Study subjects and group allocation
A prospective longitudinal study design was conducted among, fasting and non-fasting healthy subjects and diabetic patients (n=98) in Hawassa city administration, Sidama Regional State, Southern Ethiopia from February 27/2019 to April 30/2019. The subjects were Orthodox Christian communities, with age range of 30-50 years permanently living in the study area and had no plan of leaving before the completion of the study. In addition, those diabetic patients who have only a good adherence towards diabetes self-care practice were eligible for the study. The group allocation was done based on an Ethiopian Orthodox church fasting principles and dietary pattern during Lenten fasting. Fasting group (Case) have been rigorously experienced fasting for mean of 19 ± 15 years, and aiming to continue to fast and adopt to use simultaneously vegan diet and energy restriction during study period. Whereas those who have stopped fasting (fasted for 17 ± 10 years) before one year of study period and not willing to continue fasting during study period as non-fasting group (control). However, pregnant mothers or lactating mothers, and patients having chronic liver disease and or renal disease were excluded (Figure-1).
Sample size determination:
The sample size required was calculated to allow comparisons between two groups for repeated measurements (20-23). A total of 50 subjects were require per arm, assuming α = 0.05, power 0.85 a medium effect size (µ1-µ2)/α = 0.5, correlation of repeated measures (ρ) =0.6 and two time points, calculated using the following formula:
Where σ2 is the assumed common variance in the two groups, µ1-µ2 is the difference in means of the two groups; n is the number of time points and ρ is the assumed correlation of the repeated measures.
Assessments and measurements for follow-up
A digital electronic sphygmomanometer (Omron, Healthcare, Japan) was used to measure blood pressure after patients rested and completely stabilized for at least 5 min in the assessment room. Two readings of BP was taken within 2-3 min differences to maintain the accuracy of measurement and lastly the mean value was taken and documented to assess BP status. In addition, the third BP measurement was taken, when the two measurements varied by 10 mmHg within 2-3 min differences in a single study subject, and lastly the mean value of three measurements was taken to determine BP status.
Weight and height was measured when patients stood wearing light clothes and wearing no shoes. A digital electronic Adult scale (ASTO) that contain both weight scale and height scale was used to measure body weight to the nearest 0.1kilogram(kg), and height to the nearest 0.1 centimeter (cm). Moreover, body mass index (BMI) was calculated as weight in kilogram divided by squared height in meter.
Blood specimen collection and Laboratory diagnosis
About 4-5 milliliter of venous blood for prospective determination of serum appetite hormones. Blood sample was collected after 12 hour overnight fasting in the morning (from 9:00 to 9:30Am). The measurements were taken at two time points: at baseline (before start of fasting) and at last week of fasting (end time) from each study participant using serum separator tube (SST)/EP-tube. Then, the collected blood sample was allowed to form proper clot within 20-25 min at room temperature. Following this, the samples were centrifuged at 2500-3000 rotation per minute (rpm) to separate serum from clot and stored in aliquot at -80oC.
Sample transport
After collection of the blood sample, it was transported 275Km using ice pack. The analysis was done in central molecular laboratory found in Ethiopian public health institute (EPHI) in Addis Ababa, Ethiopia.
Hormone Measurements
Plasma hormone measurements were performed on blood samples in EDTA tubes that were centrifuged for 15 minutes at 4°C after collection and stored at −80°C until assayed. Thawed samples were not refrozen for other assay measurements. The appetite hormones like: insulin, glucagon and ghrelin were measured using carcinoembryonic antigen (CEA) by enzyme-linked immunosorbent assay (ELISA) immunoassay whereas leptin was measured using staphylococcal enterotoxin A (SEA) by enzyme-linked immunosorbent assay (ELISA) immunoassay. The analysis was done according to the manufacturer’s instruction (Cloud-Clone Corp., CCC, Research, WB; IHC; ICC; IP.) (24, 25). The minimum detectable limit insulin was 25.3pg/mL and intra- and inter-assay coefficients of variation were 3.0% and 8.7%. Minimum detectable limits glucagon was 20 pg/mL and intra- and inter-assay coefficients of variance was 4.3% and 7.1%. The minimum detectable limit of ghrelin was 20pg/mL and intra- and inter-assay coefficients of variance were 6.7% and 12.1%. The minimum detectable dose of leptin was less than 0.054ng/mL and intra and inter-assay coefficient was <10% and <12%.
Definitions
Socio-demographic and lifestyle factors
Standardized questionnaire was used to collect comprehensive information about socio-demographic condition, the number of years that participant fast, medical history and substance used by participants.
Alcohol consumption and smoking status were defined as subjects who had consumed any alcoholic beverage ≥1 times/week, and those who had smoked ≥10 cigarettes/week, during fasting season (26).
Physical activities also defined by participants who exercised three or more times/week for >30 min categorized as physically active (PA), 2times/week as medium PA, 1times/week as less PA and no physical activities/week as sedentary (27).
Insulin resistance was calculated using the homeostasis model assessment for insulin resistance, HOMA-IR (28, 29).
Appetite was evaluated by using VASs for hunger, fullness, desire to eat, and prospective food consumption (30).
Outcome Measurements
Body weight, BMI, blood pressure (SBP and DBP), activities of appetite hormones and HOMA-IR were assessed between baseline of fasting (week 0) and endline (last week of fasting). For each variable and study participants at each point, we calculated the change as value at last week of fasting minus value at baseline (week 0) divided by concentration at baseline, times 100 (31).
Data quality control
The questionnaires were pretested, translated and back-translated from English to Amharic to ensure the accuracy of the translation. The completion of questionnaire was frequently checked. The principal investigator gave three-day training and did close supervised of data collection. Data collection was done by trained clinical nurses; while blood sample collection and laboratory analysis was managed by laboratory technologists. In addition, at the initial day of contact, patients have got detailed information about the study and its protocol. In the study baseline and end time, blood samples were drawn from those who come only with overnight fasting.
Ethics
The protocol and the questionnaires were reviewed and cleared by the ethical review board of Natural and Computational Science of Addis Ababa University, Ethiopia (IRB/035/2018). The nature of the study was fully explained to the study participant and written consent was obtained from each participant prior the study.
Statistical analyses
The data, the optical density (OD) of appetite hormones of ELISA analyzer results, was visually checked for consistency, completeness and changed into concentration of hormones using linear fitting method of origin 8.0 version. The concentration of hormones statistically processed with IBM SPSS Statistics 23. Using Shapiron-Wilk test, normality of the mean distribution was checked. Data was summarized as mean (percent change), mean ± SD, or n (%) for continues variable were used. Furthermore, Chi-square, Fischer’s exact test used for different categorical data and student’s t-test was used to estimate of variances in means of study groups and multiple linear regression was used to determine the correlation between independent and outcome variables. Finally, a p-values < 0.05 was considered as statistical significance at 95% confidence interval (CI).