Oleanolic Acid as Therapeutic Adjuvant for Type 2 Diabetes Mellitus (OLTRAD STUDY)

TRIAL DESIGN The OLTRAD Study is a prospective, parallel group, randomized, double-blind, controlled trial designed to demonstrate that the regular intake of an OA-enriched functional olive oil is effective as an adjunct to metformin therapy (as monotherapy or in combination with other antidiabetic drugs) in the metabolic control of T2DM patients. The effect of the OA-enriched olive oil will be compared with that of the control oil, which consists of the same commercial olive oil not fortified in the triterpene.

A total of 100 volunteers of both sex will be selected from among the T2DM patients treated at the Endocrinology and Nutrition Service of the 'Virgen del Rocío' University Hospital (Seville, SPAIN).

SAMPLE SIZE CALCULATION Serum glycosylated hemoglobin (HbA1c) is adopted as the main quantitative variable for the analysis of glycemic control. The null hypothesis stated in the trial design is that the OA-based dietary intervention will reduce the baseline serum HbA1c levels of patients by at least 7%, compared to the control group. To test this one-sided hypothesis test with a 95% confidence level (α risk = 0.05) and 95% power (β risk = 0.05), a sample size of 44 volunteers per group is required. However, due to the characteristics of the study (a long-term lifestyle intervention), participant losses of up to 15% can be assumed, resulting in an adjusted sample size of 50 individuals per group.

RANDOMIZATION Once recruited, the 100 T2DM patients will be randomly assigned to one of the two study groups. Fifty individuals will be assigned to the intervention group, which will ingest the OA-enriched functional olive oil, whereas the other 50 volunteers will be assigned to the control group, which will receive the same non-enriched olive oil. At the time of admission, the study nursing staff will request by telephone the clinical coordinator of the trial (Principal Investigator 2 of the Project) the assignment of participants to the study groups (centralized randomization). Allocation to these groups will be made using computer generated tables of random numbers. Four randomization strata will be constructed by sex and age (cuttof 50 years).

INTERVENTION Participants will be instructed to ingest 55 mL/day of the assigned oil, preferably raw and freely distributed among the three main meals. Both the OA-enriched olive oil and the control oil will be delivered labeled with alphanumeric codes, the correspondence of which will only be known by the Principal Investigator 1 of the project. This ensures blinding of the clinical researchers and participants with respect to the type of olive oil assigned.

FOLLOW-UP Participants in the clinical trial will be followed up for 12 months from enrollment, according to a plan of quarterly visits.

At the time of recruitment and every three months thereafter, participants will be measured for anthropometric, blood pressure, and heart rate measurements. They will also be asked about gastrointestinal disorders or other types of complaints, and will complete a questionnaire on lifestyle (diet, physical activity, alcohol and tobacco use), medical conditions, and medication use.

Also at the beginning of participation in the study and every three months thereafter, fasting blood samples will be drawn from the cubital vein, which will be collected in sterile plastic tubes with a vacuum system. Similarly, participants will provide aliquots of their first morning urine in sterile plastic tubes. The analytical determinations in blood and urine will be carried out in the Clinical Biochemistry and Analysis Laboratory of the 'Virgen del Rocío' University Hospital in Seville (HUVR).

In addition, aliquots of plasma will be sent to the 'Instituto de la Grasa' (IG-CSIC) for complementary biochemical determinations. Laboratory technicians from both institutions will receive the samples identified with alphanumeric ID codes and, therefore, will be blinded to the intervention groups.

In these quarterly visits, volunteers will participate in sessions of the nutritional education program and will receive 6 L of the assigned oil for free. Adherence to the dietary intervention will be assessed quarterly through the Haynes-Sackett's self-reported compliance test and the return of empty bottles presumably consumed.

The primary outcome of the trial is the improvement in glycemic control, assessed by the evolution of HbA1c. As secondary results, we will obtain data on anthropometric and clinical variables, as well as on biochemical parameters of blood and urine.

ANAMNESIS AND CLINICAL EXAMINATION OF PARTICIPANTS The medical researchers of the project have access to the electronic medical records of the participants in the platform DIRAYA of the Andalusian Health Service, and will carry out the anamnesis and physical examination of the participants, which will include vital data and general medical and surgical history, as well as the pharmacological treatments followed. Information will also be obtained on lifestyle, perception of their own body image, diet, and the type and intensity of physical activity. In addition, a general clinical examination will be carried out including general appearance and inspection of hands and arms, feet and legs, skin, face, eyes, mouth, neck, abdomen, edema, lymph nodes, and vital signs (temperature, pulse, respiratory rate and blood pressure).

ANTHROPOMETRIC STUDY AND BODY COMPOSITION The anthropometric study will include the determination of total body weight, height, body mass index (BMI), as well as waist and hip circumferences. The study and evaluation of these variables will be carried out according to the guidelines of the International Society for the Advancement of Kinanthropometry (ISAK). The study of body composition will be carried out by electrical bioimpedance. Fat mass, lean mass, muscle mass, total water, bone mass, basal metabolism, and visceral fat will be quantified with this technique, using validated prediction equations adjusted for age and sex.

ASSESSMENT OF FOOD INTAKE AND NUTRITIONAL TRAINING A nutritional education program will be implemented to assess the food intake of the participants and reinforce compliance with the dietary recommendations given. To measure food intake, we will use the food intake registration form designed by the University of Navarra (Spain) and used successfully in other trials, such as the PREvención con DIeta MEDiterránea (PREDIMED) study. This form is a validated tool that quantifies food intake in terms of food portions or food groups for adults living in Spain. On the other hand, all the participants will join workshops where they will receive specific dietary recommendations for patients with T2DM, and they will be instructed on a healthy diet, the best food options and adequate portions, limiting highly processed foods, cakes, sugar, fatty foods as well as sugary and alcoholic beverages. Additionally, patients will gain information on recipes, seasonal shopping lists, and the use of olive oil for cooking and dressing. The eating habits of the participants will be analyzed quarterly by means of a 24-hour recall questionnaire. The 'NUTRIUM' software (https://nutrium.com/; Braga; Portugal) for the nutritional evaluation of the diets will be used.

BLOOD BIOCHEMISTRY IN HUVR In ulnar blood samples, determinations related to circulating lipids [total triglycerides, total cholesterol, LDL, HDL, total apolipoprotein B and lipoprotein A]; glucose homeostasis [glycemia, insulinemia, C-peptide, HOMA-IR index, HbA1c]; oxidative stress and inflammation [uric acid, bilirubin, malondialdehyde, ultrasensitive C-reactive protein, thyroid-stimulating hormone (TSH)], as well as those associated with liver and kidney damage [lactate dehydrogenase (LDH), liver transaminases (ALT, AST, GGT ) and creatinine] will be performed.

Plasma level of vitamin B12 will be also assessed. In addition, the possible appearance of liver fibrosis will be evaluated through the Hepamet Fibrosis Score, a non-invasive method that calculates a score taking into account factors such as age, sex, presence of diabetes, glucose levels, insulin, albumin, platelets, and AST. This tool, designed by the Seville Institute of Biomedicine (IBiS), is indicated to assess suspected fibrosis in patients with obesity, diabetes, metabolic syndrome, hepatic steatosis, and/or abnormal liver function markers. The method offers diagnostic reliability and a cost/benefit ratio that is superior to other methods, such as the FIB-4 and the NAFLD Fibrosis Score.

CONTINUOUS BLOOD GLUCOSE MONITORING Plasma glucose will be continuously monitored using the 'FreeStyle Libre' subcutaneous system (ABBOTT Diagnostics; Chicago, IL.; USA). The sensors will be attached to the participants at two times throughout the trial: at baseline and after 12 months of follow-up, and will collect data for 48 hours. With this system, different glycemic parameters will be determined, such as the area under the curve (AUC), the mean amplitude of the glycemic excursion (MAGE), the mean glycemia, as well as the standard deviation (SD) and the coefficient of variation (CV).

PLASMA BIOCHEMISTRY IN IG-CSIC The investigators of the IG-CSIC will perform a number of complementary determinations in plasma samples, such as the plasmatic concentrations of OA, the glutathione (GSH and GSSG) levels, the fatty acid composition, serum adipokines (ceruloplasmin, adiponectin, leptin, resistin, and ghrelin). Serum Antioxidant enzymes (superoxide dismutase, catalase) and proinflammatory cytokines (TNF-α, IL-1β, IL-6) will also be determined.

On the other hand, the VLDL fraction of plasma lipoproteins will be isolated by ultracentrifugation (230,000 × g for 18 h at 4 °C). In these particles, the total content of fatty acids, triglycerides, diglycerides, phospholipids and total Apolipoprotein B will be measured.

URINE ANALYSIS The first morning urine samples will be analyzed for the usual physical, chemical, and microscopic determinations (pH, density, glycosuria, ketones, nitrites, urobilinogen, bilirubin, sediment, leukocytes, microalbuminuria/g creatinine). The determination of these parameters will also be carried out in the facilities of the Clinical Biochemistry and Analysis Laboratory of the 'Virgen del Rocío' University Hospitals in Seville.

STATISTICAL ANALYSIS The trial will be conducted according to the intention-to-treat (ITT) principle. Qualitative variables will be expressed by their absolute and relative frequencies, whereas the quantitative ones with normal distribution will be expressed by the mean and standard deviation and those with non-normal distribution by the median and interquartile range (IQR). Comparisons between study groups for qualitative variables will be performed with the Chi-square and McNemar's tests, whereas comparisons for quantitative variables will be executed with the Student's t-test and ANOVA. The homogeneity of the populations included in the allocation groups will be evaluated using theMann-Whitney-Wilcoxon U-test. All P-values will be two-tailed at α = 0.05. Statistical analysis will be performed with SPSS 27 (IBM SPSS Statistics, NY, USA) software

Inclusion criteria

• community-residing men and women aged.
• Body Mass Index (BMI) between 25 and 39.9 kg/m2.
• diagnosed with T2DM [Following the American Diabetes Association (ADA) 2019 criteria] at least six months before being included in this trial.
• be treated with metformin (stable dose >= 850 mg/day at least three months before recruitment) as monotherapy, or in combination with other hypoglycemic agents (administration of insulin exclusively in a single basal dose), except pioglitazone and sulphonylureas.
• HbA1c below 9% at baseline, with a variation compared with a prior HbA1c from at least three months before inclusion in this trial of less than +/- 0.5 %.
• be able to give voluntary informed consent and willing to comply with all study procedures.

Exclusion criteria

• suffering from Type 1 Diabetes Mellitus or latent autoimmune diabetes in adults.
• suffering from chronic kidney disease (estimated glomerular filtration rate (eGFR) <30 ml/min/1.73m2).
• suffering from acute or chronic hepatitis, signs and symptoms of any liver disease other than non-alcoholic fatty liver disease (NAFLD), or ALT/AST ratio >3 times the upper limit of the reference range.
• To present, at the time of recruitment, allergies and intolerances associated with food consumption.
• pregnant and lactating women.
• lack of willingness to use a highly effective contraceptive method (in women of childbearing potential).
• fasting triglyceridemia > 600 mg/dL despite adequate treatment.
• grade 3 hypertension (systolic blood pressure ≥ 180 mm Hg and/or diastolic blood pressure ≥ 110 mm Hg) according to the 2018 guidelines of the European Society of Hypertension.
• use of pioglitazone or sulfonylureas.
• being treated with medications that promote weight loss (eg, Saxenda® [liraglutide 3.0 mg], Xenical® [orlistat], or similar over-the-counter [OTC] medications) within six months prior to the start of the trial.
• Being on chronic (>14 days) therapy with systemic glucocorticoids (excluding topical, intraocular, intranasal, intra-articular, or inhaled preparations) within six months prior to enrollment.
• Presenting any of the following cardiovascular conditions within 6 months prior to study entry: acute myocardial infarction, unstable angina, New York Heart Association (NYHA) class III or class IV heart failure, or cerebrovascular accident.
• Evidence, in the investigators' opinion, of significant uncontrolled endocrine abnormality (e.g., thyrotoxicosis, adrenal crisis) at baseline.
• History of active or untreated malignancy, or being in remission from a clinically significant malignancy (other than basal or squamous cell skin cancer, cervical carcinoma in situ, or prostate cancer in situ) during the last 5 years before the study entry.
• Participation in the last 30 days in a clinical trial with an investigational product [if the previous investigational product has a long half-life, 3 months or 5 half-lives (whichever is longer) should have passed].
• Being, at the time of recruitment, enrolled in any other clinical trial involving an investigational product or any other type of medical research that is not considered scientifically or medically compatible with this study.
• Presence of any hematologic condition that may interfere with HbA1c measurement (eg, hemolytic anemias, sickle cell anemia).
• History of any other condition (eg, known drug or alcohol abuse or psychiatric disorder, or any other physical or intellectual limitations), which, in the opinion of the investigator, may prevent the patient from following and completing the protocols.