This cross-sectional study was conducted in September 2007, on 200 women, to evaluate the role of air pollution on blood level of 25-OH-D, as the best indicator of vitamin D storage. Considering the degree of air pollution, we defined two separate zones, of approximately similar latitude; the east of Tehran, latitude 35/7117° N, as a significantly polluted area, and Ghazvin city, latitude 36/2693° N, a less polluted area. Ghazvin is located 165 km northwest of Tehran, in the Ghazvin Province, at an altitude of about 1800 meters above sea level, with a cold and dry climate due to its position south of the rugged Alborz range. This is while Tehran is the capital of Iran and Tehran Province, located 1139 meters above sea level with a a semi-arid, continental climate. We measured the ground levels of UVB by a Haze meter instrument, type UVB EC1 HANGER, on a completely sunny day, between 9 am and 12 pm, in 3 centers in district 13 of Tehran and 10 centers in Ghazvin. The unit of UVB measurements was watt per square meter (W/m2 ). The ground level of UVB was significantly higher in Ghazvin as compared with Tehran (mean (SE), 0.31(0.07) and 0.16(0.03) W/m2 respectively, P-value = 0.003).
All participants were selected using a randomized cluster sampling method; cases were the outpatient housewives, aged between 20 and 55 years. The Tehranian women were selected randomly from participants of the TLGS . TLGS is a population based study, conducted to determine the prevalence of non-communicable diseases among Tehranian's urban population and to develop population-based measures to decrease the prevalence and prevent the rising trend of diabetes mellitus and dyslipidemia. Ghazvinian women selected from subjects who came to public health centers for problems other than those related to vitamin D status. Based on patients' information, those excluded from this study on the basis of medical histories, including known history of malabsorption and gastrointestinal surgeries, cirrhosis, infertility, diabetes mellitus, Cushing's disease, oligomenorrhea, malignancy, and chronic renal failure. Furthermore, participants that were under anti-epileptic, corticosteroid, levothyroxine, Isoniazid, and cholestyramin therapy and those that used vitamin D ampoules in the last 6 months and finally pregnant or breastfeeding women were excluded. Eventually based on inclusion-exclusion criteria, we selected 100 women from Tehran and 100 women from Ghazvin. The project was explained to each participants and written informed consent was obtained at the time of enrollment on an institutionally approved protocol and consent form. The study protocol was approved by the research ethics committee of the Research Institute for Endocrine Sciences affiliated to Shahid Beheshti University, Tehran, Iran.
To take into account different parameters that may contribute to control of vitamin D status, two questionnaires were designed, one for nutritional evaluations (Food Frequency Questionnaires (FFQ) ) and other to assess degree of daily sun exposure. To assess the validity of FFQ, dietary data of 132 individuals were collected by means of 24-h dietary recall (24hDR), repeated twelve time; 24hDR interviews were performed every month for 12 months by the same trained dietitians according to a standard protocol. Usual dietary intake was assessed twice using a 168-item semi-quantitative FFQ (FFQ1 and FFQ2), all administered by the same trained dietitians for each participant fir assessing intra-rater reliability. The first recall was completed one month after FFQ1 administration and the last recall was completed one month before administration of FFQ2. In women, energy-adjusted and deattenuated correlation coefficients mean nutrient intake in the 24hDR and FFQ were 0.33 and 0.43 for calcium and vitamin D, respectively; the age and energy adjusted interclass correlations for calcium and vitamin D were 0.56 and 0.71 respectively . Nutritional data precisely collected, were analyzed by our nutritionists and the daily usage of Ca, Pi, vitamin D, and protein were extracted. Because the Iranian food composition table (FCT) is incomplete (limited to only raw materials and few nutrients), each food and beverage was analyzed for nutrient intake using the US Department of Agriculture's (USDA) FCT [13, 14]. We used the Iranian FCT only for food items like "kashk" which was not listed in USDA FCT. We did not have the possibilities to measure dietary phytate intake. The other questionnaire evaluated the degree of personal daily sun exposure. We inquired about their housing status (i.e. apartment or villa), time spent outdoor whether over or less than 3 days in a week, and the usage pattern of sunscreen (i.e. always or sometimes or never); each of the above questions was analyzed separately. In addition, to calculate the Body Mass Index (BMI), an important determinant of vitamin D status, the height and the weight of these women were also measured; weight was recorded, using a Seca 707 weighing machine (range, 0.1-150 kg) with an accuracy of up to 100 g, the machine being checked regularly for precision every 10 measurements. Height was measured without shoes using a tape stadiometer with a minimum measurement of 1 mm. BMI was calculated by dividing weight (in kilograms) by height squared (in meters).
For determination of 25-OH-D, Parathyroid hormone (PTH), Calcium, Phosphorus and total Alkaline Phosphatase level (ALP), 10 ml of venous blood was taken from antecubital vein of participants, between 8:00 am and 9:00 am after 12-14 h overnight fasting; all samples were centrifuged after collection and 15 min incubation at room temperature (3000 rpm, 15 min at 4°C). Sera 25-OH-D was measured using the Enzyme Immunoassay (EIA) method (25 OH VitD EIA kit, DRG, Marburg, Germany); the kit expected range 10-50 ng/ml. The assay sensitivity was 5.6 nmol/L, and intra-inter assay coefficient variation percents (CV %) were 2.7 and 3.9 respectively. Sera PTH level was determined using an enzyme-linked immunosorbent assay (ELISA) method (hPTH ELISA kit, Biosource, Belgium); the kit expected range 16-64 pg/ml. The assay sensitivity and the intra-inter assay coefficients of variation were 2 pg/ml, 6.4% and 7.8% respectively. Calcium, Phosphate and total ALP levels were measured using photometric methods (Calcium CPC, Phosphorous UV and Alkaline Phosphatase DGKC Kit, Pars Azmoun Co., Tehran, Iran). The sensitivity of assays mentioned were 0.2 mg/dl, 0.7 mg/dl and 3 U/L respectively. Intra and inter assay CV% for calcium, phosphorous and total ALP of the samples were 1.4, 2.7, 1.9, 3.1, and 1.1, 1.8 respectively.
Statistical analyses were performed using SPSS version 16.0 software. The normality of distribution was checked for all variables by Kolmogorov-smirnov analysis. If required, skewed variables were logarithmically transformed to improve normality prior to analyses, if it was needed. Normally distributed continuous variables are reported as the mean ± SD and not normally distributed continuous variables are reported as median and Interquartile (IQ) 25-75.
Student's t-test for unpaired data was used to compare groups and other than that the Mann-Whitney test was used. Correlation coefficients were calculated by Pearson's analysis. adjusted mean differences were calculated using ANCOVA. A binary logistic analysis was performed in all 200 subjects to determine the role of the city of residence per se, as an surrogate of air pollution, for having blood level of 25-OH-D less or more than 20 ng/ml following adjustment of the confounders, i.e. age (years), BMI (kg/m2), dietary usage of Ca (mg), Pi (mg), vitamin D (ug), and protein (gr), housing status (reference, villa), time spent outdoor (reference, >3 days in week), the pattern of sunscreen usage (reference, routine), and the city of living (reference, Ghazvin). Vitamin D deficiency, insufficiency, and normal cut offs considered were as 25-OH-D <10 ng/ml, 12 to <20 ng/ml, and ≥20 ng/ml, respectively . P-values <0.05 were considered statistically significant.