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European Journal of Clinical Nutrition
https://doi.org/10.1038/s41430-019-0503-0
BRIEF COMMUNICATION
Rate of change of circulating 25-hydroxyvitamin D following
sublingual and capsular vitamin D preparations
Claire E. Williams1●Elizabeth A. Williams2●Bernard M. Corfe 1,3
Received: 4 January 2019 / Revised: 30 August 2019 / Accepted: 10 September 2019
© Springer Nature Limited 2019
Abstract
Background Vitamin D is critical for skeletal health, and is increasingly associated with other pathologies encompassing
gastrointestinal, immunological and psychological effects. A significant proportion of the population exhibits suboptimal
levels of vitamin D, particularly in Northern latitudes in winter. Supplementation is advocated, but few data are available on
achievable or typical rates of change. There has been considerable interest in the potential use of sublingual sprays for
delivery of nutrient supplements, but data on efficacy remain sparse.
Methods A randomised, placebo-controlled, three-arm parallel design study was conducted in healthy volunteers (n=75) to
compare the rate of change of vitamin D status in response to vitamin D3 (3000 IU/day) supplementation in capsule and
sublingual spray preparations over a 6-week period between January and April 2017. Blood 25(OH)D concentrations were
measured after day 0, 3, 7, 14, 21 and 42 days of supplementation with 3000 IU per diem.
Results Baseline measurements show 25(OH)D deficiency (<30 nmol/l), insufficiency (31–46 nmol/l) and sufficiency
(> 50 mmol/l) in 14.9, 44.6 and 40.5% of the participants, respectively. There was a significant elevation in blood con-
centrations of 25(OH)D in both of the treatment arms (capsule p=0.003, spray p=0.001) compared with control. The
capsule and spray were equally efficacious. The rate of change ranged from 0.69 to 3.93 (capsule) and 0.64 to 3.34 (spray)
nmol/L day with average change in blood 25(OH)D levels of 2 nmol/l/day. Rates followed a simple normal distribution in
the study population (ks =0.94 and 0.82 for capsule and spray, respectively). The data suggest that rates of change are
higher in individuals with lower levels of 25(OH)D.
Conclusions A sublingual vitamin D spray is an effective mode of delivery for supplementation in a healthy population. The
data provide reference values and ranges for the rate of change of 25(OH)D for nutrikinetic analyses.
Introduction
Vitamin D is essential for the homoeostasis of calcium and
phosphate, and well known for its role in the development
and maintenance of bone health [1]. Once vitamin D has
been ingested or synthesised via sunlight exposure, it
requires activation in the liver to form 25-hydroxyvitamin
D (25(OH)D) and in the kidney to form 1,25 dihydrox-
yvitamin D (1,25 (OH)2D[2]. 25(OH)D is the most abun-
dant circulating form in the human body and is used to
determine vitamin D status. 25(OH)D levels can be defined
as; sufficient (≥50 nmol/L), insufficient (30 ≤5049 nmol/L)
of deficient (<30 nmol/L) [3,4]. There is limited research on
rates of repletion; one paper reports amounts for main-
tenance of blood 25(OH)D at 50 nmol/L requires around
11 weeks of dosing at 1000 IU vitamin D per day [5].
Hypovitaminosis is evident worldwide, and is a major
public health concern [6] leading to advocacy for
*Bernard M. Corfe
b.m.corfe@sheffield.ac.uk
1Molecular Gastroenterology Research Group, Academic Unit of
Surgical Oncology, Department of Oncology & Metabolism,
University of Sheffield, Beech Hill Road, Sheffield S10 2RX, UK
2Department of Oncology & Metabolism, University of Sheffield,
Beech Hill Road, Sheffield S10 2RX, UK
3Insigneo Institute for In Silico Medicine, The University of
Sheffield, Sheffield, UK
Supplementary information The online version of this article (https://
doi.org/10.1038/s41430-019-0503-0) contains supplementary
material, which is available to authorized users.
1234567890();,:
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supplementation in at-risk groups. Research has also shown
African Americans may require a higher dose of vitamin D
supplementation to reach optimal serum 25(OH)D con-
centrations compared with the Caucasian participants [7],
perhaps as a result of lower baseline 25(OH)D levels in this
population [8]. It is also known that serum 25(OH)D levels
is inversely associated with body fat mass [9].
Supplementation has classically been with capsule pre-
parations, but sublingual sprays are increasingly available.
There are few data available on the relative efficacy of each
type of preparation on rate of change in circulating levels.
Dose response studies using capsular delivery of vitamin D
supplementation [10–12] have shown evidence of efficiency
in increasing serum 25(OH)D levels which plateau and
begin to decrease.
This study aimed to measure and compare the rate of
change of circulating vitamin D in response to capsular or
sublingual delivery of a daily vitamin D supplement.
Methods
Study design
This was a 6-week double blind, placebo-controlled three-
arm parallel design study. The participants attended three
visits at The Medical School of The University of Sheffield.
The initial visit included anthropometrics, issue of first
batch of blood test kits and completion of a first self-test
blood sample. The second visit occurred ~2 weeks after the
initial visit for issue of further test kits and to support par-
ticipant retention in the trial. The final visit required parti-
cipants to return their preparation bottles and answer five
questions regarding the study.
Sample size and randomisation
There were no data upon which to base a power calculation.
Seventy-five healthy male and female participants were
recruited between January 2017 and February 2017, and were
randomly assigned to one of three arms: (i) active capsules and
placebo spray (n=25); (ii) active spray and placebo capsules
(n=25); (iii) double placebo (n=25). Participants were ran-
domised according to a computer-generated random sequence
using block randomisation with a block size of 9, with ran-
domisation undertaken by an independent outside source. The
allocation sequence was not available to any member of the
team until databases had been completed and locked.
Participants
The University of Sheffield Research Ethics Committee
granted ethical approval for this study (Ref: 011865).
Participants were recruited via poster advertisements at the
University of Sheffield and through a student volunteer
email list. Inclusion criteria required participants to be fit
and healthy, and aged between 18 and 50 years. Partici-
pants who reported any micronutrient supplement use
(vitamin D, multivitamin, fish oils), recent or upcoming
sunny holiday, pregnant or lactating, history of gastro-
intestinal disease, BMI > 30, diabetes, >50 years of age
were excluded. A total of 124 potential participants were
approached, of which 49 were excluded: 28 did not meet
inclusion criteria and 21 had no further contact after initial
consultation.
Participant measures
The concentration of 25(OH)D in the blood was assessed
by blood sample using a finger-prick blood spot kits at 0,
3, 7, 14, 21 and 42 days of supplementation. Blood spots
were analysed by liquid chromatography tandem mass
spectrometry (Waters TQD and Acquity UPLC) for total
blood 25(OH)D (25(OH)D2and 25(OH)D3). LC-MS was
undertaken by City Assays, Department of Pathology,
Birmingham Sandwell Hospitals NHS Trust. Previous
work has shown that this method is comparable with
other commercial assays with intra and interassay coef-
ficients of <10 and <11%, respectively [13–15].
Anthropometric measurements included: height, weight,
BMI and body fat percentage. Body fat and weight were
measured using Tanita BC-543 [16]. Skin tone was
assessed by the researcher using 1 =Caucasian, 2 =
Asian, 3 =Black.
Qualitative opinion of capsules and sprays were assessed
via exit questionnaire. Participants were asked if they had a
preference between preparations
“Did you have a preference between the two
preparations? If so which one?”
Answers were categorised as; “yes, the spray”,“yes, the
capsule”and “no preference”.
Intervention
The vitamin D3and corresponding placebos were manu-
factured by Cultech Ltd., Port Talbot, UK and provided by
BetterYou Ltd, Barnsley, UK. Preparations of vitamin D3
and corresponding placebos were provided as 15 ml sprays
and capsule. Each capsule and spray contained 3000 IU
(75 µg) of vitamin D3per dose. The content of the spray
and the capsule from the manufacturer was prepared to
97.5 µg/dose in order to maintain shelf life and to guarantee
dose. Volunteers were instructed to ingest one capsule
per day with water, and one spray orally per day for
C. E. Williams et al.
6 weeks. Compliance was measured by weighing the spray
bottles and counting the remaining capsules at the end of
the study. In total, 86% and 96.4% of participants reached
100% compliance with the spray and capsules,
respectively.
Adverse events
Two participants reported that small blisters formed on
cheek and tongue after the study began. One participant
stopped using the preparations for the duration of the study.
The second participant continued to use the preparations
throughout the intervention.
Statistical analyses
The data on vitamin D status were held by a third-party until
all other data entry was complete, spreadsheets were then
merged and analysis was undertaken at a group level with
blinding to group identity. Statistical analysis was per-
formed using the Statistical Package for the Social Sciences
(SPSS) (IBM SPSS Statistics for Windows, V.23; IBM
Corp.). Percentage change in 25(OH)D from baseline was
determined by analysis of variance (ANOVA) with Bone-
ferroni correction. Pearson’s correlations for rate of change
in 25(OH)D per day was performed. Change in 25(OH)D
over six time points were analysed by repeated measures
ANOVA (there was a high failure rate in assessments of
25(OH)D at day 42, leading to the exclusion of this time
point’s data from the main analysis). Comparisons between
percentage change in 25(OH)D from baseline in deplete and
replete participants were assessed by Mann–Whitney U
Test. Two-tailed tests were used in all analyses with the
significance value of <0.05.
Results
Baseline demographics are shown in Table 1, and a CON-
SORT is supplied in online (Supplementary Fig. 1). The
three arms were similar in numbers, age, BMI, body fat,
height, weight, skin tone, sex and baseline blood 25(OH)D
concentrations. Baseline blood 25(OH)D concentration
showed 59% of participants had insufficient/deficient vita-
min D status (<50 nmol/L).
Intention-to-treat analysis was used to evaluate the five
time points up to day 21. Kolmogorov–Smirnov test (ks)
indicates that the rate of change of 25(OH)D for both
treatment arms follow a normal distribution (p=0.200).
Raw data are available online (Supplementary Table 1).
Blood 25(OH)D concentration analysed across the time-
course in all three trial arms by ANOVA showed a sig-
nificant improvement in 25(OH)D status in those
receiving vitamin D compared with placebo. Post hoc
analyses revealed significant differences between each of
the active treatments and the placebo (capsules p=0.003,
spray p=0.001), but no difference between the active
preparations at any time point (Fig. 1a). As there are few
available data on the rates of change of ingested vitamin
D, we assessed the inter-individual and inter-preparation
difference as change in whole blood nmol/L/day (Fig. 1bi,
ii). Whilst there was a range of rates in each data set,
assessment of the distributionofrateshowedamonotonic
normal distribution for both preparations with similar
peak rates (Fig. 1biii, iv). Independent ttest was per-
formed, and found no significant difference between mean
rates of change for capsule and spray. A Mann–Whitney
Utest was used to compare differences between deplete
and replete participants within the treatment arms (replete
data was not normally distributed with a KS score of
Table 1 Demographic
characteristics and mean serum
vitamin D at baseline and exit
Capsules Placebo Spray All P-value
Participants, n25 25 25 75
Female, n14 10 15 39 0.326
Mean age ( ± SD) 22.9 (±4.82) 22.4 (±2.72) 21.7 (±3.05) 22.4 (±3.65) 0.504
BMI (kg/m2) 23.6 (±2.95) 22.7 (±2.72) 23.8 (±2.59) 23.4 (±2.77) 0.294
Body fat (%) 23.4 (±7.75) 19.1 (±5.91) 23.7 (±7.65) 22.1 (±737) 0.043
Height (m) 171.3 ±7.54) 173.5 (±10.20) 170.0 (±8.35) 171.6 (±8.77) 0.357
Weight (kg) 69.6 (±10.71) 68.6 (±12.77) 69.0 (±11.32) 69.1 (±11.48) 0.958
Skin tone 22/2/1 24/0/1 25/0/0 71/2/2 0.268
Mean serum 25(OH)D, nmol/L
(baseline)
50.7 (±19.73) 45.6 (±21.30) 54.9 (±27.84) 50.5 (±23.24) 0.381
Mean serum 25(OH)D,
nmol/L (exit)
91.35
(±19.78)
55.62 (±34.40) 95.78
(±28.03)
81.13
(±33.02)
0.001
The data are presented in means ± SD. Baseline characteristics are given along with exit serum 25(OH)D.
Significant values are p> 0.005. A one-way ANOVA was used to compare means at baseline and exit for
serum 25(OH)D
Rate of change of circulating 25-hydroxyvitamin D following sublingual and capsular vitamin D. . .
p=0.001). There was a significant difference (p=0.001)
in the percentage change of 25(OH)D between the replete
and deplete from baseline to day 21.
In order to investigate a potential homoeostatic
mechanism for 25(OH)D status, we investigated the rela-
tionship between 25(OH)D status and rate of change
(Fig. 1bv, vi). We observed inverse relationships between
baseline whole blood 25(OH)D and rates of change over
21 days using Pearson’s correlation for both the spray
(r2=0.255, p=0.012) and capsule (r2=0.351, p=0.003).
In an exit interview about preference for either the spray
or capsule for delivery, 60% preferred spray, 24% capsules
and 16% did not express a preference.
Discussion
Advocacy for vitamin D supplementation for some sub-
populations, interest in its use, availability of over-the-
counter preparations and lack of information on the factors
25(OH)D
(nmol/l)
Rate of change
(nmol/l/day)
Absolute 25(OH)D
(nmol/L)
Frequency
A
BSpray Capsules
Fig. 1 Efficacy and rates of
vitamin D uptake with differing
delivery platforms. Panel
ashows change in vitamin D
circulating levels over time in
each of the three study arms,
presented as absolute levels
(panel ai) or relative to baseline
(panel aii). Panel bshows rates
of uptake comparing spray (left
column) with capsules (right
column). Panels bi and bii show
ladder plots for individuals in
each arm of the trial plotting
difference in vitamin D between
day 0 and day 21 (the abscissa
for uptake, based on panel a).
Rates were derived as nmol/L/
day and binned into 5 nmol bins
(panels biii and biv). KS tests
showed the data were normally
distributed (capsules p=0.200,
spray p=0.200). Finally, the
rates for each individual were
correlated with the baseline
serum concentration for that
individual (panels bv and bvi).
The r2and p-values for
correlations are indicated
C. E. Williams et al.
predisposing to development of excessive levels collec-
tively identify a need for research on comparative efficacy
of preparations and the saturability of uptake. This study
used two commonly available vitamin D preparations: the
widely used capsules and a more novel sublingual spray to
investigate these factors.
Our findings show that a sublingual spray is equally
effective at raising blood 25(OH)D concentrations with no
significant difference between rate of change compared with
capsules in this study population. The study participants
reported a preference for the sublingual spray, and this
study demonstrates that this delivery platform is of com-
parable efficacy. Sublingual sprays may be particularly
advantageous in people with pre-existing malabsorption
conditions or swallowing problems. Our analysis shows for
the first time the likely rate of change in 25(OH)D and the
range of these rates, albeit in a relatively small, healthy
sample. The monotonicity of our rate distribution suggests a
limited spread of rates with no suggestions of outliers or
subpopulations; however, the relatively homogenous profile
of the study population, whilst an advantage for this pilot
exploration, is a limitation in terms of the prediction of rates
in other groups (older adults, different ethnicities). A recent
review [17] does offer suggested optimal supplementation
rates to achieve adequate serum 25(OH)D levels (75 nmol/L)
in regional, population and age-specific groups.
These data also suggest that baseline 25(OH)D status
may influence the rate of change, as a correlation between
baseline status and change exhibited a moderate inverse
relationship, furthermore the circulating 25(OH)D con-
centrations started to level off towards the end of the
intervention. This is in agreement with previous research by
Lips et al., who reported that change in serum 25OHD in
response to 6 months vitamin D supplementation was
dependent on baseline vitamin D status, with the greatest
change observed in people with the lowest baseline vitamin
D[18]. Our research complements the previous work by
undertaking an intervention over a shorter timeframe with
sampling along the timecourse, demonstrating a baseline
status-dependent response to the intervention and the pos-
sibility of a plateau effect. The mechanistic basis of this is
unclear, and it is notable that both delivery platforms exhibit
this effect, implying control in both enteric and transbuccal
absorption. Future work may address the strength of this
inferred relationship more thoroughly and identify implied
control mechanisms. This study had no data from which a
power calculation could be determined, however, the data
presented herein may prove useful for the design of pro-
spective intervention studies.
A limitation to this study is that we cannot show defi-
nitive absorption of the sublingual supplement. However,
sublingual routes of drug delivery are established in phar-
macokinetic studies [19,20]. Recent research presented by
Satia et al. found superior sublingual absorption compared
with capsules in patients with malabsorption issues [21].
Participants were given clear guidelines on how to use the
spray. Further studies should assess 25(OH)D and 1,25(OH)
D levels in localised tissues with the use of labelled D3.
Conclusions
In summary, we have shown the capsule and sublingual
spray are equally effective at delivery of a vitamin D sup-
plement. There was an overwhelming preference (64%) for
the spray over capsules for mode of supplement delivery.
Rate of change, reported for the first time, exhibits a
monotonic distribution in this population. This study saw a
reduction in 25(OH)D levels as blood 25(OH)D con-
centrations increased over 21 days in both preparations.
This suggests the oral spray has the same known mechan-
ism as the capsule for slower conversions of vitamin D3
when concentrations are higher [22]. These data illustrate
the need for further studies to explore rate of change across
mixed population groups, especially those identified as
high risk.
Funding This work was jointly supported by BetterYou Ltd and The
University of Sheffield.
Compliance with ethical standards
Conflict of interest BetterYou co-funded this PhD and provided the
supplements and placebos. This sponsor was not involved in the study
design, delivery or interpretation of the data, which was undertaken
entirely by The University of Sheffield. The authors declare that they
have no conflict of interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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