Study design
This randomized clinical trial was aimed to evaluate the effect of 8 weeks of vitamin D supplementation and water exercise on glycemic indices, lipid profile, and C- reactive protein (CRP) in women with type 2 diabetes. The sample size was determined based on the CRP level of the previous study [18] 10 participants for each group with 95% power and 5% significance. We considered 12 participants for each group due to a possible drop. The trial was ethically approved by the Ethics Committee of Kermanshah University of Medical Sciences (Ethical NO: IR.KUMS.REC.1397.656) and registered with the Iranian Clinical Trials Registry (registration number: IRCT20201223049811N1), data of registration: 12.01.2021; https://en.irct.ir/trial/53230. Written informed consent was obtained from all participants.
Participants, recruitment, and randomization
This current study was designed based on CONSORT statement for randomized clinical trials [19]. The study participants were recruited from diabetes clinic of Taleghani hospital, Kermanshah province, western Iran. Inclusion criteria were 40-60 year-old women with type 2 diabetes, no smoking, no cardiovascular disease, musculoskeletal disorders, rheumatoid arthritis, no fear of water, no supplements, especially vitamin D supplementation, and no diet in 6 months ago. Participants who were absent for more than one training session were excluded from the study, as well as, any physical injury during training, and inability to continue the training program were excluded from the study.
To assign participants to intervention or control group was applied simple random method. Using random numbers table, each participant will be assigned a number and selected randomly. Ultimately, 48 participants were assigned to four groups including: Vitamin D+ water exercise group (n=12), water exercise group (n=12), Vitamin D group (n=12), and control group (n=12). (Figure 1) The participants were completely unaware of the group they were in, therefore, they were in the same mental state.
Intervention
Water exercise group did your exercises in water during 8 weeks and every week for three sessions (24 sessions in total). Each session began from 9 to 10 in the morning for one hour in shallow part (80-120 cm) of the pool of Razi University in Kermanshah, Iran. Each water training session had three stages: the first stage, adapting to the water environment and warming up (15 minutes) including stretching movements in all joints and major muscle groups, walking forward, backward, sideways, on the heel and toe, and Jagging was in the water. The second phase of the exercise (30 minutes) included weight transfer from front to back, brisk walking in the water, sideways walking, and squats. The third stage was stretching, deep breathing and floating exercises (15 minutes). The intensity of the exercises was controlled using the Borg scale. To determine this scale, guide sheets are attached to the wall of the pool and the participants have already been trained. All exercises were performed in the indoor pool of Razi University with water temperature between 26-28 ° C. Vitamin D+ water exercise group was given vitamin D supplement (1 pearl 50000 IU Vitamin D made by Zahravi Pharmaceutical Company- Iran) weekly in addition to the mentioned exercises. Also, vitamin D group was received the vitamin D supplement (1 pearl 50000 IU Vitamin D made by Zahravi Pharmaceutical Company- Iran) weekly for 8 weeks. In total, each participant consumed 8 pearl vitamin D for two months. The control group performed only their daily activities during the study and no received the study supplement and no did the water exercise. All participant were asked to follow their usual diet, avoid from taking other supplements and physical activity during the study.
Demographic information
Demographic information and medical history of these participants were collected and recorded. These information included age, educational level, marital status, daily exposure to sunlight and medical history.
Anthropometric indices
Height was measured using standard wall-mounted stadiometer ((DETECTO, Model 3PHTROD-WM, USA) standing position without shoes with a precision of 0.1 cm. Weight was measured by (Seca, Germany) with the least clothing and without shoes. Body mass index (BMI) was calculated by dividing weight in kg by height square in meter. The non-stretched and flexible tape was applied to measure waist circumference in standing position at the level of the iliac crest at the beginning and end of the intervention [20]. Subcutaneous fat thickness of the subjects was obtained using a caliper (Lafayette skinfold caliper, Model 01127A, USA ) at three points on the triceps, under the scapula and the right leg, and by substituting the numbers obtained in the Jackson and Pollack equation of three points, the body fat mass (BFM) was calculated. In addition to anthropometric indices, a calibrated digital brachial sphygmomanometer (Beurer BM26, Germany) we used to measure systolic and diastolic blood pressure (SBP and DBP) after at least 4-5 minutes of rest in sitting position.
Biochemical indices
From each participant, blood venous samples (10ml) were obtained after an overnight (10 to 12 hours) fast at the before and after the study. The blood samples were centrifuged, and serum was stored at -80 °C until analysis. Enzymatic method was used to analysis fasting blood sugar (FBS). Serum insulin was measured using ELISA kits (French company Diaclone, French) with a sensitivity of 0.179 μN. Glycated haemoglobin (HbA1C) was analyzed by Ion exchange chromatography. Insulin resistance was determined based on the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) following formula:
HOMAIR = fasting blood sugar (mg/dl) × fasting insulin (micro U/ml)/ 405 [21].
Lipid profiles including total cholesterol (TC), high-density lipoproteins (HDL) and triglyceride (TG) concentration were measured by enzymatic kits (Pars Azmoon, Iran). LDL-cholesterol value was calculated by applying [22]. Vitamin D status based on serum levels of 25 (OH) D3 was detected by Electrochemiluminescence (ECL) method. CRP was also measured by turbidometry method. The basis of the turbidometric method is based on the formation of a complex resulting from the reaction between hs- CRP and its specific antiserum.
Dietary intake
At the beginning and end of this study, self- reported three-day food record questionnaire was received to assess the energy and nutrient intake (two days of week and the weekend) at the first and after eight week of the study. The amounts of energy and nutrients of their food intake were calculated by NUTRITIONIST 4 software using the United States Department of Agriculture Food Composition Table, which was modified for Iranian foods [23]. All data processing and entry were done by a trained dietitian.
Physical activity
The International Physical Activity Questionnaire (IPAQ) short form was applied to assess physical activity level. Total metabolic equivalent task (MET) per hour per week was calculated based on the instrument existed in the questionnaire. The validity and reliability of the questionnaire had previously been confirmed in Iran [24].
Statistical analysis
All statistical analyses were performed by SPSS software (version 23; SPSS Inc., Chicago, IL). In this study, descriptive statistics were used to describe the variables. In descriptive statistics, means, standard deviations (SD), and frequency percent were used to describe the findings. The normal distribution of quantitative data was determined by Kolmogorov-Smirnov test and the homogeneity of variances was assessed using the Leuven test. Chi-square test was used to compare qualitative variables. Two-way ANOVA was used to examine the differences between the variables before and after the intervention. Paired samples t-test was used to analyze within-group changes. P-values less than 0.05 were considered significant.