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The influence of vitamin D supplementation on melatonin status in
patients with multiple sclerosis
Daniel Golan
a,b
, Elsebeth Staun-Ram
a
, Lea Glass-Marmor
a
, Idit Lavi
d
, Orit Rozenberg
c
, Sara Dishon
a
,
Mira Barak
c
, Sophia Ish-Shalom
e
, Ariel Miller
a,e,
⇑
a
Division of Neuroimmunology & Multiple Sclerosis Center, Carmel Medical Center, Haifa, Israel
b
Department of Neurology, Carmel Medical Center, Haifa, Israel
c
Central Laboratory of Haifa and Western Galilee, Clalit Health Services, Haifa, Israel
d
Department of Community Medicine & Epidemiology, Carmel Medical Center, Haifa, Israel
e
Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel
article info
Article history:
Received 23 January 2013
Received in revised form 12 April 2013
Accepted 21 April 2013
Available online 7 May 2013
Keywords:
Chronotherapy
Interferon-b
Light
Melatonin
Multiple sclerosis
Neurohormone
Vitamin D
abstract
Background: Multiple sclerosis (MS) incidence is higher in geographic regions with less sunlight expo-
sure. Both vitamin D and melatonin are essential mediators of the effect of sunlight in health, and as such
are candidates to play a key role in MS. We hypothesized that vitamin D and melatonin may have related
influences in patients with MS.
Methods: In a randomized, double blind study of 40 IFN-btreated MS patients, 21 patients were assigned
to 800 IU of vitamin D3 per day (low dose), while 19 patients received 4,370 IU vitamin D3 per day (high
dose) for one year. Serum 25-hydroxy-vitamin-D (25-OH-D) and nighttime urine melatonin metabolite,
6-sulphatoxy-melatonin (6-SMT), were measured at baseline, 3 months and 1 year from enrolment.
Results: After 3 months supplementation, 25-OH-D levels increased and nighttime melatonin secretion
decreased significantly in the high dose group, but not in the low dose group. After 1 year, a decrease
in 25-OH-D levels, accompanied by an increase of urine nighttime 6-SMT were observed in the high dose
group. Percent change in serum 25-OH-D was significantly and negatively correlated with percent change
in urine 6-SMT after 3 months and between 3 months to 1 year. 25-OH-D levels by the end of the study
were significantly and negatively correlated to BMI.
Conclusions: Melatonin secretion is negatively correlated with alterations in serum 25-OH-D in IFN-b
treated patients with MS. The finding suggests that melatonin should be considered as a potential medi-
ator of vitamin D neuro-immunomodulatory effects in patients with MS.
Ó2013 Elsevier Inc. All rights reserved.
1. Introduction
Multiple sclerosis (MS) is the most common neurological dis-
ease associated with functional disabilities in young adults. It is
well known that MS prevalence has an impressive geographical
gradient, with significantly higher incidence with increasing lati-
tude (Simpson et al., 2011). Different sunlight exposure and the
resulting variation in vitamin D (vit D) production have been advo-
cated as the explanation for this environmental gradient (Disanto
et al., 2012; Munger et al., 2006).
Part of these effects is related to vit D mediated immunomodu-
lation such as promotion of an anti-inflammatory cytokine profile
and induction of regulatory T cells (Smolders et al., 2008, 2009). In
experimental autoimmune encephalomyelitis (EAE), the animal
model of MS, vit D is able to suppress the disease process and to
abolish EAE induction (Cantorna et al., 1996; Lemire and Archer,
1991).
Recently, it became clear that decreased sunlight exposure re-
mains significantly associated with MS risk even after controlling
for vit D levels, thus implying that vit D is not the only mediator
of the beneficial influence of sunlight on MS (Hart et al., 2011; Lu-
cas et al., 2011). Melatonin is another sunlight dependent hor-
mone, with suggested immune-regulatory effects, such as
controlling the production of cytokines and leukotrienes and regu-
lating the lifespan of leukocytes by interfering with apoptotic pro-
cesses (Radogna et al., 2010). Moreover, its anti-oxidative
properties promote scavenging of oxidative stress in inflamed tis-
sues (Radogna et al., 2010).
Both vit D and melatonin are candidate intermediates for the
influence of sunlight on MS (Mehta, 2010). We hypothesized that
these two hormones might have mutual relationship because they
0889-1591/$ - see front matter Ó2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bbi.2013.04.010
⇑
Corresponding author. Address: Division of Neuroimmunology & Multiple
Sclerosis Center, Carmel Medical Center, 7 Michal Street, Haifa 34362, Israel. Tel.:
+972 4 8250 851; fax: +972 4 8250 909.
E-mail addresses: millera@tx.technion.ac.il,milleras@netvision.net.il (A. Miller).
Brain, Behavior, and Immunity 32 (2013) 180–185
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are both light-dependent mediators and share immunomodulatory
properties. Thus, homeostatic mechanisms might exist to keep
them both coordinated. Accordingly, we aimed to assess the effects
of vit D supplementation on melatonin secretion in patients with
MS.
2. Methods
2.1. Patients
The study took place at north Israel, latitude 32.8°north.
Relapsing remitting MS (RRMS) patients who attended our clinic
for routine follow-up from November 3rd, 2010 to March 4th,
2011 were offered to participate. Inclusion criteria were: at least
18 years of age, IFN-btreatment of at least 4 months duration,
25-hydroxy-vitamin-D (25-OH-D) blood levels below 75 nmol/l,
EDSS score up to 7 and signed informed consent.
Exclusion Criteria consisted of abnormalities of vit D related
hormonal system other than low dietary intake or decreased sun
exposure. Participation after enrolment was terminated in case of
withdrawal of IFN-btreatment from any reason, pregnancy, hyper-
calcemia or patient’s decision. The study was approved by the Hel-
sinki Committee of Carmel Medical Center, Haifa, Israel. All
participants signed informed consent. The trial was registered at
ClinicalTrials.gov ID: NCT01005095.
2.2. Study groups and randomization
There were two interventional groups. The ‘‘high dose’’ group
was orally treated with a bottle containing 75,000 IU of cholecalcif-
erol (vit D3) solution every 3 weeks plus 800 IU of vit D3 by daily
tablets (total of 4370 IU/d). The ‘‘low dose’’ group received 800 IU
of vit D3 by daily tablets, along with the content of a bottle of pla-
cebo solution every 3 weeks (total of 800 IU/d).
After cholecalciferol (vit D3) is ingested, part of it is stored in fat
tissue and is subsequently gradually released for about 3 months.
Part of the ingested and storage released cholecalciferol undergo
25-hydroxylation in the liver (Bringhurst et al., 2008). The half-life
of 25-OH-D is about 2–3 weeks (Bringhurst et al., 2008). Basic
pharmacology suggests that circulating half life is a suitable dosing
interval for a drug and that after five half lives serum levels are at
steady state (Buxton, 2006). Because of the long half life of 25-OH-
D it is expected that after 3 months supplementation, at steady
state, administration of 75,000 IU every 3 weeks plus 800 IU daily
of vit D3 will produce the same 25-OH-D serum levels as if
4370 IU per day of vit D3 had been given. The equivalence of daily
and monthly vit-D3 administration has been shown empirically
(Binkley et al., 2011; Ish-Shalom et al., 2008).
The assignment to groups was randomly set in advance, accord-
ing to recruitment order. vit D supplementation was double-blind
in this study – both participants, physicians and investigators were
unaware of the ingredients of the solution bottles.
2.3. Evaluations
Patients’ characteristics including age, gender, ethnicity, BMI
(Body mass index), MS disease characteristics and use of anti
depressants were registered at recruitment. Health related quality
of life (HRQoL) was evaluated at baseline and at the completion of
the study, using the ‘Functional assessment of MS’ questionnaire
(FAMS), which is divided to six subscales: mobility, symptoms,
emotional well-being (depression), general contentment, think-
ing/fatigue, and family/social well-being (Cella et al., 1996). The
depression score runs between 0 and 28. High score denotes de-
creased mood.
Serum 25-OH-D and calcium levels were measured at baseline
and after 3 months and 1 year from enrolment. Total vit D was
measured by chemiluminescent immunoassay (CLIA) technology
and was tested on the Liaison analyzer (DiaSorin S.p.A., Italy) for
the quantitative determination of 25-hydroxy-vitamin-D.
Urine was collected over a 12-h nighttime (19:00–07:00) period
at baseline, after 3 months and by the end of 1 year. Aliquots were
frozen at 80 °C until analyzed. Nighttime Melatonin metabolite
6-sulphatoxy-melatonin (6-SMT) levels were measured using a
highly specific ELISA assay (IBL International, Biotest Ltd., Israel).
Night durations at each date of urine collection were calculated
as the time between sunset and sunrise at the city of Haifa, which
were obtained by using the ‘suncalc.net’ application. (Agafonkin,
2009).
2.4. Statistical analysis
Data analysis was performed using the SPSS statistical package
(SPSS Inc., Chicago, IL, USA). Comparisons of patients’ characteris-
tics between study groups were done by Student’s t-test or
Mann–Whitney test, according to data distribution. Categorical
variables were compared using Chi square or Fisher’s exact test.
Comparisons of serum 25-OH-D and urine 6-SMT at various times
were done by mixed model ANOVA with Bonferroni adjustment for
multiple comparisons. This procedure takes into account the intra-
correlation of repeated measurements carried out at the same sub-
ject and does not exclude subjects with incomplete data at follow-
up. Changes in FAMS depression scores in each dosage group were
analyzed by Wilcoxon Signed Rank test. Changes in night duration
between baseline and 3 months were compared with paired Stu-
dent’s t-test. Spearman’s Correlation analysis was used to estimate
the association between percent change in serum 25-OH-D and
percent change in overnight urine 6-SMT. Pearson’s Correlation
analysis was used to estimate the associations between BMI and
25-OH-D, BMI and 6-SMT, age and 6-SMT and between FAMS
depression scores and 6-SMT. Summary statistics are given as
mean ± standard deviation and median and (range).
3. Results
Flow chart of the study is shown in Fig. 1. Patients’ characteris-
tics at recruitment are provided in Table 1. There were no signifi-
cant differences between baseline characteristics of the dosage
groups. 14 patients dropped out (‘censored’) before the completion
Fig. 1. Study flow chart.
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of the intended 12 months follow up. Notably, all premature with-
drawals took place after the 3 months’ evaluation. Censored pa-
tients in the high dose group were closer to the time of MS
diagnosis. Censored patients in the low dose group were mainly
males whereas those with complete follow up were mainly fe-
males. Apart from these, no other significant differences in baseline
characteristics were noted between the censored and uncensored
patients in the two intervention groups.
Serum 25-OH-D levels at baseline, 3 months and 1 year in the
two dosage regimens are presented in Fig. 2. Serum 25-OH-D levels
at both 3 month and 1 year were significantly higher in the high
dose group [F(1,35.4) = 21.6, P< 0.0001]. In patients receiving the
high dose, 25-OH-D levels were significantly above baseline at all
time points [t(18) = 9.29, P< 0.0001 at 3 months, t(15.5) = 3.91,
P= 0.003 at 1 year], and within 3 month reached levels above
75 nmo/l, considered sufficient by the American society of endocri-
nology (Holick et al., 2011). Notably, a significant decrease in ser-
um 25-OH-D was found between 3 months and 1 year of follow
up [t(13.5) = 2.66, P= 0.038]. 5 out of 13 patients (38%) assigned
to the high dose group, reached the end of the study with 25-
OH-D levels below 75 nmol/l.
Low dose vit D supplementation resulted in significantly in-
creased 25-OH-D levels compared to baseline only at 3 months
[t(20) = 5.72, P< 0.0001], however average level did not reach
75 nmo/l. A trend towards reduced 25-OH-D levels at 1 year
compared to levels after 3 months, was also apparent in this dos-
age group, albeit not statistically significant [t(18.7) = 2.17,
P= 0.09].
All participants’ 25-OH-D levels by the end of the study were
significantly negatively correlated to BMI [Pearson’s r=0.45,
P= 0.015].
Urine melatonin metabolite levels in the two intervention
groups are shown in Fig. 3. One patient, in the low dose group,
had 414% increases in urine melatonin metabolite at 1 year com-
pared to 3 months. This increment was higher than the median
change in urine 6-SMT at that period, by 10.9 times the inter-quar-
tile range. This patient had severe limb ataxia, which might have
hampered the ability to collect urine accurately. Therefore, 6-
SMT data from this patient was excluded from analysis, as an
outlier.
Despite randomization, significantly higher baseline 6-SMT
levels were found in the high dose vit-D group. [t(37) = 2.06,
P= 0.05]. None of the registered characteristics at baseline, as
shown in table 1, could account for the difference in baseline
6-SMT levels between the groups. No correlation was observed
between baseline depression score and baseline 6-SMT levels
[Pearson’s r= 0.045, P= 0.8]. However, baseline 6-SMT levels
from all participants, were significantly and negatively correlated
with age and BMI [Pearson’s r=0.51, P= 0.001 and r=0.39,
P= 0.014, respectively].
Table 1
Patients’ baseline characteristic
a,b
.
Low dose group
800 IU/d
N=21
High dose group
4370 IU/d
N=19
All
N=21
Complete follow up
N=13
Censored
N=8
All
N=19
Complete follow up
N=13
Censored
N=6
Age 44.7 ± 10.7
43.6
(27–64)
46.3 + 9.2
44.6
(32–59)
42.1 ± 13
40.1
(27–64)
46.5 ± 11
46.9
(23–64)
47.7 + 11.6
47.5
(23–64)
44.1 ± 10.1
42.5
(32–61)
Gender (M = Male, F = Female) 8 M
13 F
2M
c
11 F
6M
c
2F
5M
14 F
3M
10 F
2M
4F
Ethnicity Jews 16
Arabs 5
Jews 10
Arabs 3
Jews 6
Arabs 2
Jews 18
Arabs 1
Jews 13
Arabs 0
Jews 5
Arabs 1
Time from MS diagnosis (years) 9.3 ± 7.4
8.1
(0.4–32.9)
9.9 ± 8.3
8.3
(0.7–32.9)
8.4 ± 6
7.2
(0.4–19)
6.9 ± 6
4.6
(0.3–19.1)
8.7 ± 6.3
d
8.3
(1.7–19.1)
3.0 ± 2.7
d
2.3
(0.3–7)
BMI
e
26.8 ± 6.6
25.7
(18.3–45.8)
26.2 ± 7.4
24.2
(18.3–45.8)
27.8 ± 5.3
28.7
(19.2–36.3)
25.1 ± 5.8
24.6
(17.4–41.9)
25.2 ± 6.2
24.6
(17.4–41.9)
25.0 ± 5.6
24.9
(17.4–33.8)
IFN-btreatment duration (months) 61.3 ± 46
59.1
(3.1–144.1)
58.5 ± 47.2
53
(6.2–143)
65.8 ± 46.8
60.9
(3.1–144.1)
44.6 ± 33.8
33.3
(3.0–96.7)
51.6 ± 34.5
54.6
(7.5–96.7)
29.7 ± 29.6
18.8
(3–69.6)
EDSS 3.6 ± 2.2
4.5
(0–6.5)
3.8 ± 2
4.5
(1–6.5)
3.2 ± 2.4
3.3
(0–6)
3.3 ± 2.1
3
(0–7)
3.4 ± 2.4
3
(0–7)
3.1 ± 1.5
2.8
(1–5)
Depression score (FAMS) 20.1 ± 5.5
21
(9–28)
20.9 ± 5.7
22
(12–28)
19 ± 5.5
20
(9–25)
20.4 ± 7
22
(5–28)
19.3 ± 7.3
22
(5–27)
22.7 ± 6.2
25.5
(13–28)
Number using anti depressants 3 3 0 4 3 1
Night duration (hours) 13.6 ± 0.4
13.7
(12.4–14)
– – 13.7 ± 0.3
13.8
(13.1–14)
––
a
Summary statistics are mean ± standard deviation and median (range).
b
There were no significant differences between baseline characteristics of the dosage groups. Baseline characteristics of censored patients and those with complete follow
up in each dosage group were not statistically different except:
c
Significantly different gender distribution in the low dose group (P= 0.02).
d
Significantly different times from MS diagnosis in high dose group (P= 0.05).
e
BMI = Body mass index, calculated as body weight (kg) divided by the square of height (m). All participants’ 25-OH-D levels by the end of the study and 6-SMT at baseline
were negatively correlated to BMI [Pearson’s r=0.45, P= 0.015 and Pearson’s r=0.39, P= 0.014, respectively].
182 D. Golan et al. / Brain, Behavior, and Immunity 32 (2013) 180–185
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Urine 6-SMT was significantly decreased after 3 months of high
dose vit D supplementation [t(18) = 3.33, P= 0.008], however by
the end of the study nighttime 6-SMT secretion returned to base-
line levels [t(13.4) = 0.89, P= 0.78]. In contrast, melatonin metabo-
lite levels were not changed after 3 and 12 months follow up in the
low dose group [t(19) = 1.12, P= 0.55 and t(16.1) = 0.24, P= 1.0,
respectively].
The duration of the nights, in which urine were collected, at
3 months was 11.7 ± 1.2 h, significantly shorter compared to their
duration at baseline (13.7 ± 0.3 h) and at 1 year (13.6 ± 0.4 h)
[t(38) = 10.5, P< 0.0001].
The percent increase in serum 25-OH-D was significantly corre-
lated to the percent decrease in urine 6-SMT after 3 months (Spear-
man’s rho = 0.32, P= 0.05) (Fig. 4). Interestingly, percent decrease
in 25-OH-D between 3 months and 1 year was significantly corre-
lated to a corresponding increase in urine 6-SMT secretion (Spear-
man’s rho = 0.4, P= 0.05) (Fig. 5). Vitamin D and melatonin data
from both dosage groups were used for correlation analysis.
Depression scores after 1 year, in both dosage groups, were not
significantly changed from baseline. No gender effect was observed
on either 25-OH-D or 6-SMT levels. There was no significant differ-
ence in either 25-OH-D or 6-SMT levels between males and fe-
males at all time points, and regardless of supplementation
schedule.
4. Discussion
The effect of vit D on MS disease activity in IFN-btreated pa-
tients is gaining increasing attention in recent years, but has yet
Fig. 3. Nighttime urine 6-sulphatoxy-melatonin (6-SMT). Nighttime (12 h) urine 6-
SMT levels were significantly decreased after 3 months of high dose vit D
supplementation (P= 0.008), however by the end of the study nighttime 6-SMT
secretion returned to be insignificantly different from baseline levels. Melatonin
metabolite levels were not significantly changed in the low dose group.
Fig. 4. Correlation between increase in serum 25-OH-D and decrease in urine
melatonin metabolite, baseline to 3 months. The percent increase in serum 25-OH-
D was significantly correlated to the percent decrease in urine 6-SMT after
3 months.
Fig. 5. Correlation between decrease in serum 25-OH-D and increase in urine
melatonin metabolite, 3 months to 1 year. The percent decrease in 25-OH-D
between 3 months and 1 year was significantly correlated to a corresponding
increase in urine 6-SMT secretion.
Fig. 2. Serum 25-hydroxy-vitamin-D levels. With high dose, 25-OH-D levels were
significantly above baseline at all time points. Notably, a significant decrease in
serum 25-OH-D was found between 3 months and 1 year of follow up (P= 0.038).
Low dose supplementation resulted in significantly increased 25-OH-D levels
compared to baseline only at 3 months.
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to be clarified. Recently, two prospective observational studies ad-
dressed this question, reaching conflicting results. Stewart et al.
found that increasing 25-OH-D was related to lower relapse rate
only in patients that were on IFN-btreatment (Stewart et al.,
2012). Løken-Amsrud et al. reported decreased odds for new MRI
lesions with increasing 25-OH-D levels only in MS patients that
were not on IFN-b(Løken-Amsrud et al., 2012). Interestingly, the
first study is from Tasmania, latitude 40–44 south, while the later
is from Norway, latitude 58–63 north, implying that sunlight
dependent mediators, other than vit D, may confound the associa-
tion between 25-OH-D and MS disease activity in IFN-btreated pa-
tients (Ascherio and Marrie, 2012). One possible such mediator is
melatonin, which is both sunlight-dependent (Brzezinski, 1997)
and an immunomodulator (Radogna et al., 2010).
The exact role of melatonin in MS is yet to be elucidated. Prior
reports have documented melatonin levels to be inversely corre-
lated with MS disease activity (Sandyk and Awerbuch, 1992,
1993a), and we have recently reported that IFN-btreatment in-
creases melatonin levels in treatment naïve patients with MS (Mel-
amud et al., 2012) as well as proposed application of Melatonin-
related chronobiological concepts to MS care (Glass-Marmor
et al., 2007, 2009).
Melatonin has both anti-inflammatory and pro-inflammatory
effects, depending on the context, such as the stage of inflamma-
tion (Radogna et al., 2010), making its potential influence on MS
complex to predict. Among melatonin’s pro-inflammatory proper-
ties, with potential negative consequences in MS, is enhancement
of inflammatory cytokine production, e.g. IL-2, IL-6 and (IFN)-
c
,
which was demonstrated in cultured human mononuclear cells
(Garcia-Maurino et al., 1997). This finding was attributed to the
upregulation of cytokine gene expression (Liu et al., 2001). On
the other hand, melatonin has also anti-oxidative properties (Re-
iter et al., 2008), which may exert favorable neuroprotective effects
in diseases such as MS, where axonal loss is part of the pathogen-
esis and seems to be induced, at least partially, by oxidative stress
(Gonsette, 2008). Neuroprotective influence of melatonin was re-
cently demonstrated in hypoxic-ischemic models of brain injury
(Robertson et al., 2013).
Our data show that the high dosage regimen is more effective
than low dosage in increasing 25-OH-D serum levels, at least for
short term. Our patients’ average EDSS score was about 3.5, imply-
ing that most of them were not housebound and could have been
influenced by outdoor sun exposure. Therefore, the decline in 25-
OH-D at 12 months in both dosage groups can be partly attributed
to winter at that time (baseline and 12 months examinations were
carried out at November to March). Annual variation in 25-OH-D
levels, with decreased levels at winter, has been reported in Israel
(Saliba et al., 2012). Additionally, attrition in patients’ compliance
with the vit D supplementation regimen cannot be ruled out.
BMI was found to be associated with decreased 25-OH-D levels
at 1 year, despite supplementation. This finding is in line with pre-
vious reports of diminished response to vit D supplementation in
obese patients (Saliba et al., 2013; Gallagher et al., 2012). Our pa-
tients’ BMI was also negatively correlated with melatonin secretion
as was their age. Both findings in agreement with prior research
(Wetterberg et al., 1999; Schernhammer et al., 2006).
In this study an inverse association was found between changes
in serum 25-OH-D levels and changes in urine melatonin metabo-
lite. The overnight secretion of melatonin decreased after 3 months
of high dose vit D supplementation in IFN-btreated MS patients.
Thereafter, along with a decrease in serum 25-OH-D levels, an
accompanying increase of melatonin secretion at night was ob-
served. In fact, the course of changes in nighttime melatonin secre-
tion was the mirror image of the trend in serum 25-OH-D levels.
These findings are derived from a clinical trial, in which vit D
supplementation preceded melatonin decreased secretion, thus
implying that the observed association may be causal.
Nights in which urine was collected, were on average 2 h short-
er after 3 months. It is unlikely that night shortening was the rea-
son for the observed decreased 6-SMT excretion, because it should
have influenced both dosage groups to the same extent, and not
only the high dose group. Furthermore, most prior studies found
that nighttime melatonin levels in humans are stable throughout
the year, regardless of the photoperiod (Bojkowski and Arendt,
1988; Kennaway and Royles, 1986; Wetterberg et al., 1999). Sea-
sonal variation in night hours in Israel is modest, much unlike
the major fluctuations in polar region. Indeed, studies which have
reported seasonal variation in nighttime melatonin secretion were
generally conducted at polar or sub-polar latitudes (Ruhayel et al.,
2007; Stokkan and Reiter, 1994).
The synthesis and release of melatonin from the pineal gland is
stimulated by darkness and inhibited by light. Photic information
from the retina is transmitted to the pineal gland through the sup-
rachiasmatic nucleus of the hypothalamus (Brzezinski, 1997). It is
tempting to hypothesize that 25-OH-D can bring a ‘message of
light’ to the pineal gland and consequently decrease melatonin
synthesis. Such a biological cascade and interconnectivity between
the vit D and the neuro-endocrine/hormonal (melatonin) system is
yet to be depicted.
Vit D receptor protein is expressed throughout the human
brain, including the hypothalamus, and found on most neurons
and on some glia cells (Eyles et al., 2005). 1
a
-Hydroxylase, the en-
zyme responsible for the formation of the active vit D from 25-OH-
D, is also present in the brain, and is strongly expressed in the
supraoptic nucleus of the hypothalamus (Eyles et al., 2005; Harms
et al., 2011). These findings support the possibility that 25-OH-D
and the pineal gland of the central nervous system could indeed
influence each other, as part of a vit D – related neuro-endo-
crine-immune network (Fernandes de Abreu et al., 2009).
Our results must be interpreted with caution. We did not fully
control for potential confounders of the alleged association be-
tween vit D and melatonin. Depressed mood is known to be asso-
ciated with decreased melatonin synthesis in MS (Akpinar et al.,
2008; Sandyk and Awerbuch, 1993b). Although no significant dif-
ference was observed in the depression subscale of the FAMS be-
tween the dosage groups, this cannot be regarded as full
evaluation of an affective disorder. Furthermore, we did not evalu-
ate for circadian rhythm disorders, which may shift the melatonin
secretion curve and influence the nighttime 6-SMT levels (Pandi-
Perumal et al., 2007). Randomization in clinical trials usually re-
sults in balanced representation of confounders, but the small sam-
ple size and high dropout rate in this trial, increases the possibility
for unbalanced representation of unrecognized confounders be-
tween the two intervention groups.
Another concern is the higher baseline 6-SMT level, which was
observed in the high dose group. Baseline characteristics, including
age, BMI and depression scores, were similar for the two dosage
groups. Therefore, the most plausible explanation is a chance
occurrence of unbalanced randomization, which was caused by
small sample size and the known high inter-individual variability
of human 6-SMT levels (Wetterberg et al., 1999). Because of this
natural variability, the change in 6-SMT level within an individual
is more important than the difference between groups of individu-
als. The importance of using subjects as their own control when
studying changes in melatonin excretion was advocated by others
(Bojkowski and Arendt, 1988).
Repeated measurement in the same subjects tends to give lower
values if they were initially high. This phenomenon, ‘regression to-
wards the mean’, probably did not contribute to the described re-
sults because an increase in 6-SMT, back towards baseline levels,
184 D. Golan et al. / Brain, Behavior, and Immunity 32 (2013) 180–185
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For personal use only. No other uses without permission. Copyright ©2016. Elsevier Inc. All rights reserved.
significantly correlated to the decrease in 25-OH-D, was noted
after the initial decrease in 6-SMT.
Despite the caveats the observation of decreased melatonin
secretion following vit D supplementation and the apparently mir-
ror trends of vit D and melatonin are novel and important, as both
hormones are light-dependent and have immunomodulatory prop-
erties. If this observation is verified in larger trials, alteration in
melatonin level should be taken into account as a potential medi-
ator of the neuro-immunomodulatory effects of vit D in patients
with MS. As many other factors can influence melatonin secretion,
this may potentially explain some of the variation in the clinical
and immunologic response to vit D in MS patients. The mutual ef-
fects of melatonin and vit D on immune-competence and neuro-
protection should be further assessed.
Acknowledgments
This study was supported by Merck Serono – Israel and Bayer
Schering – Israel. Vitamin D supplements and placebos were do-
nated by CTS – Israel and Altman – Israel.
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