There have been a wide range of 25-OHD levels in studies from countries in the Middle Eastern region; possibly attributed to methodological and genetic variances [
16‐
19]. The low levels of 25-OHD in our pediatric population were found in contrast to the reports of US NHANESIII study, where mean 25-OHD levels in over 2800 adolescents, aged 12–19 years, were between 26 and 36 ng/ml [
15]. The inverse correlation between 25-OHD and iPTH levels have also been demonstrated previously in different studies [
20‐
24]. In our study, the relationship between 25-OHD and iPTH was exponential and statistically significant (iPTH = 65.326 × 25-OHD
-0.28,
r = 0.347,
p < 0.001). This finding is unique when compared to adults [
25‐
29]. In our previous study we searched for the vitamin D cut-off level mainly in adults at 10 year intervals, while in this study we searched for vitamin D versus iPTH relationship at all pediatric ages [
30]. Discussion over oscillations between 25-OHD and iPTH have also been published previosly and due to these studies there is still controversy over optimum 25-OHD level recommendations in the guidelines [
31,
32]. Due to the fact 25-OHD levels show seasonal variances, determination of reference intervals can not be made accurately. Hill et al. analyzed the the relationship between serum 25-OHD and PTH in children and adolescents in the United States to determine the inflection point of 25-OHD for maximal suppression of PTH. This was in contrast to adults where the relationship between 25-OHD and PTH was linear with no inflection point [
33]. In our study, using regression analysis, the 25-OHD level above which iPTH levels did not decrease was 25 ng/ml. In other studies values for this plateu vary from 16 to 60 ng/mL which may partially be explained by differences in ethnicity, age, gender, assays used and most importantly the method used to implement curve fitting and derive plateau [
34‐
37]. Assessment of Vitamin D status can be assayed using radioimmunoassays, competetive protein binding assays (CPBA), high pressure liquid chromatography (HPLC), and liquid chromatography-tandem mass spectrometry. Because of an inter-assay variation these methods may yield different results; varying by up to 25% (at lower serum levels) and 10% with intra-assay. The optimal serum vitamin D concentration has not yet been established, and may change across different life stages. In our study 25-OHD levels were measured by a very sensitive method; LC MS being the most accurate method for 25-OHD level measurement. The cross sectional analysis of NHANES (National Health and Nutrition examination Survey) 2003–2006, which included 14,681 patient data older than 6 years, demonstrated that optimal 25-OHD level, defined as estimated maximum iPTH supression, does not occur until at least 25-OHD level is equal or higher than 40 ng/ml [
38]. This is higher than our population based plateau. There were some limitations in our study, as it does lack body mass index, as well as other contents of fat as major determinants of 25-OHD. Secondly, as this study was a data-mining study, it could not unravel other predictors of low 25-OHD levels. These include polymorphisms of vitamin D metabolism pathways, other medical consequences, clothing habits, geographical location, weather conditions, skin color, hours of sun exposure, use of sun screen, use of calcium and vitamin D supplementation. Neverthless, our study does provide valuable insight on the grounds that it was based on retrospective data gleaned from tha largest data set in a pediatric population, in the literature to date. Due to the difficulty in maintaining adequate 25-OHD serum levels throughout the pediatric age while at the same time reacting to seasonal adjustments, more studies are needed to better understand the clinical implications of hypovitaminosis D and consequently, make recommendations for vitamin D supplementation.