found no difference due to timing of the doses. Ish-Shalom et al. (2008) suggested that the attenuated response to monthly dose in the Chel et al. (2008) study may have been due to poor compliance with a powdered monthly supplement compared with pills used for their daily and weekly doses. An alternative explanation is that only a lower (Chel et al., 2008) and not a higher (Ish-Shalom et al., 2008) dose is influenced by timing of the vitamin D supplement. Thus far, studies suggest that weekly and daily dosing give similar serum 25OHD responses.

Serum 25OHD comprises the sum of 25OHD₂ and 25OHD₃. Because of the widespread use of both vitamin D₂ and vitamin D₃ in the United States and Canada, analysts must measure both 25OHD₂ and 25OHD₃ in order to provide the total 25OHD level in serum. This is in contrast to the situation in Europe where there has been a tradition of using only vitamin D₃ and where commercial methods that purport to measure only 25OHD₃ are available.

In North America, several assay types are currently in use, each with strengths and weaknesses (Makin et al., 2010). The two most common types of assays are

• Antibody-based methods, which use a kit or an automated clinical chemistry platform; and

• Liquid chromatography (LC)-based methods, which use automated equipment featuring either UV or mass spectrometric (MS)-detection.

As discussed below, both these methods are equivalent in terms of measuring the physiologically relevant parameter (total 25OHD level in serum), but there remains controversy over the performance of these assays in clinical and research laboratories. Moreover, reports in the literature for serum 25OHD measures should be interpreted with care, taking into account the type of assay employed, use of automation, year of analysis, and context of the analysis.

Assays for total 25OHD level in serum have existed for four decades since the metabolite was first discovered (Blunt et al., 1968). The earliest assays were competitive protein-binding assays (CPBAs), based upon the ability of either 25OHD₂ or 25OHD₃ to displace [³H]25OHD₃ from the plasma binding protein, DBP (Belsey et al., 1971; Haddad and Hahn,