Reply: Neither human nor mouse is hypercalcaemic with 250 nmol/l 25-hydroxyvitamin D

Sir, We would like to reply to the comments by Dr Vieth (2019) regarding our recent publication describing a clinical deterioration upon high-dose vitamin D supplementation in a murine model of multiple sclerosis (Häusler et al., 2019). We have noted the concerns about our approach to measure serum vitamin D concentrations. First, we would like to emphasize that our system to measure 25-hydroxyvitamin D [25(OH)D] serum levels using the Chromsystems MassChrom® 25-OHVitamin D3/D2 in Serum/Plasma Kit was, at that time, approved and certified by the Centers for Disease Control and Prevention (2019). This system has been validated for serum vitamin D measurements up to 250 µg/l or 625 nM (S.A.S. Corporation, 2019), which is well within the range we have analysed. Taking the concern of a possible underestimation very seriously, we have now included an analysis specifically requested by the author. We created additional data by analysing the serum samples of animals fed with the respective vitamin D diets and measuring them using an LC/MS approach involving solid phase extraction (online-SPE), as proposed by Dr Vieth. As indicated in Fig. 1A and B, these independent measurements detected serum vitamin D concentrations corresponding to those published in our original article. These additional data confirm that the initial measurement of vitamin D levels was accurate and correctly associated with a rise in serum and urine calcium. Determination of serum 25-OH-vitamin D3 upon vitamin D supplementation using an additional, independent method. Mice were fed a diet containing low (Low; <5 IU/kg food), standard (Std; 1500 IU/kg food) or high vitamin D concentrations (Hi; 75 000 IU/kg food). Serum 25-hydroxyvitamin D3 [25(OH)D] concentrations were measured using (A) liquid chromatography-tandem mass spectrometry or (B) liquid chromatography-tandem mass spectrometry (LC-MS) with solid-phase extraction 15 weeks after diet onset (data presented as mean ± SEM). (A) n = 13–15, (B) n = 3; **P < 0.01, ***P < 0.001; two-tailed t-test. Panel A from Häusler et al. (2019). The study cited by Dr Vieth to underscore his concern that mice may require higher doses of vitamin D to generate hypercalcaemia used an immunoassay to measure vitamin D concentrations. This approach has been described by Gervasoni et al. (2013) as follows: ‘Immunoassay methods are the most used for their rapidity, despite that several studies reported problems of reproducibility and interferences’. Accordingly, we believe that our measurement, as now confirmed by an independent additional method, is more accurate. Additionally, in the study by Rowling et al. (2007), the calcium concentrations in the chew were different for the control diet (2200 IU vitamin D, 2% Ca2+) and vitamin D-adjusted diets (1000, 10 000 or 20 000 IU vitamin D, 0.5% Ca2+). This might explain why there was a sudden drop of serum calcium in their transgenic mouse strain receiving the low vitamin D diet (Fig. 2 in Rowling et al., 2007) that was non-existent at increasing doses. This points towards a vitamin D-dependent increase in serum calcium that, however, could not reach hypercalcaemic levels because of a simple lack of available calcium. We took the expressed, instructive critique very seriously and have proved that our serum vitamin D measurements were indeed reliable and correct. Therefore, we thank Dr Vieth for his help in further consolidating our central hypothesis. The data that support the findings of this study are available from the corresponding author, upon reasonable request. D.H. is supported by the Startprogramm of the Universitätsmedizin Göttingen. M.S.W. receives research support from the National Multiple Sclerosis Society (NMSS; PP 1660), the Deutsche Forschungsgemeinschaft (DFG; WE 3547/5-1), from Novartis, TEVA, Biogen-Idec, Roche, Merck and the ProFutura Programm of the Universitätsmedizin Göttingen. S.S.Z. is supported by grants from the National Institutes of Health (1 RO1NS092835-01; 1 R01 AI131624-01A1; 1 R21 NS108159-01), the NMSS (1 RG1701-26628) and the Maisin Foundation. M.S.W. is serving as an editor for PLoS One.

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