Prevalence of Vitamin D De�ciency and Its Associated Risk Factors Among Rural Population of the Northern Part of the Persian Gulf

Background: Accumulating evidence indicates that vitamin D de�ciency has been increased globally since the last two decades. However, the majority of these studies concerned on cities and there is scant information regarding the prevalence of vitamin D in rural areas. The main aim of this study was to investigate the prevalence of vitamin D de�ciency and its associated risk factors among rural population in Bushehr province which has the longest border with the Persian Gulf. Methods: The rural inhabitants with ≥ 25 years old from three mountainous, plain, and seashore areas of Bushehr province were selected through a strati�ed multi-cluster random sampling method. After obtaining the participants’ demographic and anthropometric data and their past medical history, serum 25-hydroxy vitamin D was measured using ELISA. Results: A total of 1806 (means±SD, 46.30± 14.22 years old) rural subjects (34.84% males and 64.88%) participated in this study. The prevalence of vitamin D de�ciency, insu�ciency and su�ciency were 27.87%, 50.39% and 21.74%, respectively. The de�ciency of vitamin D in women was higher than men (OR=1.27, 95% CI: 1.05 to 1.54, P=0.040). There was a positive signi�cant correlation between age and serum vitamin D levels. Men with vitamin D de�ciency had higher BMI (P=0.008); this association was not observed among women (P=0.74). There was no signi�cant difference between the food items consumption frequencies, and vitamin D status (P>0.05). The mountainous, and plain areas had the highest and lowest vitamin D levels, respectively. Conclusion: Although, Bushehr province is located in a sunny part of Iran, the prevalence of vitamin D de�ciency was high among its rural population. The shift of their life styles patterns and rapid industrialization in these rural areas may be responsible. Therefore, the enrichment of dietary sources with vitamin D and the use of vitamin D supplements are recommended to tackle the high prevalence of vitamin D de�ciency in the rural population of the northern part of the Persian Gulf.


Introduction
The de ciency of vitamin D has been reported from all parts of the world, and there is a signi cant association between low serum levels of vitamin D and communicable and non-communicable diseases [1].Accumulating evidence has shown that vitamin D presents bene cial effects on our tissues and organs.
Beside this, the more vitamin D concertation means the less occurrence of cancer and the related mortalities.In addition, vitamin D de ciency has signi cant association with high blood pressure, type I diabetes, multiple sclerosis, rheumatoid arthritis , and other autoimmune diseases [2].
Although there is no consensus for de ning, and optimal levels of serum 25-hydroxy vitamin D, vitamin D de ciency has been de ned at cut point serum levels of less than 20 ng/ml; because at this level, parathyroid hormone begins to increase.Hence this outpoint is considered the physiologic de nition for vitamin D de ciency [3].
Insu cient sun light exposure, age-related decrease in vitamin D synthesis in skin, and low dietary intake are attributing factors for circulating levels of this vitamin.Therefore, vitamin D supplementation and su cient sun light exposure can protect most of the people from vitamin D de ciency.Five to ten minutes of direct sun light exposure on the arms and legs between 10:00 AM to 3:00 PM during spring, summer, and autumn can prevent vitamin D insu ciency [4].
Because of the signi cant effect of vitamin D on bone mineral homeostasis, bone mineral density, and nally pick bone mass, several large multi-central interventional studies with vitamin D supplementation are under investigations in all over the world [5].
Even though there is a limited amount of vitamin D in plants, a su cient amount of vitamin D3 could be found in sh oil, calf of liver, cheese, and eggs yolk [6].In addition, acute liver failure, nephrotic syndrome, renal disease, rickets and hyperparathyroidism could be considered as etiologies for vitamin D insu ciency [7].The geographical location, methods of skin coverage, skin color, and consumption of foods with lack of vitamin D are prominent causes for vitamin D insu ciency [6].
In a survey study which was done on American population from 2001 to 2006 (National Health , and Nutrition Examination Survey), it was shown that 32 percent of participants had serum vitamin D level less than 20 ng/ml [8].The studies in developing countries have shown that osteoporosis, increased risk of falling, and bone fracture were associated with an increase in the prevalence of vitamin D de ciency [9].Moreover, the past two decades studies elucidated a high prevalence of vitamin D de ciency in China, Turkey, Saudi Arabia, and Iran which varied between 30 to 93 percent [10].
Vitamin D de ciency was reported in 75.1% of females and 72.1% of males from the Iranian Multi-Centric Osteoporosis Studies (IMOS).The result of this study was consistent with those results that had been reported with other studies from the Middle East region.[11].A comprehensive epidemiological and ecological descriptive study on vitamin D status in Iran showed that the mean of vitamin D concentration was 25.4 ng/ml which was in the range of vitamin D de ciency [12].
The high prevalence of vitamin D de ciency among the Iranian population may be due to the shift of Iranian lifestyle patterns towards the urbanization and industrialized lifestyles resulting in low sunlight exposure that accentuates the low dietary intake of vitamin D in this country [11].In addition, air pollution is an important affecting factor for vitamin D de ciency in the large cities [13].Although similar prevalence rates for vitamin D de ciency in urban and rural areas of Asia have been reported, in some studies, the urban subjects in comparison to the rural subjects had more prevalence of vitamin D de ciency [5].
In Iran, the majority of studies have been done in urban areas and capital cities [10,14,15].There is a lack of information about the prevalence of vitamin D de ciency and its serum levels in rural areas [10] Therefore, the limited information about vitamin D in rural areas may prevent to design strategies to combat vitamin D de ciency in Iranian rural subjects who are rapidly changing their lifestyle towards urbanization and industrialization.Hence, this phenomenon may accelerate the situation of vitamin D de ciency among this rural population.The aim of this study was to investigate the prevalence of vitamin D de ciency and its associated risk factors among rural population in Bushehr province which has the longest border with the Persian Gulf.

Community sampling:
Bushehr province which is located in the northern part of the Persian Gulf, was divided in three geographical (the northern, the central, and the southern) parts.Each geographical part was then divided into three mountainous, plain, and seashore areas in order to assess the effect of latitude, dietary habits, and socio-economic status of inhabitants on their serum vitamin D levels.The sample size of each part was proportional to the size of each population.The household was selected as the sampling unit which were randomly selected systematically using census data.
A total of 1806 rural inhabitants with ≥ 25 years old were selected.The exclusion criteria included being non-native, inability to give blood sample, disability to answer the questions, and being less than 25 years old.The participants completed a questionnaire including their anthropometric, demographic data, past medical history, gravidity, menopausal status, nutritional conditions and supplementary consumptions as well as their drug history.

Examinations:
Examination were conducted in the primary health care centers attached to Bushehr University of Medical Science (BUMS).Height, and weight were measured using stadium meter.Heavy outer garments, and shoes were removed before measuring heights, and weights.Body mass index (BMI) was calculated.Waist circumference was de ning at the mid waist level between the costal margin and iliac crest.Hip circumference was measured at the greater trochanters.A 3 milliliter (ml) sample of blood was taken from all participants, and all samples were promptly centrifuged, and sera were separated, and kept frozen at -80 until they were used.
Serum vitamin D levels were measured using a commercial ELISA kit (Immunodiagnostic Systems Limited, UK).The reportable range of the assay was 6.5-100 ng/ml.Its limit of detection was 2.7 ng/ml.The range of inter-assay %CV of the kit was 1.9 to 3.7 ng/ml.Serum 25-hydroxyvitamin D levels were de ned su cient if they were between 30-100 ng/m, insu cient if varied between 20-<30 ng/ml, and de cient at levels of <20 ng/ml.

Statistics:
The statistical analysis was performed using STATA version 14.The Kolmogorov-Smirnov test (KS-test) was used to determine the normality of data distribution.For descriptive data, frequency, mean, median, and standard deviation were used.The statistical signi cance of variables between groups was analyzed using X 2 test.A univariate and multivariate analysis was performed to evaluate the association between serum 25-hydroxyvitamin D (dependent variables) and other co-factors including (age, sex, marital status, anthropometrics data, sun-light exposure, sun protection factor (SPF), dietary, and supplementary consumption).P<0.05 was considered statistically signi cant.

Results
A total of 1806 rural subjects including 631 (34.84%) mens and 11759 (64.88%) womens, participated in the study.The characteristics of the participants were shown in table 1.The subject ages ranged from 23 to 94 years old (mean ± standard deviation 46.30± 14.22 years).The gure 1 were shown that the highest age groups vitamin D de ciency belong to 30-39 years old(Figure1).The prevalence of vitamin D de ciency and insu ciency were 27.87% (505 subjects), 50.39% (913 subjects), respectively.A total of 394 subjects (21.74%) had su cient serum vitamin D levels.
The de ciency of vitamin D in women was higher than men (OR=1.2795% CI: 1.05 to 1.54, P=0.040).Furthermore, 537 (29%) of the participants (22% of women, and 42% of men) had sun exposure more than 15 minutes per-day 2 to 3 times per week.There was a signi cant difference for sun-light exposure among su cient, insu cient and de cient groups (Table 1).
The prevalence of consumption of oral calcium, vitamin D calcium + vitamin D, and parenteral vitamin D were 37 (2.04%), 239 (13.20%) , 32 (1.77%) , 7 (0.39%), respectively.The negative consumption of tuna and curd was found for a half of the participants, while 72% of them reported positive consumption of egg.Also, more than fty percent of the participants reported more than the median consumption of dough and milk.Furthermore, 148 (29.37%) and 327 (18.54%) of the participants reported to have history of hypertension and diabetes mellitus, respectively.
There was also a signi cant association between age and serum vitamin D levels (P≤0.0001).
The rural subjects between 30 to 39 years old and elderly participants of the study with more than 80 years old had the highest and lowest amounts of vitamin D de ciency, respectively.
In men, their serum levels of vitamin D was increased with increasing their age, while by increasing their height, weight, and BMI, it was decreased.However, in women, only age had a positive association with vitamin D serum levels.In addition, men with vitamin D de ciency had higher BMI (P=0.008); this association was not observed among women (P=0.74).No association could be found between waist to hip ratio in relation to the status of vitamin D levels (P>0.05)(Table 2).In addition, there was no signi cant difference between the food items consumption frequencies, and vitamin D status (P>0.05)(Table 3).
There was a signi cant difference between the score of gravidity and vitamin D status (P<0.0001).However, there was no signi cant association between the months of breast feeding, and serum vitamin D levels (P=0.212).However, 292(46%) of subjects with vitamin D de ciency, 196 (31%) of subjects with the insu cient, and 143(22%) of subjects with insu cient serum vitamin D levels reported that they had history of breast feeding; however, no signi cant differences could be found among them (P=0.087).
The mountainous, and plain regions of the rural areas had the highest, and lowest vitamin D levels, respectively.

Discussion
In this population-based study which was done in all rural districts of Bushehr province, it was shown that a half of the rural participants had vitamin D de ciency, more than one to ve of the participants had vitamin D insu ciency, and one to ve of total population had su cient vitamin D levels.Unfortunately, there are limited studies on rural population in Iran regarding vitamin D levels, and most of the studies have been done on the capital cities; therefore, it is impossible to compare vitamin D serum levels of rural and urban populations.The only study that compared the vitamin D serum levels was done on Guilan province in the northern part of Iran which compared 750 postmenopausal women in rural and urban areas and demonstrated that vitamin D de ciency was more common in urban than rural subjects [16].This difference among rural and urban subjects was also observed in other countries [17].In a systematic review and metaanalysis study in Africa it was shown that the mean of serum vitamin D levels in urban places was less than the rural areas [18].This difference could be explained with different lifestyles, jobs, and habitual conditions because the rural inhabitants live more outdoor and expose to more sun-light with resulting with more absorption of vitamin D by their skin; in another aspect, the urban lifestyle patterns cause less amount of vitamin D absorption via sun-light exposure or less amount of dietary vitamin D intake through food habits [16,18].
Although, in the current study, we could not compare the prevalence of vitamin D de ciency in urban and rural regions, we found that the prevalence of vitamin D de ciency in rural areas was at least similar to those prevalence rates that has been reported from Bushehr city ( the capital of Bushehr province ), the middle east and south east of Asia ,and China [10,[19][20][21].In two systematic reviews and metaanalyses from Iran, it was reported that more than a half of the Iranian population, especially those who live in capital cities, had vitamin D de ciency [14,15].The observed high prevalence of vitamin D de ciency in rural areas in the current study may indicates that the lifestyle of these villagers has been changed due to industrialization, and also their nutritional habitus has converted to the urban styles.These changes of life style among villagers of Bushehr province may have induced their mean vitamin D serum levels to approach to their urban counterparts.
In our study, we found a linear relationship between serum vitamin D levels and sun-light exposure.In the European population, it has been revealed that sun exposure of 18 percent of body surface area for 15 minutes per-day, 2 to 3 times per week is su cient for absorbing vitamin D [22].However, in the current study, only 29 percent of participant had ful lled the above criteria.This low level of sun-exposure in the rural areas may be due to the mentioned changes of lifestyle towards the urban patterns.The cultural factors may also have an effect on the amount of villager's sun-light exposure because of the types of their clothing which cover their arms and legs for all seasons.
The effect of body coverage on circulating vitamin D is so important that in sunny countries such as Saudi Arabia and the United Arab Emirates, a high prevalence of vitamin D de ciency could be found; likewise, in Iranian sunny cities such as Zahedan and Isfahan, a high vitamin D de ciency have been reported.[23, 24] [25, 26].
In another aspect, the effect of sun exposure on vitamin D serum levels could be ascribed by its seasonal patterns of sun-light exposure; in winter, we can expect to obtain less amount of vitamin D by decreasing in sun-light exposure [26].In the current study, all the serum samples were obtained during winter; therefore, the effect of seasonal patterns could not be evaluated.However, it seems that there is a complex interaction between the effect of season and bio-cultural factors.
Surprising, the mean serum vitamin D was highest in winter and lowest in the summer in a sunny country like the United Arab Emirates [23].The climate of the United Arab Emirate is very similar to Bushehr province in the northern parts of the Persian Gulf.It could be postulated that the high temperature of these places induces people live most of their times indoor to escape the hot condition during summer times.Hence, they receive less amounts of sun-light exposure, leading to the lowest range of serum vitamin D levels during summer.In order to elucidate the complex interaction of cultural, and bio-environmental factors on circulate vitamin D levels, more studies during different seasons are warranted.
In this study, an association between vitamin D serum levels and age was found.It was interesting that the minimum serum levels of vitamin D was observed in 30 to 39 years old age group; and surprisingly the highest levels of vitamin D were found among rural subjects who had more than 80 years old.In an Iranian meta-analysis the highest prevalence for vitamin D de ciency was found among the 20 to 50 years old age group [15].In contrast, in a previous study, children and elder people had the highest prevalence of vitamin D de ciency [27].In Iran, due to not including older people in the previous population based studies about the prevalence of vitamin D de ciency, a comparison would not be possible [14].
The consumption of multivitamins and vitamin D pills by the elderly may lead to a lower prevalence of vitamin D in this age group in comparison to the younger participant in our study.Another contributing factor for this difference may be the changes of life styles of younger people that induce them to choose living and working indoor places with resulting in less sun-light exposure.In consistent with our nding, a positive correlation between age and vitamin D serum levels was found in Zahedan city (the capital of Sistan , and Balochestan province in the south east of Iran) [14,15].Likewise, the younger age group had a higher prevalence of vitamin D de ciency than older age group in Isfahan city ( the capital of Isfahan province in the center of Iran) [26].Taken together, the change of lifestyle patterns and the trend of younger people to stay in indoor places, and living in apartments may explain this difference for prevalence of vitamin D de ciency among different age groups.
In our study, the prevalence of vitamin D de ciency was more common in men than women.The results of two meta-analyses studies from Iran showed that the prevalence of vitamin D de ciency, like other Asian countries, was more common in women than men [14,15].In a trend prevalence study of vitamin D deviancy during 1990-2010 in Iran, it was also reported that women gained a higher vitamin D de ciency than men year-over-year [28].
There are contradictory results about vitamin D de ciency in relation to sex in the world.In contrast to a previous study in America, no difference could be found between sex groups in relation to vitamin D de ciency in NHANES 2001NHANES -2004 [29] [29]; but, in a later study in America , women had a higher vitamin D levels than men [30].In Africa, the prevalence of vitamin D de ciency was higher in women than men [18].In the United Arab Emirates (UAE), the prevalence of vitamin D de ciency was similar in both sexes [23].It has been suggested that cultural and religious factors might be the causative factors for the observed higher prevalence of vitamin D among Muslims women; for instance, in Lebanon, the Muslim women had lower vitamin D serum levels than the Christian women [31].These contradictory results indicate that other contributing factors beyond veiling should be considered to explain this difference.
Obesity is another condition that has a connection both to the patterns of life style and vitamin D serum levels [32].The body fat content has a reverse correlation with vitamin D concentration.This inverse correlation may be due to the decrease bio-availability of vitamin D3 from dietary sources and skin because of the deposition of vitamin D in body fat compartments [33]; even the elder subjects with high body fat and higher body mass index have lower levels of 25-hydroxy vitamin D levels [34].Other factors contribute to the inverse relationship between body mass index and vitamin D levels, such as decreased mobility in obese people, which reduces their exposure to sunlight [32].
In our study, men with vitamin D de ciency had a higher anthropometric index (BMI) than men with su cient vitamin D levels, while no difference was observed between the two groups in women; therefore, other contributing factors beyond obesity should be considered that modify the effect of obesity, such as intake of vitamin D supplements and the number of pregnancies.
Although the current study is one of the largest studies that investigated vitamin D de ciency in the Iranian rural subjects, it has some limitations.One of these limitations is non-repetition of measurements for vitamin D levels during different seasons.The physical activity, and their smoking status of the participants were not also assessed.However, the evaluation of nutritional status as well as sun exposure which were addressed in this study could be ascribed as one of its strengths.
Another strength of the study is its design so that the effect of the latitude (mountainous, plain and coastal places) of living areas on the level of vitamin D could be evaluated.
In this study, the northern half part of the rural mountainous area of Bushehr province had a higher mean of vitamin D level than the plains and coastal areas.There was no signi cant difference in daily consumption of dairy, and milk products between mountainous area, and other parts this province.

Conclusion
In conclusion, our study showed that there is a high prevalence of vitamin D de ciency among the rural population of Bushehr province, this prevalence is similar to those reported from the urban areas in other parts of Iran.Therefore, the observed higher mean vitamin D level in the mountainous area in comparison to the plains and costal area of Bushehr province may be due to presence of different patterns of life style in these areas.
Although, Bushehr province is located in a sunny part of Iran, the prevalence of vitamin D de ciency was high among its rural population.The shift of their life styles patterns and rapid industrialization in these rural areas may be responsible.Hence, in order to combat vitamin D de ciency, enrichment of dietary sources with vitamin D, and health promotion with emphasis on healthy life with increasing physical activity may be suggested as effective strategies.These strategies have been considered in Asian countries [5].In India, the programs of enrichment of a dietary source with vitamin D, supplementation of vitamin D plus calcium, and inclusion of local forti ed food items were launched by local governmental agencies, and other state holders.[35] In Iran, a community-based iterventional trail of vitamin D fortied milk has been started [36].The results of this trail could be applied in different parts of the coutry to tackle vitamin D de ciency in the near future.

Table 1 :
This study was conducted in agreement with the declaration of Helsinki and Iranian national guidelines for ethics in research.written informed consent was obtained from all participants prior to study enrolment.participation was voluntary and each participant could withdraw consent at any time without any consequence.Data collected are stored in a re-identi able form by national id code.The study was approve in the ethical committee by a grant from National Institute for Medical Research Development (NIMAD).Muka T, Zillikens MC, Voortman T, Uitterlinden AG, Hofman A, Rivadeneira F, Kiefte-de Jong JC, Franco OH: Vitamin D and body composition in the elderly.Clinical Nutrition 2017, 36(2):585-592.34.Suryanarayana P, Arlappa N, Sai Santhosh V, Balakrishna N, Lakshmi Rajkumar P, Prasad U, Raju BB, Shivakeseva K, Divya Shoshanni K, Seshacharyulu M: Prevalence of vitamin D de ciency and its associated factors among the urban elderly population in Hyderabad metropolitan city, South India.Annals of Human Biology 2018, 45(2):133-139.35.Kamboj P, Dwivedi S, Toteja G: Prevalence of hypovitaminosis D in India & way forward.The Indian journal of medical research 2018, 148(5):548.The demographic and anthropometric data of the rural population in the northern part of the Persian Gulf

Table 2 :
The association among age, anthropometric indices and vitamin D status in the northern part of the Persian Gulf

Table 3 :
The consumption of food items (grams per day) across vitamin D groups in the northern part of the PersianVariableVitamin D de cient Vitamin insu cient Vitamin D de cient P-Value