Underestimation of Serum 25-Hydroxyvitamin D by the Nichols Advantage Assay in Patients Receiving Vitamin D Replacement Therapy - Reply

An author of one of the studies cited in the above letter and a representative of Nichols Institute Diagnostics respond: In their letter, Leventis et al. compare methods for 25-hydroxyvitamin D [25(OH)D] measurements. In light of recent reports, and given the complexities of accurately measuring the physiologically relevant species among metabolites of vitamin D, we consider that the title and the conclusions of their letter may be potentially misleading. One of the methods they studied is based on antibody recognition and isotopic detection (DiaSorin), whereas the other is based on vitamin D-binding protein recognition and chemiluminescence detection (Nichols Institute Diagnostics). These important differences may be responsible for the different results. We communicated to Nichols Advantage assay users in customer bulletins issued on June 15, 2004, and December 6, 2004, that our method gives lower results than do liquid chromatography–mass spectrometry and other assays, particularly in patients treated with vitamin D2. In those bulletins, we advised customers that samples tested with the assay may yield results lower than actual 25(OH)D values because of potential underrecovery of 25(OH)D2. We also advised our customers of a study showing that, after treatment of patients with 25(OH)D2, the assay gave measured increases in 25(OH)D concentrations (1). In their letter, Leventis et al. also indicate that in the posttreatment samples, both of the assays that they studied detected an increase in 25(OH)D concentration compared with baseline. Representatives of the Vitamin D External Quality Assessment Scheme, in a Technical Brief published in this journal (2), indicated that the Nichols Advantage assay does detect 25(OH)D2, although with lower absolute values. We do not dispute that different vitamin D assays may yield different results. One possibility is that the natural vitamin D-binding protein in the Nichols Advantage assay quantifies the physiologically relevant metabolite(s) of vitamin D2 better than does an antibody-based assay. This has led us to start investigating the identities of metabolites generated by vitamin D2, using HPLC and mass spectrometric methods. A recent report by Heaney et al. (3) is noteworthy in that it reflects substantially more rapid metabolism or clearance of the vitamin D2 metabolites. Further research is necessary to uncover the identities of vitamin D metabolites and their clinical significance. Until this is done, conclusions such as those drawn in the letter of Leventis et al. should be considered as preliminary and premature. Michael F. Holick is affiliated with Nichols Institute Diagnostics as an Academic Associate.