nutrients

Article
Vitamin D Supplementation Modestly Reduces
Serum Iron Indices of Healthy Arab Adolescents
Mohammad S. Masoud, Majed S. Alokail, Sobhy M. Yakout, Malak Nawaz K. Khattak,
Marwan M. AlRehaili, Kaiser Wani and Nasser M. Al-Daghri *
Chair for Biomarkers of Chronic Diseases, Biochemistry Department, College of Science, King Saud University,
Riyadh 11451, Saudi Arabia; [email protected] (M.S.M.); [email protected] (M.S.A.);
[email protected] (S.M.Y.); [email protected] (M.N.K.K.); [email protected] (M.M.A.);
[email protected] (K.W.)
* Correspondence: [email protected]; Tel.: +966-(11)4675939




Received: 3 October 2018; Accepted: 27 November 2018; Published: 2 December 2018


Abstract: Vitamin D deficiency has been shown to affect iron status via decreased calcitriol production,
translating to decreased erythropoiesis. The present study aimed to determine for the first time
whether vitamin D supplementation can affect iron levels among Arab adolescents. A total of 125
out of the initial 200 Saudi adolescents with vitamin D deficiency (serum 25(OH)D < 50 nmol/L)
were selected from the Vitamin D-School Project of King Saud University in Riyadh, Saudi Arabia.
Cluster randomization was done in schools, and students received either vitamin D tablets
(1000 IU/day) (N = 53, mean age 14.1 ± 1.0 years) or vitamin D-fortified milk (40IU/200mL) (N = 72,
mean age 14.8 ± 1.4 years). Both groups received nutritional counseling. Anthropometrics, glucose,
lipids, iron indices, and 25(OH)D were measured at baseline and after six months. Within group
analysis showed that post-intervention, serum 25(OH)D significantly increased by as much as 50%,
and a parallel decrease of −42% (p-values <0.001 and 0.002, respectively) was observed in serum
iron in the tablet group. These changes were not observed in the control group. Between-group
analysis showed a clinically significant increase in serum 25(OH)D (p = 0.001) and decrease in iron
(p < 0.001) in the tablet group. The present findings suggest a possible inhibitory role of vitamin D
supplementation in the iron indices of healthy adolescents whose 25(OH)D levels are sub-optimal
but not severely deficient, implying that the causal relationship between both micronutrients may be
dependent on the severity of deficiency, type of iron disorder, and other vascular conditions that are
known to affect hematologic indices. Well-designed, randomized trials are needed to confirm the
present findings.

Keywords: serum iron; vitamin D; adolescents; Arab; vitamin D supplements

1. Introduction
Through the last decade, vitamin D has gained considerable interest in health and biomedical
research [1]. Globally, vitamin D deficiency is widespread and is considered a pandemic [2]. The Middle
East and North African regions, including the Kingdom of Saudi Arabia (KSA), are not spared from
this micronutrient deficiency, and in fact have among the highest rates of vitamin D deficiency in
the world [3,4]. Among the most common risk factors for vitamin D deficiency in the Middle-East
include female gender and their clothing style, multi-parity, sedentary lifestyle, urban living and
socio-economic status for adults, and longer than average breastfeeding as well as low dietary
vitamin D and calcium intake in children [5].
Vitamin D is involved in the proliferation and differentiation of bone marrow stem cells and
may play a role in red cell production [6]. Vitamin D can also potentially affect circulating iron

Nutrients 2018, 10, 1870; doi:10.3390/nu10121870 www.mdpi.com/journal/nutrients

Nutrients 2018, 10, 1870 2 of 11

status by promoting erythropoiesis and by suppressing hepcidin expression [6]. Lower levels
of pro-inflammatory cytokines and hepcidin increases iron bioavailability for erythropoiesis and
hemoglobin synthesis by preventing iron sequestration in macrophages [7]. On the other hand,
iron deficiency damages intestinal absorption of fat soluble vitamins, including vitamin D [8].
Similar to vitamin D deficiency, iron deficiency is also endemic and is, in fact, the most common
micronutrient deficiency globally [9]. Adolescent girls are at high risk for iron deficiency because of diet
and blood loss during menstruation [10]. Furthermore, according to the World Health Organization,
the two main risk target groups for iron deficiency are pre-school children and young women [11,12].
It is also highly prevalent in infants, adolescents, and pregnant women and is believed to account
for 75% of all types of anemia in the world, affecting 30% of population [13]. In the Middle East,
the prevalence of anemia among women of child-bearing age is 47% in Egypt, 16% in Lebanon, 26.7% in
the United Arab Emirates (UAE), and 40% in KSA [14].
Several cross-sectional studies confirm the association of vitamin D status and serum iron. In a
large-scale study involving 2526 Korean children and adolescents, they observed that the occurrence of
both vitamin D deficiency and anemia were significantly higher in females than males and concluded
that the positive correlation between vitamin D and iron may be through the suppressive action
of vitamin D in decreasing levels of hepcidin, an iron regulatory hormone [15]. Their results were
consistent with the previous observations of Smith and colleagues among African Americans [7].
To date, observational and functional studies suggest a positive relationship between vitamin D
status and serum iron, but interventional studies are lacking to prove causality. The recent
meta-analysis of Azizi-Soleiman and colleagues indicated that iron supplementation trials conducted
so far failed to improve vitamin D status [16], and limited data is available whether the reverse is
true. Thus, the present interventional study aims to determine for the first time whether a vitamin D
supplementation of six months duration can influence iron status among vitamin D deficient Saudi
Arab adolescents.

2. Materials and Methods

2.1. Study Design and Participants

This was a 6-month follow up study involving 200 apparently healthy Saudi adolescents (100 boys
and 100 girls) aged 13–17 years (overall mean age 14.1 ± 1.1 years; overall mean body mass index
(BMI) 21.2 ± 0.8 kg/m2 ) with known vitamin D deficiency (serum 25(OH)D < 50 nmol/L) [17] at
baseline and without medical conditions, such as asthma, hypertension, diabetes, liver, and renal
diseases. The participants were taken from the Vitamin D School Project database of the Prince Mutaib
Chair for Biomarkers of Osteoporosis (PMCO), King Saud University in Riyadh, Saudi Arabia [18,19].
In brief, the Vitamin D School Project is a collaborative project between King Saud University and
the Ministry of Education in Riyadh, Saudi Arabia, ascertaining the beneficial effects of 1000 IU/day
vitamin D supplementation and other vitamin D correction strategies, including vitamin D-fortified
milk consumption and overall public health awareness in raising vitamin D levels. The project database
includes information on more than 1000 students and teachers recruited from 34 different schools in the
central region of Riyadh during winter-spring season (November–May 2014–2015), when sun exposure
for optimum vitamin D3 production in Riyadh was observed to be shorter (10 A.M.–before 2 P.M.)
than summer (9 A.M.–3 P.M.) [20]. Government-run school hours were from 6:30 A.M. until 1–2 P.M.,
Sunday-Thursday. Cluster randomization was done in the 34 schools. This type of randomization
was done to prevent ‘contamination of allocation’, defined as participants in the control group being
aware of the interventions given in the test group and adopting it themselves [21]. In the case of the
present study, contamination of allocation can occur if both groups are in the same school, since the
students in the control group can be influenced by peers/classmates to switch to the tablet group
instead. Students from schools assigned to the milk (control) group were allocated to receive daily
200 mL of milk (per 100 mL contains 4.52 g carbohydrates, 3.22 g proteins, 3.0 g fats, 113 mg calcium,
Nutrients 2018, 10, 1870 3 of 11
Nutrients 2018, 10, x FOR PEER REVIEW 3 of 11

40 IUmg
113 of calcium,
vitamin D, 40102
IU ofIUvitamin
of vitamin A, and
D, 102 IU of58vitamin
kcal) forA,6and
months. Thefor
58 kcal) milk provided
6 months. Thewas
milk previously
provided
shown to have no effects in serum 25(OH)D levels [18]. Students
was previously shown to have no effects in serum 25(OH)D levels [18]. Students from from schools assigned to theschools
tablet
group received ®
assigned to the1000
tabletIU/day
groupvitamin
receivedD1000
supplementation
IU/day vitamin (VitaD1000 , Synergy Pharma,
D supplementation (VitaD1000Dubai, UAE)
®, Synergy
daily for 6 months. These interventions were monitored by their respective
Pharma, Dubai, UAE) daily for 6 months. These interventions were monitored by their respective teachers and parents who
were assigned to ensure that they were carried out daily in schooldays and
teachers and parents who were assigned to ensure that they were carried out daily in schooldays and weekends, respectively,
for the entirerespectively,
weekends, duration of for the the
study. Ethical
entire approval
duration of thewas obtained
study. Ethicalfrom the Ethics
approval was Committee
obtained from of the
the
College of Science Research Center, King Saud University, Riyadh, Saudi Arabia
Ethics Committee of the College of Science Research Center, King Saud University, Riyadh, Saudi (Ref No. 15/0502/IRB;
Project
ArabiaNo. (RefE-15-1667), in accordance
No. 15/0502/IRB; withNo.
Project the principles
E-15-1667),inin theaccordance
Declaration withof Helsinki, as well asinwith
the principles the
the
Declaration of Helsinki, as well as with the guidelines on good clinical practice. Prior toconsent
guidelines on good clinical practice. Prior to inclusion in the study, written informed inclusionwas in
acquired
the study, from parents,
written as wellconsent
informed as assent
was from the students.
acquired from parents, as well as assent from the students.
The
Thecohort
cohortused
used in in
thethe
present study
present werewere
study randomly selected
randomly from the
selected fromschool database
the school of 2 groups
database of 2
(tablet group (N = 100); milk (control) group (N = 100)). Baseline characteristics
groups (tablet group (N = 100); milk (control) group (N = 100)). Baseline characteristics of both of both groups are
groups
found in Supplementary Table S1. From the baseline assessment, significant
are found in Supplementary Table S1. From the baseline assessment, significant differences were differences were found in
the prevalence
found of severe vitamin
in the prevalence of severe D deficiency
vitamin D(25(OH)D
deficiency < 25 nmol/L)<(tablet,
(25(OH)D 47%, versus
25 nmol/L) (tablet,control, 28%;
47%, versus
pcontrol,
= 0.01) as well as baseline serum iron (p < 0.001), making the groups incomparable
28%; p = 0.01) as well as baseline serum iron (p < 0.001), making the groups incomparable for for prospective
analysis.
prospectiveAs extremely
analysis. low levels of 25(OH)D
As extremely low levels canofalter the overall
25(OH)D can metabolic profile, metabolic
alter the overall participants with
profile,
severe vitamin D deficiency (25(OH)D <25nmol/L) were excluded in the
participants with severe vitamin D deficiency (25(OH)D <25nmol/L) were excluded in the analysis analysis (N = 28 from the
control, and N = 47 from the tablet group). The final overall sample size was N
(N = 28 from the control, and N = 47 from the tablet group). The final overall sample size was N = 125.= 125. A flowchart has
been provided
A flowchart in been
has Figure 1.
provided in Figure 1.

Figure1.1.Study
Figure Studyflowchart.
flowchart.VDD,
VDD,vitamin
vitaminDDdeficiency.
deficiency.

2.2. Anthropometric and Biochemical Assessment

Information on anthropometrics (height, weight, body mass index, waist and hip circumference,
waist-hip ratio, systolic and diastolic blood pressure) were extracted from the database to include
Nutrients 2018, 10, 1870 4 of 11

2.2. Anthropometric and Biochemical Assessment

Information on anthropometrics (height, weight, body mass index, waist and hip circumference,
waist-hip ratio, systolic and diastolic blood pressure) were extracted from the database to include
values at baseline and after 6 months intervention. Anthropometrics were taken by an assigned
research physician using a standard scale (Digital Pearson Scale, ADAM Equipment Inc., Oxford,
CT, USA) for the assessment of height (cm) and weight (kg) measured in light clothing and without
shoes. Waist and hip circumferences were measured using standard tape measure. Blood pressure
(mmHg) was measured twice using a mercurial sphygmomanometer and the appropriate pediatric
cuff. The average was noted. Body mass index (kg/m2 ) and waist-hip ratio (WHR) were calculated
accordingly. Biochemical parameters for baseline and after 6 months, such as glucose, lipid profile
(triglycerides, total cholesterol, LDL- and HDL-cholesterol), and calcium, were also retrieved from the
database. Morning blood extraction was done twice (at baseline and after 6 months) for each participant
after fasting for 8 h. Blood samples were centrifuged and delivered immediately in pre-labeled plain
tubes, placed on ice, to King Saud University in Riyadh, Saudi Arabia, for storage and routine analysis
using a biochemical analyzer (Konelab, Espoo, Finland).

2.3. Vitamin D and Iron Indices

Serum 25(OH) D was measured using COBAS e-411 automated analyzer (Roche Diagnostics,
Indianapolis, IN, USA) in a DEQAS-certified laboratory (PMCO). Colorimetric ferrozine-based assay
was used to measure iron and total iron-binding capacity in serum samples using a spectrophotometer.
Transferrin saturation (%) was calculated as serum iron (µg/L)/total iron-binding capacity (TIBC)
(µg/L) × 100.

2.4. Data Analysis

A G*power calculator was used for sample size determination. Using repeated measurement
analysis, the observed effect size was 0.40 for a total sample size of 125, and the actual observed
power was >0.85. Data were analyzed using SPSS (version 21) (IBM, Armonk, New York, USA).
Continuous data were presented as mean ± standard deviation (SD) for variables following
Gaussian variables, and non-Gaussian variables were presented in median (minimum-maximum).
All continuous variables were checked for normality using Kolmogorov-Smirnov test, and non-normal
variables were log-transformed. Categorical variables were presented in percentages (%) and
Chi-square tests were performed. Independent t-test and paired t-test were used to check mean
differences between group and time points respectively. Repeated measures analysis of co-variance
(ANCOVA) was done to compare control and tablet groups. A p-value of <0.05 was considered
statistically significant.

3. Results
Table 1 shows the general characteristics of the control and tablet groups after exclusion of
participants with severe vitamin D deficiency at baseline. The control group had a higher systolic
blood pressure than the tablet group, although this was borderline significant (p = 0.053). The rest of
the baseline anthropometrics, biochemical indices, as well as serum 25(OH)D, calcium and iron indices
were not significantly different between groups.
Nutrients 2018, 10, 1870 5 of 11

Table 1. Baseline characteristics of intervention and control groups.

Parameter Tablet Control p-Value

N 53 72
Males (%) 30 (56.6) 40 (55.6) 0.68
Anthropometrics
Age (years) 14.1 ± 1.0 14.8 ± 1.4 0.09
BMI (kg/m2 ) 22.8 ± 5.8 22.9 ± 6.2 0.96
Waist circumference (cm) 78.2 ± 15.8 80.1 ± 16.8 0.60
Hip Circumference (cm) 92.3 ± 13.8 94.5 ± 15.9 0.50
Waist-Hip Ratio 0.80 ± 0.1 0.80 ± 0.1 0.98
Systolic Blood Pressure (mmHg) 116.2 ± 12.9 122.5 ± 16.6 0.05
Diastolic Blood Pressure (mmHg) 70.6 ± 11.6 71.1 ± 13.6 0.88
Routine Biochemical Indices
Glucose (mmol/L) 5.2 ± 0.6 5.4 ± 0.7 0.17
Triglycerides (mmol/L) 1.2 ± 0.6 1.3 ± 0.6 0.43
Total Cholesterol (mmol/L) 4.7 ± 0.8 4.5 ± 1.0 0.22
LDL-Cholesterol (mmol/L) 2.9 ± 0.7 2.5 ± 0.8 0.06
HDL-Cholesterol (mmol/L) 1.1 ± 0.3 1.3 ± 0.3 0.10
Calcium (mmol/L) 2.0 ± 0.1 1.9 ± 0.3 0.11
Vitamin D and Iron Indices
25(OH)D (nmol/L) 34.6 ± 6.4 37.2 ± 7.5 0.09
Iron (µmol/L) # 18.2 (3–41) 21.5 (8–39) 0.09
83.4
Transferrin Iron-Binding Capacity (µmol/L) # 83.6 (28–99) 0.35
(19–102)
Transferrin Saturation (%) # 23.9 (3–71) 26.3 (2–70) 0.91
Note: # presented as median (interquartile range); p-value significant at <0.05.

Table 2 shows the changes in the anthropometrics and routine biochemical indices over time.
Within-group comparisons in the tablet group showed significant increases in waist circumference
(p = 0.01) and waist-hip ratio (p < 0.001). There was also a significant decrease in glucose levels
(p = 0.038) and triglycerides (p = 0.015) over time, parallel to the improvement, although borderline
significant, in high density lipoprotein (HDL)-cholesterol levels (p = 0.06). Within-group comparison
in the control group showed an overall increase in anthropometrics, including weight (p = 0.006),
BMI (p = 0.09), hips (p = 0.049), and waist-hip ratio (p = 0.011). There was also a significant decrease in
HDL-cholesterol levels over time in the control group (p = 0.008). Between-group comparisons showed
a clinically significant difference in systolic blood pressure (p = 0.006), glucose (p = 0.029), triglycerides
(p = 0.059), and HDL-cholesterol (p = 0.005) in favor of the tablet group. The rest of the between-group
comparisons were not significant (Table 2).
Table 3 shows the effects of vitamin D supplementation in vitamin D status and iron indices over
time. Within-group comparison showed a significant increase in 25(OH)D levels in the tablet group by
as much as 50% (p < 0.001). Also in the tablet group, a significant decrease in iron levels was observed
(−42%; p = 0.002), as well as in transferrin saturation (p = 0.01), parallel to the significant increase
in TIBC (8.5%; p = 0.01). No significant changes were found in the control group. Between-group
comparisons revealed a clinically significant increase in 25(OH)D levels in favor of the tablet group
(p = 0.001) as well as a clinically significant reduction in iron (p < 0.001) and transferrin saturation
levels (p = 0.005) (Table 3).
Nutrients 2018, 10, 1870 6 of 11

Table 2. Changes in anthropometric and clinical parameters at baseline and follow-up.

Parameter Tablet Control

Tablet Effects
N 53 72
Baseline Follow-Up p-Value Baseline Follow-Up p-Value p-Value
Anthropometrics
Weight (kg) 55.9 ± 17.3 56.3 ± 18.6 0.65 62.1 ± 19.3 65.0 ± 22.9 0.006 0.07
BMI (kg/m2 ) 22.8 ± 5.8 22.9 ± 6.2 0.59 22.9 ± 6.2 23.9 ± 7.5 0.09 0.69
Waist circumference (cm) 78.2 ± 15.8 82.2 ± 17.1 0.01 80.1 ± 16.8 81.4 ± 18.0 0.29 0.87
Hip circumference (cm) 92.3 ± 13.8 91.0 ± 13.4 0.17 94.5 ± 15.9 92.4 ± 14.4 0.049 0.56
Waist-Hip Ratio 0.8 ± 0.1 0.9 ± 0.1 <0.001 0.8 ± 0.1 0.9 ± 0.1 0.011 0.67
SBP (mmHg) 116.2 ± 12.9 113.9 ± 12.3 0.27 122.5 ± 16.6 121.4 ± 13.0 0.61 0.006
DBP (mmHg) 70.6 ± 11.6 69.8 ± 12.6 0.68 71.1 ± 13.6 69.9 ± 15.5 0.6 0.92
Routine Biochemical Indices
Glucose (mmol/L) 5.2 ± 0.6 5.0 ± 0.5 0.038 5.4 ± 0.7 5.2 ± 0.7 0.15 0.029
Triglycerides (mmol/L) # 1.0 (0.3–3.1) 0.9 (0.3–2.3) 0.015 1.2 (0.3–3.1) 1.3 (0.4–3.0) 0.45 0.059
Total Cholesterol (mmol/L) 4.7 ± 0.8 4.7 ± 0.8 0.84 4.5 ± 1.0 4.6 ± 1.0 0.22 0.33
LDL-Cholesterol (mmol/L) 2.9 ± 0.7 2.8 ± 0.7 0.62 2.4 ± 0.8 2.5 ± 0.7 0.53 0.74
HDL-Cholesterol (mmol/L) 1.1 ± 0.3 1.3 ± 0.3 0.06 1.3 ± 0.3 1.1 ± 0.2 0.008 0.005
Calcium (mmol/L) 2.0 ± 0.2 1.9 ± 0.2 0.07 1.9 ± 0.5 1.8 ± 0.5 0.44 0.062
Note: # presented as median (min-max); significant at p < 0.05.

Table 3. Changes in vitamin D and iron indices at baseline and follow-up.

Tablet (N = 53) Control (N = 72)

Parameters Intervention Effects
Baseline Follow-Up p-Value Baseline Follow-Up p-Value
25(OH)D (nmol/L) 34.6 ± 6.4 51.9 ± 13.0 <0.001 37.2 ± 7.5 37.9 ± 10.6 0.69 0.001
Iron (µmol/L) # 18.2 (2.1–40.9) 11.5 (1.3–49.5) 0.002 21.5 (8.1–39.5) 21.7 (8.7–38.0) 0.86 <0.001
TIBC (µmol/L) # 83.4 (18.7–102.8) 90.5 (78.9–102.5) 0.01 83.6 (28.0–99.5) 84.9 (52.7–99.5) 0.90 0.42
Transferrin Saturation (%) # 23.9 (2.1–70.8) 12.3 (1.4–48.7) 0.001 26.3 (1.2–70.7) 25.1 (10.3–80.3) 0.70 0.005
Note: # presented as median (min-max); significant at p < 0.05.
Nutrients 2018, 10, 1870 7 of 11

4. Discussion
The present interventional study evaluated the changes in circulating serum iron levels and other
iron indices in a cohort of Saudi adolescents with suboptimal vitamin D levels before and after six
months of daily 1000 IU vitamin D supplementation, as compared to controls. The present study is
the first among Arab adolescents in prospectively determining the association between vitamin D
and iron status. Among the highlights of the study are the clinically significant decrease in serum
iron and transferrin saturation levels post-intervention in the tablet group, parallel to the significant
improvements observed in blood pressure, glucose, and selected lipids, also in favor of the tablet group.
The counterintuitive effect of vitamin D supplementation in serum iron levels observed in the
present study is in alignment with the observations of Doudin and colleagues conducted among
>5000 healthy German adolescents of similar age groups, in that vitamin D levels seems to have an
inhibitory role in hematological parameters, including hemoglobin where the bulk of iron is stored [22].
On the other hand, the clinical trial done by Madar and colleagues on healthy adults found that
while four months of vitamin D3 (25 µg or 1000 IU) supplementation did not significantly affect
any of the iron markers, the observed percentage changes post-intervention in hemoglobin (−0.6%),
ferritin (−35%), and iron (−5.9%) were all trending downwards, similar to the present study [23].
Other clinical trials also found no significant changes in iron indices among healthy adults despite
mega-doses of vitamin D3 [24,25]. Jastrzebska and colleagues have even taken into consideration the
influence of physical activity and intermittent training since it can potentially affect vitamin D and iron
metabolism, yet no significant differences were still found in the hematological parameters (Hb, Hct,
and ferritin) of athletes given 5000 IU of vitamin D daily for eight weeks over those who did not receive
supplementation [26]. All these previous studies, including the present one, suggest that vitamin D
correction is unlikely to improve, if not reduce, iron indices, at least in apparently healthy populations.
Given the negative results of previous clinical trials among healthy subjects and the inhibitory
effects found in the present study, it appears that the role of vitamin D supplementation in improving
iron stores may be limited to those with certain metabolic conditions, such as those with poor vascular
and renal function [27,28]. Suboptimal levels of both vitamin D and iron are biomarkers of ill health,
and the hypothetical association appears to be reciprocal, as clinical observations demonstrated the
role of 1, 25(OH)D in erythropoiesis, and the participation of iron is essential in the second activation
of vitamin D in order to be functional [29,30]. The effect of vitamin D in reducing iron levels as
observed in the present study, at least in participants who are apparently healthy and with no known
vascular diseases, seem to support the mechanistic role of vitamin D as a chemopreventive agent in
inhibiting erythropoiesis and angiogenesis, that in turn, suppresses proliferation of certain types of
cells, including cancer cells [31].
Other findings include a general improvement in glucose and select lipids in favor of the tablet
group. The mean serum vitamin D also significantly increased and almost reached a sufficient level
(vitamin D ≥ 50 nmol/L) at follow-up. These changes were in alignment with previous vitamin D
studies done in the KSA adolescent population [18,19]. Changes in selected anthropometric measures
in both groups can be partially explained by dietary intake and physical activity which, unfortunately,
were not taken into account in the present study.
The present findings should be interpreted taking into consideration its limitations. The present
study is not a randomized controlled trial, and as such, several biases are evident due to
non-randomization of participants and the lack of a better placebo group. Differences in baseline
characteristics between the tablet and the control groups were minimized by removing all participants
with baseline severe vitamin D deficiency. This finally gave a more comparable baseline metabolic
profile as the 25(OH)D range was narrowed down (25–50 nmol/L).
Another major limitation is that the control group was given vitamin D-fortified milk, and dairy
products have been observed to affect iron absorption due to their calcium content. The effects of dairy
products in iron absorption, however, is still debatable since several intervention studies showed no
significant change in iron indices from dairy product consumption [32,33]. Current recommendations,
Nutrients 2018, 10, 1870 8 of 11

however, in milk consumption without affecting vitamin D and iron stores in children are 2 cups
(500 mL) per day [34]. The control group in the present study were consuming only 200 mL/day.
Serum calcium were also unaffected in both groups. More importantly, the vitamin D and calcium
content in the milk products sold in Saudi Arabia are much lower than what the labels claim to be [35].
Other factors, such as dietary intake as a whole, as well as vitamin D intake and sunlight exposure,
were also not taken into consideration and can significantly influence vitamin D status independent
of the intervention assigned. However, epidemiologic observations done in Arab adolescents of
Riyadh show that the majority have darker complexion, are fully clothed during outside activities
(especially females), and prefer sunlight exposure before 10 A.M. [36]. These factors significantly
reduce any clinically meaningful vitamin D conversion through sunlight exposure in this age group,
especially in the present study, which was conducted during winter time when optimum sun light is
not only reduced, but the best time to get sun exposure also falls well within their school hours. Other
important parameters could not be analyzed, such as hepcidin and ferritin. Hepcidin, in particular,
as a master regulator for iron absorption, has been shown to distinguish iron deficiency anemia and
anemia of inflammation [37,38], with the latter type of anemia possibly benefiting more from vitamin
D correction than the former [39,40].
Despite these limitations, the study remained sufficiently powered and adds value, as it
documents the modest but significant effects of vitamin D supplementation in terms of influencing
iron status in a relatively understudied ethnic population and age group. To the best of our knowledge,
this is also the longest intervention trial done to determine changes in serum iron levels secondary
to vitamin D supplementation. As the majority of the limited interventional studies also yielded
negative results, given the clear association between vitamin D deficiency and risk of anemia [41,42],
identifying which type of anemia will benefit from vitamin D supplementation might give more
conclusive evidence.

5. Conclusions
In conclusion, a six-month vitamin D supplementation of 1000 IU/day significantly improved
vitamin D status and consequently decreased serum iron levels among Saudi adolescents whose
25(OH)D levels are sub-optimal but not severely deficient. The study adds to the growing literature
of the inhibitory and limited effects of vitamin D correction in the iron status of healthy individuals.
The identification of the cause of iron deficiency is essential as to which demographics will benefit
the most from vitamin D supplementation in terms of improving iron status. Well-designed and
adequately powered randomized controlled trials including other iron indices, such as hepcidin, are
encouraged to confirm present results.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/10/12/1870/

s1, Table S1: Baseline characteristics of intervention and control groups before exclusion of participants with
severe vitamin D deficiency (25(OH)D <25nmol/L).
Author Contributions: Conceptualization, M.S.M., M.S.A., S.M.Y., and N.M.A.; methodology, M.S.M., M.S.A.,
S.M.Y., and N.M.A.; formal analysis, M.N.K.K., M.M.A., and K.W.; investigation, M.S.M., M.M.A., and S.M.Y.;
writing original manuscript, M.S.M.; writing-review and editing, M.S.A., S.M.Y, K.W; funding acquisition, N.M.A.
Funding: This study is supported by the Deanship of Scientific Research Chairs, Chair for Biomarkers of Chronic
Diseases, Department of Biochemistry College of Science in King Saud University, Riyadh, Saudi Arabia.
Acknowledgments: The authors are grateful to Syed Danish Hussain for the statistical analysis provided and to
Dr. Yousef Al-Saleh for his clinical inputs.
Conflicts of Interest: The authors declare no conflict of interest. The funders, including the company who
provided the vitamin D supplements, had no role in the design of the study; in the collection, analyses,
or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
Nutrients 2018, 10, 1870 9 of 11

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