Vitamin D deficiency and anemia frequently coexist [1
]. A number of mechanisms could explain this association [1
] and recent data provide evidence for a direct role of vitamin D in the suppression of hepcidin: the primary regulator of systemic iron homeostasis. Hepcidin inhibits and ultimately degrades ferroportin, the transmembrane protein that transports iron, and therefore controls the amount of iron absorbed in the intestine and released from cellular storage. Hepcidin is suppressed when iron status is low to maximize dietary iron absorption and the release of iron from stores. Conversely, inflammation causes an increase in hepcidin concentration that reduces dietary iron absorption and the capacity for iron egress from cells, resulting in decreased hemoglobin concentrations [6
In vitro work has shown a 1,25-dihydroxyvitamin D (1,25(OH)2
D; the active metabolite of vitamin D)-induced, dose-dependent decrease in hepcidin expression [7
]. The identification of the vitamin D response element (VDRE) on the human hepcidin promoter also supports the direct effect of 1,25(OH)2
]. Recent in vivo studies in healthy adults further support an effect of both single and longer-term high doses of vitamin D on hepcidin regulation. An increase in 25-hydroxyvitamin D concentration (25OHD; the status marker of vitamin D) of 68 to 109 nmol/L 72 h after an oral dose of 100,000 IU vitamin D2
was accompanied by a 34% decrease in hepcidin concentration (n
= 7) [8
]. A further study found a 73% decrease in hepcidin concentration one week after a single dose of 250,000 IU vitamin D3
= 18) [9
]. In early-stage, chronic kidney disease patients, the percentage increase in 25OHD concentration after 3 months of vitamin D3
supplementation (50,000 IU vitamin D3
/week) was associated with an inverse change in hepcidin concentration (n
= 38) [7
]. In addition to the effects on hepcidin concentration, vitamin D may also have immunomodulatory effects which may in turn alter hepcidin expression and iron status [10
Pregnant women are at high risk of anemia [13
] due to the high iron demands of pregnancy, particularly in low-resource settings, and data suggest associations between low hemoglobin, low iron status, and adverse birth outcomes [15
]. Improvements in iron stores and hemoglobin concentrations are associated with increases in birth weight [16
]. There is conflicting evidence as to whether C-reactive protein (CRP), as a marker of inflammation, rises [17
] or remains the same during pregnancy [18
]. However, inflammation may be relevant in pregnant women affected by conditions such as obesity or pre-eclampsia, known to be associated with inflammatory states [19
]. Low vitamin D status may be a contributing factor to iron deficiency anemia via direct effects on hepcidin or through potential inflammatory effects of low vitamin D status.
The effect of vitamin D supplementation on iron status has not been investigated in pregnant women. Through the mechanisms outlined above, adequate vitamin D status may be necessary for optimal hepcidin function and may help to provide protection against iron deficiency, potentially offering a complementary approach to combat anemia during pregnancy.
The aim of this current study was to examine if daily vitamin D3 supplementation (cholecalciferol, 1000 IU/day) in pregnancy suppresses hepcidin concentration and/or affects iron body stores (ferritin) and inflammation (CRP and α1-acid glycoprotein (AGP)) compared to placebo.
Antenatal supplementation with vitamin D3 (cholecalciferol, 1000 IU/day) in women due to give birth between March and May in the UK, increased 25OHD concentrations in the supplement group from early to late pregnancy by 17 nmol/L while the placebo group experienced a significant seasonal decrease in 25OHD concentrations (−11 nmol/L). Despite a 40 nmol/L difference between the two groups by late pregnancy, no group differences were seen in hepcidin, ferritin, or markers of inflammation.
Rates of iron deficiency (ferritin <15 µg/L) increased significantly in both groups to ~70% by late pregnancy, while hepcidin, CRP, and AGP concentrations decreased significantly. This decrease in ferritin is highly likely to indicate a corresponding decrease in hemoglobin concentrations and an increase in anemia prevalence [17
], although this was not verified due to a lack of stored whole blood samples. In addition, the decrease in hepcidin is likely to reflect the increased requirement for iron mobilization from stores and iron intestinal absorption as iron demands increase across pregnancy.
As with other studies, this study highlights the finding that iron deficiency is often associated with lower vitamin D concentration [2
]. This was seen in late pregnancy where 79% of women with 25OHD <25 nmol/L were also iron deficient compared to 61% of women with iron deficiency and 25OHD >25 nmol/L.
Smith et al. randomized 28 healthy individuals in Atlanta, Georgia, USA, to receive one oral bolus of vitamin D3
(250,000 IU) or placebo [9
]. Similar to our study, 75% of participants had 25OHD concentrations <50 nmol/L (20 ng/mL) at baseline and the bolus dose increased 25OHD concentrations by ~21 nmol/L in the treatment group at the 1 week follow-up. Unlike our study, however, Smith et al. found that those in the treatment group had a ~73% decrease in hepcidin concentration with no significant change detected in the placebo group and no change in ferritin or inflammatory markers in either group, 1 week post treatment. HAMP
, the gene encoding hepcidin, is known to contain the vitamin D response element in its promoter region [8
]. Smith et al. speculated that the vitamin D bolus dose resulted in a direct suppression of HAMP
gene expression and therefore a reduction in hepcidin concentration rather than indirectly acting through inflammation [9
The major differences in study design between the study by Smith et al. and the current study were (1) population (non-pregnant versus pregnant), (2) mode of vitamin D supplementation (250,000 IU bolus versus 1000 IU/day), (3) follow-up time (1 week versus 19 weeks), and (4) Smith et al. observed no changes in 25OHD concentration, markers of iron status, or markers of inflammation in the placebo group over time. The metabolic demands of pregnancy and pregnancy-related changes in vitamin D and iron metabolism (and their binding proteins) [28
] may influence vitamin D–hepcidin–iron interactions. In addition, the kinetics and metabolism of bolus versus frequent, lower doses of vitamin D are different [30
]. Therefore, while the overall change in 25OHD concentration in the treatment arm of the two studies was similar at follow-up, it is possible that the effect of vitamin D supplementation on hepcidin depends on the dose and/or frequency of vitamin D given.
No group difference was found in hepcidin or ferritin concentrations in late pregnancy or in change in hepcidin or ferritin between early and late pregnancy. However, there was a significant group interaction between 25OHD concentration and hepcidin and between 25OHD and ferritin in late pregnancy. Interestingly, in late pregnancy, the positive relationships between 25OHD and hepcidin and between 25OHD and ferritin that were seen in the placebo group were not observed in the vitamin D3
group suggesting an effect of supplementation on this relationship. Therefore, there may be a relationship between 25OHD concentration and iron metabolism when 25OHD concentrations are lowest and not at higher concentrations. This finding differs from that of Thomas et al. who reported no relationship between 25OHD and hepcidin concentrations in non-supplemented adolescent pregnant women from the USA at ~26 weeks gestation and at delivery [1
]. However, whilst vitamin D status in early pregnancy and in the supplemented group in late pregnancy were similar to the Thomas study, late pregnancy vitamin D status in the placebo group was notably lower, and further supports a concentration-dependent relationship between vitamin D and iron metabolism. Other non-hepcidin mediated mechanisms may explain associations observed between vitamin D and iron status. Thomas et al. found that the relationship between 25(OH)D and hemoglobin concentration was mediated by erythropoietin (EPO), with an inverse relationship between 25(OH)D and EPO in mid-gestation and at delivery [1
The current study found that CRP decreased as pregnancy progressed, in contrast to some studies that have suggested an increase [17
] or no change in CRP throughout pregnancy [18
]. The impact of inflammation on vitamin D status [26
] and the potential of vitamin D to mediate the immune response and temper inflammation is of interest [10
]. However, we found no effect of vitamin D supplementation on CRP or AGP, suggesting that vitamin D3
supplementation did not impact inflammatory pathways.
There were a number of limitations to this analysis. Firstly, whilst ferritin concentration is the standard World Health Organization measure of iron deficiency [24
], its interpretation, in common with other markers of iron status, can be affected by inflammation and pregnancy-related hemodilution. Whilst there is uncertainty over ferritin cut-offs for iron deficiency in pregnancy, as a marker of change in iron status, ferritin remains a sensitive biomarker for body iron stores [31
]. Hepcidin has been suggested an alternative marker of iron status in pregnant women [17
] but additional confirmatory research is required. Secondly, other biomarkers of iron status, erythropoiesis, inflammation or vitamin D (i.e., 1,25(OH)2
D) were not measured and so alternative mechanisms were not explored. Thirdly, increases in plasma volume as well as metabolic and hormonal changes during pregnancy may confound interpretation of biomarkers of nutritional status. However, the placebo controlled nature of the study should have accounted for most of these changes. Fourthly, we observed a difference between groups in the proportion of women who took iron-containing supplements in late pregnancy which was higher in the vitamin D3
group. However, the difference was small (n
= 3) and the method used to record supplement use was simple; whilst the brand of the supplement was recorded and coded for the presence or absence of iron in its formulation, the frequency and length or supplement use was not recorded and therefore it was not possible to determine the amount of iron in the supplement nor the amount of supplement consumed. Finally, 94% of women in this substudy were of white, self-reported ethnicity and the participants were screened for low and high 25OHD concentration before inclusion in the trial. It is possible that a larger effect on hepcidin would be detected at lower 25OHD concentrations.