This protocol study was guided by the SPIRIT recommendations (Figure 1 – flowchart of study). The proposed prospective, randomized, placebo-controlled, double-blind trial has been duly registered (http://www.ensaiosclinicos.gov.br/rg/; trial registry number: RBR-6yj8sj/UTN number: U1111-1217-9237, July 23, 2018).
The clinical trial will be conducted in accordance with the precepts stipulated in the Declaration of Helsinki and the project received approval from the Human Research Ethics Committee of the Federal University of Pernambuco (certificate number: 2.464.997 from 01/01/2018). All participants will be recruited from the psychiatric outpatient clinics of the University of Pernambuco and Federal University of Pernambuco. We will evaluate 224 male and female adults (18 to 60 years of age) with depression under routine care at the psychiatric clinics for a period of six months.
Individuals between 18 and 60 years of age with a diagnosis of depression (score > 6 points on the Montgomery-Asberg Depression Rating Scale)  will be recruited independently of sex or gender. The participants will need to have a diagnosis of depression provided by a psychiatrist and based on the ICD-10 , such as F33.0 (recurring mild depression), F33.1 (recurring moderate depressive disorder), F33.2 (severe recurring depressive disorder without psychotic symptoms, including recurring major depression, endogenous depression and manic-depressive psychosis in the depressive form without psychotic symptoms), F33.3 (severe depressive disorder with psychotic symptoms, including major depression with psychotic symptoms, psychotic depression, manic-depressive psychosis in the depressive form with psychotic symptoms), F33.8 (other recurring depressive disorders), F33.9 (recurring severe depressive disorder without psychotic symptoms, including recurrent major depression without specification or unipolar depression).
The patients may be taking an antidepressant prescribed by their psychiatrists. No restriction will be imposed regarding the type of antidepressant medication. The severity of the disease may be mild, moderate or severe, as evaluated by the psychiatrist.
Individuals with cognitive impairment (determined using the Mini-Mental Health Examination ), those already supplementing with vitamin D, those with a past history of psychiatric comorbidities, chronic kidney disease, hypercalcemia or neoplasms and pregnant or nursing women will be excluded from the study.
Two pharmacists will perform the randomization and will be aware of which patients are using vitamin D and which are using the placebo. The participants will be divided into two groups (A and B). The main researcher of the clinical trial will interview all participants and fill out the clinical charts. A nurse will administer the vitamin D and placebo but will not know which substance is being given to the participants. The patients and investigator will be unaware of which capsule (vitamin D or placebo) the patients are taking. This procedure is necessary to avoid observation bias during the clinical-laboratorial follow up and maintain complete impartiality in the evaluation of the effects.
All instructions will be precise and written in the form of a manual to ensure the execution of all procedures determined for the clinical trial, including patient recruitment, allocation to the study groups, administration of the intervention, record systems, criteria for interrupting the intervention, etc. All activities to be performed during the clinical trial will be previously established in the form of a task list distributed to the investigation team.
The initial evaluation will consist of the filling out of the identification chart, sociodemographic inventory, standardized medical chart with patient history, physical and cardiological examinations, a questionnaire addressing the practice of activities involving exposure to sunlight, standardized statement of informed consent, a structured interview addressing depression with the use of the Montgomey-Asberg Depression Rating Scale, a scale addressing the risk of suicide and the recording of the results of laboratory and cardiological exams. The researcher will accompany the examinations and administration of the depression and suicidal risk scales.
The laboratorial exams will be conducted at the laboratory of the hospital in which the study will be developed. Venous blood (10 ml) will be collected from a peripheral vein for the determination of fasting glucose, cholesterol (total and fractions), urea, creatinine, total proteins, albumin, homocysteine, ultrasensitive C-reactive protein, lipoprotein A, ionic calcium and phosphorus as well as hormonal levels of total and free testosterone, estradiol, estriol, luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone, triiodothyronine, free tetraiodothyronine, cortisol, concentration of 25-hydroxyvitamin D and parathyroid-stimulating hormone upon admission to the study and every ninety days for a total period of six months. Urine will be collected every 90 days for the determination of 24-hour calciuria and microalbuminuria. The following laboratorial equipment will be used for the determinations: Architect C8000 for vitamin D and hormone concentrations and CMD 800 with ISE (Wiener Group) for the biochemical concentrations.
Cardiological and imaging exams (electrocardiography, Doppler echocardiography, stress test, electrocardiographic monitoring [24-h Holter] and Doppler ultrasound of the carotid and vertebral arteries) will be performed at the hospital in which the study will be developed.
When the participants return with the exam results, simple randomization (heads or tails) will be performed for the allocation to the different groups: 112 individuals in the vitamin D3 group and 112 in the placebo group. A nurse at the clinic will administer the vitamin D3 and placebo. The capsules will have the same size and color (compounded for the clinical trial) and the nurse will be blinded to the allocation of the patients to the different groups.
The researchers will evaluate the participants every 90 days. Each evaluation will involve the clinical and cardiological examinations with the electrocardiogram and laboratorial exams selected for the study. The final evaluation will occur after six months of intervention. The participants will be submitted to psychiatric (Montgomery-Asberg Depression Rating Scale)  and Columbia-Suicide Severity Rating Scale , clinical and cardiological examinations as well as the laboratorial exams, electrocardiography, Doppler echocardiography, stress test, 24-h Holter and Doppler ultrasound of carotid and vertebral arteries.
As the region in which the clinical trial will be conducted is sunny throughout the entire year, with an absence of well-defined climatic seasons, seasonality will not exert an influence on exposure to sunlight or the synthesis of vitamin D .
Calculation of sample size
The following were considered for the determination of the sample size:
a) The comparative objective will be an improvement in the depression score among the patients taking and those not taking vitamin D. We considered the success percentages obtained in a study on vitamin D , in which 69.2% and 65% improvements were found in the depression scale scores, respectively;
b) 5.0% acceptable rate of error; 80.0% power; ratio of 1.00 between the two groups.
The necessary sample size for each group was 112 patients (total of 224 patients). This calculation was performed using the EPI-INFO program, version 6.04.
Reduction of risk of bias
The dose of vitamin D for supplementation considered for the study (50.000 IU weekly) meets the pre-established criteria in previous trials that have presented positive results in the reduction of depressive symptoms . The aim of considering this variable is to minimize the potential bias. Vitamin D3 is quite safe when administered with the prescribed posology. Doses up to 10,000 IU per day for five months induced no signs of toxicity, such as hypercalcemia and hypercalciuria [30,31].
Collection and management of data, including meta-data
The characteristics and procedures adopted for each test group are described below:
Group 1 – Intervention: 112 participants will receive vitamin D3 supplementation (ADDERA D3, MANTECORP Laboratory) at a dose of 50,000 IU per week for six months.
Group 2 – Placebo: 112 participants will receive a placebo capsule (compounded by the pharmaceutical laboratory of the Federal University of Pernambuco) once a week for six months.
The groups will be submitted to five evaluations on the first day and after six months of intervention:
1- Questionnaire addressing sociodemographic characteristics: The questionnaire will be administered to the participants in interview form and filled out by the researcher. The following data will be recorded: identification number attributed to the participant, age, sex, schooling, marital status, ethnicity and religion.
2 – Brazilian Economic Classification Criteria (BECC) – 2011 established by the Brazilian Association of Research Companies : The BECC is an economic segmentation instrument that uses a survey of residential characteristics (presence and quantity of household items of comfort and degree of schooling of the head of the family) to differentiate the population (Appendix C). Points are attributed to each household characteristic and the total is used to classify economic strata from highest to lowest class: A1, A2, B1, B2, C1, C2, D, E .
3 – Severity of depression: determined through a structured clinical interview:• The Montgomery-Asberg Depression Rating Scale  will be administered at the beginning of the study and after six months of intervention.
4- Assessment of Risk of Suicide: The Columbia-Suicide Severity Rating Scale  will be administered to assess presence and intensity of suicidal ideation and suicidal behavior.
5- Cardiovascular risk factors: a) Systemic arterial hypertension – based on systolic and diastolic blood pressure levels ; b) Dyslipidemia (total cholesterol total, HDL, LDL, triglycerides) ; c) Coronary artery disease: coronary calcium > 100th or > 75th percentile for age or sex, cardiac stress test or myocardial scintigraphy with signs of myocardial ischemia ; d) Cerebrovascular accident (stroke): clinical condition or suggestive imaging exam; e) Obstructive peripheral arterial disease: ankle-brachial pressure index < 0.9; arterial stenosis detected by ultrasound of lower limbs ; f) Carotid atherosclerosis: stenosis/thickening of carotid artery ; g) Heart failure: left ventricular hypertrophy or reduction in systolic function of the left ventricle or diastolic dysfunction detected by Doppler echocardiography ; h) Arrythmia: detected by resting electrocardiography, 24-hour Holter or stress test; i) Diabetes mellitus: reduction in glucose tolerance and resistance to insulin (fasting glucose, A1C hemoglobin, oral glucose tolerance test, basal insulin, insulin curve, homeostatic model assessment) [38,39]; j) Serum markers of cardiovascular risk (ultra-sensitive C-reactive protein, homocysteine, lipoprotein A) ; k) Hyperthyroidism or hyporthyroidism (ultra-sensitive TSH, free T4, free T3) [40,41]; l) Chronic kidney disease: albuminuria, measured by glomerular filtration rate and calculated based on the Chronic Kidney Disease Epidemiology Collaboration ; m) Hyperparathyroidism (HPT, phosphorus) .
The data will be analyzed using statistical measures: mean and standard deviation values for numerical variables and percentage frequencies for categorical variables. The inter-group comparisons will be performed using either the Student’s t-test or Mann-Whitney test for numerical variables and Pearson’s chi-square test for categorical variables. The intra-group comparisons of initial and final values will be performed using either the paired Student’s t-test or the Wilcoxon test for paired data for numerical variables and McNemar’s test for categorical variables.
The unpaired and paired t-tests will be used in situations for which the data exhibit normal distribution, whereas the Mann-Whitney test and Wilcoxon test for paired variables will be used in situations in which normality has been rejected. The Shapiro-Wilk test will be used to determine the normality of the data and Levene’s F test will be used to determine equality of variances.
The margin of error used in the decisions of the statistical tests will be 5% and 95% confidence intervals will be calculated. The data will be entered onto an EXCEL spread sheet and the Statistical Package for the Social Sciences (SPSS version 23) will be used for all statistical calculations.
A statistician will perform the calculations and statistical analyses of the participants taking substances A and B. The blinding will only be lifted at the end of the statistical analysis.
The present clinical trial is in accordance with Resolution nº 466/2012 of the Brazilian National Board of Health, which establishes norms for research involving human subjects, and received approval from the UFPE ethics committee through Platform Brazil (certificate number: 79347717.0.0000.5208) on January 11, 2018. Recruitment (screening of patients) was initiated on April 30, 2019. Thus, the study is currently in its initial phase (ongoing). The clinical trial (intervention) is expected to begin on October 1, 2019, as stipulated in the registry: RBR-6yj8sj /. UTN Number: U1111-1217 -9237.
- Therapeutic effects of vitamin D on depression, evaluated using the Montgomery-Asberg Depression Rating Scale;
- Therapeutic effects of vitamin D on cardiovascular risk factors, evaluated using the scoring system proposed by the American College of Cardiology (ACC) and American Heart Association (AHA) [44,45] for the primary prevention of the risk of acute myocardial infarction, death by coronary heart disease, heart failure, fatal and non-fatal stroke in a period of 10 years. The new scoring system provides specific estimates based on current age, sex, race, blood pressure, total cholesterol, HDL cholesterol, LDL cholesterol, diabetes, smoking status (current smoker, ex-smoker or never smoked), in treatment for systemic arterial hypertension, in treatment with statins and in treatment with aspirin. After including these data, the calculator estimates risk for cardiovascular disease in the next 10 years, which is categorized as low (< 5%), borderline (5 to 7.4%), intermediate (7.5 to 19.9%) or high (≥ 20%).
- Therapeutic effects of vitamin D for reducing the risk of suicide, evaluated using the Columbia-Suicide Severity Rating Scale.
- Reduction of cardiovascular risk factors described in subsection “Collection and management of data, including meta-data”.
Analysis plan and measurement of results
- Hypertension – reduction in systolic or diastolic blood pressure by 10 mmHg ;
- Dyslipidemia – reduction in total cholesterol: 100 mg/dL; increase in HDL: 10 mg/dL; reduction in triglycerides by 50 to 100 mg/dL ;
- Coronary artery disease: improvement in myocardial ischemia; reduction in coronary calcium score ;
- Obstructive peripheral arterial disease: improvement in ankle-brachial pressure index < 0.9; arterial stenosis detected by deep-vein thrombosis ultrasound of lower limbs ;
- Carotid atherosclerosis: reduction in intima media thickness or degree of carotid stenosis ;
- Heart failure: reduction in degree of left ventricular hypertrophy or increase in systolic function of left ventricle or improvement and/or reversal of diastolic dysfunction, detected using Doppler echocardiography ;
- Arrythmia: reduction in number of events of arrythmia, detected by 24-h Holder or stress test;
- Diabetes mellitus, reduction in glucose tolerance and insulin resistance; improvements in fasting glucose, A1C hemoglobin, oral glucose tolerance test, basal insulin, insulin curve, homeostatic model assessment [38,39];
- Serum markers of cardiovascular risk: reductions in ultra-sensitive C-reactive protein, homocysteine and lipoprotein A ;
- Hyperthyroidism or hypothyroidism: improvement in serum levels of ultra-sensitive TSH, free T4 and free T3 [40,41];
- Chronic kidney disease: reduction in albuminuria, measured by glomerular filtration rate and calculated based on the Chronic Kidney Disease Epidemiology Collaboration ;
- Hyperparathyroidism (HPT): reduction in or normalization of serum levels of HPT .