doi:10.

1093/brain/awz389 BRAIN 2020: 143; 1–2 | e10

LETTER TO THE EDITOR

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

Darius Häusler,1, Sebastian Torke,1, Evelyn Peelen,2 Thomas Bertsch,3 Matthias Weber,4
Marcus Heilmann,3 Marija Djukic,1,5 Roland Nau,1,5 Catherine Larochelle,2

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Scott S. Zamvil,6 Wolfgang Brück1 and Martin S. Weber1,7


These authors contributed equally to this work.

1 Institute of Neuropathology, University Medical Center, Göttingen, Germany

2 Department of Neurosciences, Centre de recherche de l’Université de Montréal (CRCHUM), Montréal, Québec, Canada
3 Institute of Clinical Chemistry, Laboratory Medicine and Transfusion Medicine, Nuremberg General Hospital, Paracelsus Medical
University, Nuremberg, Germany
4 LaborDiagnostik-Karlsruhe GmbH, Clinical Mass Spectrometry, Karlsruhe, Germany
5 Department of Geriatrics, Evangelisches Krankenhaus Göttingen-Weende, Göttingen, Germany
6 Department of Neurology, University of California, San Francisco, CA, USA
7 Department of Neurology, University Medical Center, Göttingen, Germany

Correspondence to: Martin S. Weber, MD

Department of Neuropathology, Department of Neurology, University Medical Center,
Georg August University, Robert-Koch-Str. 40, 37099 Göttingen, Germany
E-mail: martin.weber@med.uni-goettingen.de

Sir, the respective vitamin D diets and measuring them using

We would like to reply to the comments by Dr Vieth
(2019) regarding our recent publication describing a clin-
ical deterioration upon high-dose vitamin D supplementa-
tion in a murine model of multiple sclerosis (Häusler
et al., 2019). We have noted the concerns about our ap-
proach 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 MassChromÕ 25-OHVitamin D3/D2 in
Serum/Plasma Kit was, at that time, approved and certi-
fied by the Centers for Disease Control and Prevention
(2019). This system has been validated for serum vitamin
D measurements up to 250 mg/l or 625 nM (S.A.S.
Corporation, 2019), which is well within the range we
have analysed. Taking the concern of a possible underesti-
mation 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
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.
The study cited by Dr Vieth to underscore his concern
that mice may require higher doses of vitamin D to gen-
erate 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 rapid-
ity, despite that several studies reported problems of re-
producibility and interferences’. Accordingly, we believe
that our measurement, as now confirmed by an independ-
ent additional method, is more accurate. Additionally, in

Advance Access publication December 17, 2019

ß The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
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e10 | BRAIN 2020: 143; 1–2
Figure 1 Determination of serum 25-OH-vitamin D3 upon
vitamin D supplementation using an additional, independ-
ent method. Mice were fed a diet containing low (Low; 55 IU/kg
food), standard (Std; 1500 IU/kg food) or high vitamin D concen-
trations (Hi; 75 000 IU/kg food). Serum 25-hydroxyvitamin D3
[25(OH)D] concentrations were measured using (A) liquid chro-
matography-tandem mass spectrometry or (B) liquid chromatog-
raphy-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 5 0.01, P 5 0.001; two-tailed
t-test. Panel A from Häusler et al. (2019).
the study by Rowling et al. (2007), the calcium concen-
trations 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 to-
wards a vitamin D-dependent increase in serum calcium
that, however, could not reach hypercalcaemic levels be-
cause 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.
Data availability
The data that support the findings of this study are avail-
able from the corresponding author, upon reasonable
request.
Letter to the Editor
Funding
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
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Maisin Foundation.
Competing interests
M.S.W. is serving as an editor for PLoS One.
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