The results of this study demonstrated a significant Gender differences of vitamin D level with a higher mean value in males (23.90 ± 16.41) ng/ml than females (21.24 ± 15.65) ng/ml. About 53% of males and 58% of females who were participated in this study were suffering from vitamin D deficiency. The severity of vitamin D deficiency is more common in females 17% than males (7%) of the whole participants. Regarding free vitamin D level, (95%) of the participants had normal FD3 level and a non-significant effect of gender was noticed with a slightly larger values males than females.
Lower 25(OH) D levels were also observed in females, as compared to males in [19, 20]. Similarly, many other previous studies were done in different Governorate of Iraq. A total of 3520 subjects were enrolled in a study done in central Laboratory in Suleimani, consisting of 574 men and 2946 women, it showed a significant difference between the mean values of vitamin D level in males (16.23 ± 12.86) which is higher than females (13.32 ± 11.6) ng/ml . Another group of volunteers were tested for their vitamin D levels at Kufa district, the results indicated a significant higher level of vitamin D in males (24.56 ± 5,52) as compared to the females (14.37 ± 1086) ng/ml .
The mean serum level of vitamin D in 120 postmenopausal women studied at Karbala governorate was 13.5ng/ml, whereas a child bearing group of 100 women was 17.3ng/ml. The males in the same study showed higher mean level of vitamin D 18.5ng/ml for the young ages and 14.6 ng/ml for elderly .
One other study was done in Erbil city with the involvement of 10823 subjects of different ages and both genders. 75% of the participants where females, the mean value of vitamin D level in females was (14.67 ± 22.14) ng/ml which is lower than its corresponding level in males (15.05 ± 16.88) ng/ml .
After a revision of all of the above-mentioned studies done in different governorates of Iraq and comparing it to the results of the current study, all studies agreed that there are a significant gender differences of vitamin D level and the males had higher mean levels of vitamin D than females. Interestingly, the data of this study showed better vitamin D status of both genders compared to other Governorates, besides that, the results of vitamin D in females where very close to that of males and much higher than the females studied in other Governorates of Iraq. The outdoor activity of males in addition to excess adipose tissue of females compared with males were suggested as a causal factor of gender variations .
Gender-associated variations in 25-OH Vitamin D levels also could be related to androgen-associated changes in the concentration of Vitamin D-binding protein, precursor production by skin, and 25-hydroxilation in the liver . Moreover, it probably reflects the cultural and religious practices leading to less skin exposure in women than in men .
When the subjects were divided based on ages in this study, a non-significant variation on the mean levels of TD3 and FD3 was observed. Vitamin D deficiency is more prevalent in younger ages whereas older ages specifically more than 50 years old, about 50 % of the involved subjects in this group they had sufficient vitamin D level and the rest had either insufficient or deficient vitamin D level. Similarly, the results of FD3 according to different ages indicated that 5% of the subjects had a non-significant low value which is most frequently occurs in the younger ages between (16–25) years old. Furthermore, a significant positive association was shown between age and both TD3 and FD3 levels. Bischof and co-workers in 2006 also found similar association of ages with 25-OH-vit D.
When the subjects in Sulaymaniyah city/ Iraq were grouped according to their ages into five groups(15years/group), Abdullah and his collaborators in 2018 found that the mean vitamin D level increased gradually from 8.6ng/ml in the first and younger subjects (16–30) years to 14.8ng/ml in older (> 80) years. Their explanation was that elders may have more healthy foods and their exposure to sunlight more frequently than younger . Another research group, in their study which was carried out in Iran indicated that serum level of 25-hydroxyvitamin D did not decline with age, their finding was considered very similar to this work .
To confirm the above findings, the mean values of TD3 and FD3 were calculated at different age groups, the least TD3 level was noticed in the youngest groups (16–25) years old then increased gradually with maximum level seen in the older group (> 56) years old. The males showed slightly higher TD3 levels than females with in the same age group with the older age group give higher level, they had sufficient vitamin D level. Regarding FD3 level, also the highest level noticed was among males (10.67 ± 2.54) pg/ml in (> 56) years old and among females in (51–55) years age group (10.90 ± 4.09) pg/ml. Furthermore, FD3 level was increased gradually and proportionally to increasing ages. Its worthy to mention here that this is the first-time aging effect on the level of TD3 and specifically FD3 was studied in details and this result could be considered as the first one in this aspect.
However, our results agree with the above-mentioned studies but disagree with a previous study which was demonstrated that aging affect vitamin D level through different aspects, among them, decreased calcium absorption, intestinal resistance to circulating 1,25(OH)2D, decreased vitamin D receptors, decreased production of 1,25(OH)2D by the aging kidney, decreased skin production of vitamin D and deficiency of the substrate to produce vitamin D .
A non-significant increase in TD3 and FD3 with an increase in BMI was observed, males had higher levels of vitamin D as compared to the females with in the same group also was indicated. The highest mean level of TD3 among males in obese group (29.62 ± 24.67) and among females in overweight group (22.47 ± 17.62). Similar results were also found in FD3 according to BMI specifically in females, anon significant increase in its level proportionally to BMI was clearly resulted with the lowest level seen in the underweight subjects (5.88 ± 4.40).
Whether the storage of vitamin D and 25(OH)D in adipose and other tissues is of clinical importance is uncertain and previous study found that the stored amount has only negligible effects on serum 25(OH)D levels [30, 31]. It was reasonable that vitamin D metabolites are gradually released from adipose or other tissues into the circulatory system to prevents serum 25(OH)D from falling to critically low levels during the winter . It’s very important to mention here that neither BMI nor body weight levels reflect the percentage of body fat. To clarify this point, athletes’ persons may have high BMI and may be considered overweight or obese while they have low total fat mass .
It was expected that obese persons have the tendency to produce more vitamin D in the skin than normal weight persons since they have larger body surface area with the same amount of provitamin D (7-dehydrocholesterol) per unit body surface area . Its worthy to mention that regardless of the increase in total vitamin D3 level occurred with the increase in BMI, but the increase was not significant which is similar to the results obtained in a prospective study conducted in Korea with the involvement of 1080 subjects and another local study at Kufa University / Iraq involved 273 individuals, both studies revealed a non-significant association between BMI and serum levels of vitamin D [35, 36].
The results regarding vitamin D status in smokers of this study were unexpected, Smokers had a higher but non-significant level of TD3(26.95 ± 19.01) ng/ml and FD3 (9.47 ± 4.94) pg/ml than nonsmokers (22.14 ± 14.59) ng/ml and (7.87 ± 4.32) pg/ml respectively. Moreover, a positive association was found between smoking with both total and free vitamin D3. Higher serum levels of 25OHD were also found by  in smokers at the time of study than in never smoked and the differences remains unchanged even after correction for the confounders age, sex, physical activity, vitamin D supplementation, season of blood sampling and BMI. Accordingly, in another previous study done in America on 805 women aged between 18–33 years, they found that smokers had higher level of 25OHD than nonsmokers . The above studies confirmed our finding with an exception which is different assessment method was used in each study.
It was suggested that smoking may be less indicator of serum 25OHD level. However, smoking has become more regulated in Iraq, specifically in our region. Persons who want to smoke most go outside the building. In doing so, their exposure to sunlight will increase which in turn lead to an increase production of vitamin D among smokers similar to the finding of .
Several studies reported lower serum 25(OH)D levels in smokers [39, 40], while others find no significant differences in serum 25(OH)D levels between smokers and non-smokers [41, 42].
The body covering varies with women who wear a Hijab (can be colorful), from a loose robe to various types of modern clothing, with skin covering minimum to the wrist, including legs. It allows for the entire face to be revealed, while continuing to cover the hair . This style is the most common covering in Duhok city.
Clothes are a main blocker to sun exposure and therefore 25(OH)D production in skin. Females with western style wearing have higher levels of 25(OH)D than those wearing hijab. Sun exposure to uncovered face and hands as in hijab dressed females is not enough for vitamin D synthesis as it was found by .
A strong correlation between the levels of 25(OH)D and clothing was reported by Mallah and his coworkers in Jordanian women . Also, a lower serum 25(OH)D level was measured in Tunisia with lower mean level of veiled compared to non-veiled women was indicated . The prevalence of vitamin D deficiency was higher in the covered than the uncovered females’ students at Istanbul Medipol University and that vitamin D level is associated with clothing style and age at which the females started wearing hijab as it was proved by .
Regarding the results of this study, firstly, the prevalence of severe deficiency, deficiency, insufficiency and sufficient vitamin D level was higher in the covered group than the uncovered women were detected, it was expected result since the number of the covered women were doubled the uncovered women involved in this study. Secondly, the mean serum level of total vitamin D in the veiled women was non-significantly higher than the unveiled women with in the same group which was unexpected result and inconsistent with all of the above-mentioned studies. This result could be attributed to the dress style of the veiled women involved in this study which was carried out in Duhok city. The dress style of the veiled women is very similar to the style of the unveiled women, both they wore a western dress style with a single difference that the veiled covered their hair. Similarly, a non-significant variation was also observed between three groups of women wearing different dress styles in a study done by  in Jordan which was on the same line of our finding.
However, the results of our work were in accordance with those stated by  who demonstrated a non-significant Difference of vitamin D levels among hijab users and non-users.
Regarding free vitamin D3 level, as there are no previous studies on the impact of the dress style of females on free vitamin D level, the current result could be considered as the first one in this respect. There is a significant increase in FD3 level in the veiled women (9.12 ± 4.64)ng/ml as compared to unveiled ( 6.16 ± 3.73)ng/ml. Furthermore, there is a significant positive correlation between FD3 level and dress style whereas a nonsignificant correlation was found with TD3 level.
The results of the present work demonstrated that 63% of the subjects, their daily exposure to sunlight was 30 minutes, yet they weren’t protected from becoming TD3 deficient. 30% and 26% of them they were deficient and severe deficient respectively. Also, within a group whom their exposure to sunlight was for 30 minutes/day, the majority of them constituted (69%, 61% and 58%) within their subgroups had severe deficient, deficient and insufficient vitamin D respectively. Strangely, 33% of the participants in the group of exposure for > 1 hour to sunlight, they suffered from severe TD3 deficiency.
Regarding FD3 results versus daily exposure to sunlight, just 6% of the participants had abnormally low results of FD3 and most of them (14 out of 23) their exposure to sunlight was for 30 minutes/day and 8 of the rest they exposed for more than 1 hour to sunlight. Its worthy to mention here that the above-mentioned differences in total and free vitamin D level according to the daily exposure to sunlight were statistically non-significant, but there is a negative significant association between daily exposure to sunlight with FD3 level. That mean a highly exposure, led to a decrease in FD3 level. This result could be considered as a new result in this respect. Although it is well documented that sunlight-induced vitamin D synthesis in the skin is the major source of vitamin D, the precise impact of habitual sunlight exposure on vitamin D status remains to be further explored, due to several factors that can affect the efficacy of dermal vitamin D production .
The results shown above were in agreement with the results of others [49, 50] demonstrated no statistically significant differences between sun exposure and serum vitamin D level. The finding of Binkley and his colleagues was very similar to ours, they reported that individuals have different responsiveness to UVB radiation, causing some to have low vitamin D status despite abundant sun exposure . However, our findings contradicted several previous studies in Southern and Northern Europe as well as other regions of the world, which reported a significant effect of sunlight exposure on 25(OH)D levels [52, 53]. it can be concluded in agreement with , that there are factors not yet well understood which can restrict skin production of vitamin D in response to UV radiation and we have to accept the concept that vitamin D deficiency is not due exclusively to inadequate UV exposure.
The true importance of free form of vitamin D has yet to be established through clinical studies mainly pregnancy, fertility, renal and liver diseases . Since a number of clinical conditions alter the correlation between free and total vitamin D, a question was raised previously whether the assessment of vitamin D status might be improved by measuring free D3 level instead of or together with total D3 level . Similar question was indeed our motive to find the correlation between total versus free 25(OH)D but in apparently healthy individuals not clinical conditions in the current study. Up to our knowledge, the above question was not answered previously and the present data could be helpful and the first one in this respect.
Now, after passing through all the studied factors that can affects the levels of total and free D3, it can be concluded that always all the factors had similar impact on both parameters. They were increased with BMI, males had higher mean values of total and free D3 than females, both were increased with ages (elderly had higher TD3 and FD3 than younger), smoking increases both, wearing hijab also had similar effect on both parameters in addition to daily exposure to sunlight which also had similar effect on both. Finally on comparison between free versus total mean values, our data indicated that among the deficient subjects (TD3 level < 20ng/ml) who participated in this study (208), 5% of them had abnormal FD3 level. On the other hand, among the 95 subjects whom were sufficient in TD3 level(> 30ng/ml), 9% of them they had abnormal level of FD3.