## 3 Overview of Vitamin D
### Measurement, Definition and Prevalence of Vitamin D Deficiency  
Measuring 1,25(OH)\({}_{2}\)D\({}_{3}\) in serum is impractical due to a number of factors including its short half-life (hours), its highly lipophilic and unstable properties, and because it is present at picomolar concentrations within the circulation (a thousand-fold less than 25(OH)D) [51]. The formation and concentration of 1,25(OH)\({}_{2}\)D\({}_{3}\) is also not directly regulated by vitamin D intake. Rather, it is regulated by other factors (such as serum PTH) and, even in the presence of severe vitamin D deficiency, 1,25(OH)\({}_{2}\)D\({}_{3}\) levels may be normal or elevated due to an upregulation of the 1\(\alpha\)-hydroxylase enzyme. Serum 25(OH)D is thus currently considered the best indicator of vitamin D status [51].  
Several commercially available methods are used for measuring vitamin D status, including competitive protein binding assays (CPBA), radioimmunoassays (RIA), enzyme-linked immunoassays (EIA or ELISA), random access automated assays using chemiluminescence technology (RAAA or chemiluminescent immunoassay [CLIA]), high-performance liquid chromatography (HPLC), and what is now considered to be the gold standard: liquid chromatography-tandem mass spectrometry (LC/MS) [7; 51].  
The blood concentration of 25(OH)D used to define deficiency remains an area of debate. The Institute of Medicine (IOM) [52] and National Institutes of Health (NIH) [53] suggest that circulating 25(OH)D concentrations \(<\)50 and \(<\)25 nmol/L are considered deficient and severely deficient, respectively. These definitions would translate to 40-98% of pregnant women globally being deficient (25(OH)D \(<\)50 nmol/L), with 15-84% being severely deficient (25(OH)D \(<\)25 nmol/L) [54]. However, there is currently no consensus regarding optimal vitamin D levels during pregnancy, and the amount of supplementation required remains relatively unknown [19]. Epidemiological studies have postulated a U-shaped exposure-risk relationship between serum 25(OH)D concentrations and several disease outcomes (i.e., increased risk at both low [\(<\)30 nmol/L] and high [\(>\)75 or \(>\)125 nmol/L] concentrations) [55]. These U-shaped curves may reflect biologically meaningful differences such as genetic variants, or may stem from confounding factors such as sun exposure or the use of vitamin D supplements by participants prior to study enrolment. Although both explanations are plausible, further investigations are needed to confirm these observations and how they may influence biological responses to vitamin D.  
Low vitamin D status is prevalent across all age-groups, geographic regions, and seasons [56]. Vitamin D concentration in blood varies cyclically over the course of the year in relation to genetic (gender, ethnicity, skin pigmentation, VDR polymorphisms) and environmental factors (sun exposure and sun safety practices, diet, food-fortification, use of vitamin D supplements). Data from the National Health and Nutrition Examination Surveys (NHANES) demonstrate a three-fold increase in the prevalence of severe vitamin D deficiency over the past 10-13 years in North America, with nearly 36% of the US population being deficient [57]. Similarly, an estimated 31% of adults in Australia have 25(OH)D concentrations \(<\)50 nmol/L, increasing to more than 50% during winter-spring and in people residing in Southern states [58].  
The rising prevalence of vitamin D deficiency is due to several lifestyle changes including the global obesity epidemic and increase in sedentary indoor lifestyles, more common use of sunscreen and protective clothing due to fears of skin cancer, limited sources of vitamin D-rich foods or fortified food products, as well as insufficient amounts of vitamin D in dietary or prenatal supplements. Of concern is that populations with the greatest physiological needs for vitamin D, such as pregnant women, neonates, and children tend to be at highest risk for vitamin D deficiency [59].