle mass; fat mass; and body cell mass.

The individuals who participated in this study were instructed to fast for four hours before the exam and to refrain from drinking coffee or tea the night before and from drinking alcohol or performing physical activities for eight hours before the exam.

The guarantee of ethical aspects was based on the principles described in the Declaration of Helsinki and the Nuremberg Code, respecting the standards for research involving human beings (Resolution n 466/2012) of the National Health Council, guaranteeing confidentiality with regard to identification of the researched, with signature of Free and Informed Consent Form (TCLE), presented in two copies, one of the copies was given to the participant and the other remained with the researcher for control. Through the ICF, the participant was informed that the research could generate some benefits for individuals with lipodystrophy, in terms of treatment guidance, in addition to contributing to the development of a nutritional assessment and treatment protocol. On the other hand, there were clarifications about the possible risks and discomforts resulting from the study, such as embarrassment, physical damage resulting from the use of assessment equipment such as radiometer and bioimpedance, in addition to possible allergies or gastrointestinal discomfort resulting from the use of supplementation, highlighting as a measure of mitigating risks and ensuring confidentiality in relation to identity, with the use of data only fulfilling the objectives described in the study, calibration and maintenance of the equipment to be used, with a detailed explanation of the preparation criteria for carrying out the tests, exposed through language clear and accessible, making periodic calls to check possible gastrointestinal discomforts, such as vomiting and diarrhea, in the supplementation groups. It is noteworthy that the privacy of the participants was respected, that is, the name or any other data or element that could identify the individual in any way was kept confidential, with no financial loss as a research participant. They were also informed that the individual could refuse to participate in the study, or withdraw consent at any time, without the need for justification, and if they wished to leave the research, they would not suffer any harm to the care they received at the outpatient clinic.

This study included people living with HIV/AIDS; adults of all sexes, over the age of 18 years, who were undergoing outpatient monitoring and antiretroviral therapy (ART).

All individuals who were intolerant to omega-3 were excluded from our sample, as were those with vulnerabilities and/or associated diseases that negatively interfered with the predilected phase angle values (such as severe obesity, i.e., BMI ≥ 40 Kg/m²), with edema or ascites, neurological diseases that compromised their understanding of this study, use of a pacemaker or metal prostheses/or theses, kidney diseases, use of diuretics, and Indigenous people, pregnant or lactating women, and those in the supplementation group who failed to adhere to treatment.

Based on the positive opinions of the ethics and research committee, a brief pilot study was developed with six participants in two consecutive consultations to standardize and adjust the measurement instruments to be adopted. Then, our research took place in three stages by elective consultations at a lipodystrophy outpatient clinic.

In the first stage, an initial consultation between the researcher and patients took place, consisting of the proper guidelines of this study, informed consent form signing, completion of patients’ sociodemographic and clinical protocol, and transfer of these guidelines for the second consultation with the supply of printed material, in addition to comments on the preparation criteria for the bioimpedance exam.

In the second consultation, the Guidance (GC) and Supplementation Groups (SG) were randomly defined. Then, the bioimpedance test was carried out for SG and their anthropometric measurements were recorded. These participants were given dietary guidance + supplementation – adapted from Cortes (2014) [6] containing 2100 mg of omega-3 (1270 mg de EPA and 840 mg of DHA) [6] , divided into three capsules/day (to be taken two after lunch and one after dinner) for three months. For this intervention, VITHA^© omega-3, duly certified by IFOS—an international quality and safety certification (FSSC 22000)—was used, containing 10 mg of vitamin E, which helps to prevent lipid peroxidation. The bioimpedance exam, anthropometric measurements and dietary guidelines were provided to this group.

Every month during treatment, patients were contacted by phone for monitoring, during which possible adverse effects in the intervention group were followed up. SG participants were told to take their supplementation packages for the next consultation so we could evaluate their adherence to the proposed treatment.

In the last consultation, three months after the beginning of treatment, post-treatment biochemical and anthropometric data were collected and bioelectrical impedance was reassessed.

To compile and analyze our results, a database was built in Microsoft Excel 2010 and analyzed using the statistical package BioEstat 5.0 and Jamovi 2.3.18.

The overall phase angle was averaged. Phase angle per group and at different moments (T0 and T1) were described in a table with means and standard deviations. The T-test for independent samples was used to assess possible statistically significant differences between groups at T0, whereas the Wilcoxon test was applied to evaluate the significance at T0 and T1 of each group.

Body composition variables were displayed in tables. The Guidance (GC) and Supplementation Groups (SG) at T0 were compared by the T-test. The related T0 and T1 samples for each group were evaluated by the T-test.

Phase angle according to gender was displayed in a table and the T test for independent samples was applied to it.

Our sample consisted of 63 individuals of all genders divided into groups, one received a supplementation intervention with omega-3 and the other, only dietary guidance.

As for the sociodemographic profile of the supplementation group (n = 32), we found a 56.25% prevalence of women (n = 18) and a 43.75% (n = 14) of men. Most men fell into the 60 - 70 age group (n = 7/50%), whereas most women into the 50 - 60 age group (n = 7/38.89%).

Regarding the guidance group’s sociodemographic profile (n = 31), we observed that most individuals were men (n = 19/61.29%), rather than women (n = 12/38.71%). Most men were aged 50 - 60 (n = 5/26.32%) and 60 - 70 years (n = 5/26.32%), whereas women showed a higher prevalence of the age group between 50 - 60 years (n = 5/41.67%).

Table 1 describes our analysis of phase angle in the supplementation group at T0 and T1, with a significantly higher body cell mass at T1 (22.71% ± 6.12 SD) than at T0 (21.79% ± 5.98 SD) (p-value = 0.01). We found no significant differences in fat and lean mass values between T0 and T1.

Table 2 shows our analysis of the body composition variables for the guidance group at T0 and T1, with a significantly lower body cell mass at T1 (21.78% ± 4.24 SD) than at T0 (22.89% ± 4.30 SD) (p-value = 0.01). We found significant differences in fat and lean mass between T0 and T1.

We calculated phase angle values for the entire sample. We obtained a mean initial value of 6.45˚ ± 1.06 SD.

After comparing phase angles before and after the intervention, we compared the initial phase angle of our groups, finding no significant difference between them (p = 0.316). Our analysis of phase angles at T0 and T1 for the guidance group showed no statistically significant differences between phase angles at T0 (6.70˚ ± 1.2˚), and T1 (6.50˚ ± 1.4˚) (p-value = 0.108). However, when we evaluated the phase angle in the supplementation group, we observed a significantly higher phase angle at T1 (6.65˚ ± 1.67˚) than in T0 (6.5˚ ± 1.02˚) (p-value = 0.006). Table 3 shows these data.

Regarding our phase angle analysis by gender, Table 4 shows the mean phase angle for men (6.87˚ ± 1.06 SD), significantly higher (p-value = 0.001) than for women (6.01˚ ± 0.88 SD).

T-test*. Source: Field Research – 2022.

T-test*. Source: Field Research – 2022.

*T-test for independent samples ** Wilcoxon test. Source: Field Research - 2022.

T-test*. Source: Field Research - 2022.