To study whether a free supply of vitamin D2 drops to 6-week-old infants together with tailor-made information handouts improves the vitamin D status after 7 weeks in the intervention group compared to a control group.
In this cluster randomized controlled trial in eight child health clinics in Oslo, Norway, 66 healthy infants with Pakistani, Turkish or Somali background were included. The intervention group received daily supplementation of vitamin D drops containing 10 μg (400 IU) of ergocalciferol (vitamin D2) with a tailor-made information brochure about vitamin D and its sources, and instruction on how to administer the drops. They were compared to a control group receiving usual care. The principal outcome measure was increase in serum 25-hydroxyvitamin D (S-25OHD) 7 weeks later. S-25OHD was analyzed by high-performance liquid chromatography-ultraviolet-mass spectrometry.
Total 78% (n=51) of the included infants completed the study. At follow-up, S-25OHD was significantly higher in the intervention group than in the control group (93.5 versus 72.7 nmol l−1, P=0.03). The mean increase in S-25OHD adjusted for baseline was 28 nmol l−1 (95% confidence interval 10.9–45.2, P=0.002) higher in the intervention group than in the control group. Among exclusively breastfed infants at baseline, S-25OHD increased by 32.3 nmol l−1 (P=0.035) in the intervention group compared to control group.
Free supply of vitamin D drops to 6-week-old infants together with tailor-made information handouts significantly improved the vitamin D status of infants with immigrant background compared to usual care.
Small children grow fast and have a high demand of calcium to build their skeleton. An association has recently been reported between maternal vitamin D status during pregnancy and a deficit in bone mineral accrual in their offspring at the age of 9 years (Javaid et al., 2006). Vitamin D deficiency is a major cause of rickets. Rickets was almost eliminated from Norway during the 1960s. However, in recent years a substantial number of cases of rickets among immigrant children have been reported. During year 2000, 65 children with rickets, most of these with a Pakistani background, were registered at Norwegian pediatric hospitals (Brunvand and Brunvatne, 2001). In addition, several studies have reported a high prevalence of serious vitamin D deficiency among immigrants in Norway (Meyer et al., 2004; Holvik et al., 2005). In one study, a quarter of Pakistani immigrant women aged 30 years had severe vitamin D deficiency defined as secondary hyperparathyroidism (Meyer et al., 2004). Also in other Western countries a high prevalence of vitamin D deficiency has been reported in non-Western immigrant populations (Lips, 2007). This has led to an increased awareness of vitamin D deficiency as a problem among immigrants. Exclusively breastfed infants have been reported to be at higher risk of developing vitamin D deficiency (Atiq et al., 1998; Kreiter et al., 2000; Andiran et al., 2002; Dawodu et al., 2003; Thomson et al., 2004). Although breastfeeding is optimal for infants, and both World Health Organization (Global Strategy for Infant and Young Child Feeding, 2003) and the Norwegian health authorities recommend exclusive breastfeeding for all children (only breast milk up to the age of 6 months), the vitamin D content of breast milk is very low and does not cover the vitamin D requirements of the infants (Gartner and Greer, 2003). In Norway, information about vitamin D is routinely given at the child health clinics, and intake of vitamin D supplements is encouraged. However, it is uncertain to what extent this advice is followed by mothers of immigrant background.
The main objective of the present trial was to study whether a free supply of vitamin D drops to 6-week-old infants, together with tailor-made information handouts, improves the vitamin D status (greater increase in serum 25-hydroxyvitamin D (S-25OHD) concentrations) at the age of 3 months in the intervention group compared to a control group. The infants included had immigrant background from Pakistan, Turkey and Somalia.
Material and methods
Recruitment and randomization
Following a pilot study at a health center in Drammen (a town near Oslo) in December 2003, the recruitment of child health clinics for the main study started in March 2004. Total 12 of the 25 child health clinics in Oslo, including those with high proportions of immigrant clients, were recruited. Eight of these clinics agreed to participate while four declined. To randomize at the individual level would have been problematic as the public health nurses would then have to carry out both the intervention and usual care at the same time. Randomization was carried out at the child health clinic level. The eight clinics were paired based on estimates of the number of children belonging to each clinic to secure approximately equal numbers of eligible children in each group. Within each pair, the names of both child health clinics were placed in a box and one was drawn by an independent person. Each clinic drawn was allocated to the intervention group. The public health nurses at the child health clinics were requested to invite all mothers with Pakistani, Turkish or Somali background who came for a routine 6-week check of their infants to participate in the study. Those willing to participate signed a form of consent and were included in the study. For those who declined to participate, the reasons for not participating were noted. A total of 119 mothers and their infants with Pakistani (n=72), Turkish (n=24) and Somali background (n=23) affiliated to the eight different child health clinics in Oslo were asked to participate in the study.
Mothers who came to the intervention child health clinics received free vitamin D drops. They also received a small brochure specially designed for this intervention study describing the importance of vitamin D, including simple illustrations and information translated into Urdu, Turkish and Somali with instructions on how to administer the drops to their infants (tailor-made information brochure). Vitamin D drops have been registered for sale in Norway since the year 2000. The drops are oil based and contain ergocalciferol (vitamin D2). The mothers were instructed to give their infant five drops totally containing 10 μg (400 IU) vitamin D2 per day. One small bottle containing 340 drops was handed out at baseline and the mothers were asked to note any unusual event that occurred during the period, such as forgetting to administer the drops. This intervention group was compared to a control group receiving usual care. Usual care is a traditional children health care provided by public health nurses and consists of oral information about vitamin D and recommendation of vitamin D supplementation to the infants. The recommended daily dosage of vitamin D for children from 4 weeks is 10 μg (The Norwegian Directorate of Health and Social Affairs, 2001). Most frequently cod liver oil is recommended but there can also be other vitamin D supplements.
A priori, and according to conventional power calculation, we estimated that 18 persons in each group would give the study an 80% power at the 5% significance level to detect a 15 nmol l−1 difference in increase in S-25OHD between the two groups. However, taking into account that cluster randomization would reduced the power, we estimated that a study with a total of 48 participants distributed at eight child health clinics corresponded to an individually randomized study with 18 participants in each group given an intraclass correlation of 0.05 (Hanley et al., 2003).
Blood samples (capillary) were collected from the infants at baseline and at follow-up in Oslo (60°N) between March 2004 and February 2006 by staff at the child health clinics. The health staff received training on blood taking from infants and used a standard instruction manual. The blood was centrifuged within 30 min and serum specimens were frozen at −70 °C until analyzed. S-25OHD2 and S-25OHD3 were determined by high-performance liquid chromatography-ultraviolet-mass spectrometry (HPLC-APCI-MS) at AS VITAS. Human plasma (150 μl) was diluted with 2-propanol-containing hexadeuterio 25-hydroxyvitamin D3 as internal standard and butylated hydroxytoluene as an antioxidant. After thorough mixing and centrifugation, an aliquot was injected from the supernatant into the HPLC system. HPLC was performed with an HP 1100 liquid chromatograph (Agilent Technologies, Palo Alta, CA, USA) interfaced by atmospheric pressure electrospray ionization to an HP mass spectrometric detector operated in single ion-monitoring mode. Vitamin D analogues were separated on reversed-phase HPLC column. A calibration curve was made from analysis of albumin solution enriched with known vitamin D concentrations. Recovery was >95%, the method was linear from 5 to 400 nM at least, and the limit of detection was 1–4 nM. RSD was 5.8 (29.4 nM) and 5.2% (73.6 nM). The lab is a part of vitamin D quality assessment scheme (www.vitas.no).
Background information about the infants and their mothers including anthropometry, breastfeeding practices, introduction of complementary feeding and the use of cod liver oil and other vitamin/mineral supplements, years of education and number of years living in Norway, was collected by public health nurses at baseline and at follow-up using an interview-administered questionnaire including food frequency questions that have previously been validated (Lande et al., 2003). Information about compliance and acceptability of the study was collected at follow-up.
Statistical analyses were performed with SPSS version 11.0 for Windows and descriptive analyses for all variables were calculated and the two groups were compared at baseline. We compared the increase in S-25OHD (the sum of S-25OHD2 and S-25OHD3) in the infants from baseline to follow-up by analysis of covariance (ANCOVA). Adjustment was made for baseline S-25OHD (Vickers and Altman, 2001). As the randomization was made at the health-center level, we first performed a multilevel analysis. As this analysis showed there was no clustering effect, further analyses were performed by conventional ANCOVA. The main analyses were executed by a statistician blinded for the grouping of the participants, and who was not otherwise involved in the study. Additional analyses were performed adjusting for gender, ethnicity and the mother's educational background.
The study was recommended by the regional committees for medical research ethics, and approved by the Norwegian Board of Health and Data Inspectorate.
Overall, 66 (55.5%) mothers and their infants were included in the study between March 2004 and February 2006, and 78% (n=51) of these completed the study and were included in the final analysis. The primary reasons for nonparticipation were moving back to their countries of origin, frequent moving within the city, having sick children or many children and poor communication skills.
Dropout analysis revealed no differences between dropouts and those who completed the study (data not shown).
Figure 1 shows the flow of child health clinics and infants through the trial. There were no significant differences between the background variables in the intervention and control groups (Table 1). The only exception was that the proportion of infants reported to take supplements daily at baseline was higher in the control group (65 versus 8.7%). A slightly higher proportion of infants were exclusively breastfed in the intervention group than in the control group. The infant formulas used by those not exclusively breastfed were all fortified with vitamin D3 (100 ml infant formula contains 1 μg of vitamin D3). We did not try to influence the use of infant formula. Maternal education and the number of years living in Norway were equally distributed between the two groups. Although the control group had higher S-25OHD at baseline, this difference was not significant (P=0.09). Mean S-25OHD at baseline was not significantly different in infants included during the winter months (October–March) compared to those included during the rest of the year (46.9 versus 51.7 nmol l−1, P=0.64).
S-25OHD concentrations at baseline and at follow-up are presented in Table 2. At follow-up, S-25OHD was significantly higher in the intervention group than in the control group (93.5 versus 72.7 nmol l−1, P=0.03; Figure 2). The mean increase in S-25OHD adjusted for baseline was 28 nmol l−1 (95% confidence interval 10.9–45.2, P=0.002) higher in the intervention group versus the control group. Gender, mother's ethnic background or level of education was not related to the increase of S-25OHD.
At baseline, 59 (13 of 22) and 45% (13 of 29) of infants in the intervention and control groups had S-25OHD <50 nmol l−1, while at follow-up 34% (10 of 29) of infants in the control group and 14% (3 of 22) infants in the intervention group still had S-25OHD <50 nmol l−1 (Figure 2).
Exclusive breastfeeding and serum 25-hydroxyvitamin D
In infants exclusively breastfed at baseline, S-25OHD increased by 32.2 nmol l−1 (P=0.035) in the intervention group compared to the control group, and at follow-up the whole distribution of S-25OHD was shifted to the right in the intervention group (Figure 3). At the follow-up 18 infants were either exclusively (n=11) or partially breastfed (n=7) in the intervention group, while in the control group 22 infants were either exclusively (n=13) or partially breastfed (n=9). Among exclusively breastfed infants at follow-up, the mean increase of S-25OHD adjusted for baseline in the intervention group versus the control group was 29 nmol l−1 (P=0.056), and 58% of the exclusively breastfed infants in the control group still had S-25OHD <50 nmol l−1 (range 5–43 nmol l−1).
At the end of the study we asked the mothers in the intervention group whether they had administered five drops of vitamin D daily to the infants and 91% responded ‘yes’. We analyzed S-25OHD3 and S-25OHD2 separately both at baseline and at the follow-up in both groups. In the intervention group mean S-25OHD2 increased by 45.9 nmol l−1 (Table 2), and 86% of the infant in the intervention group had detectable levels of S-25OHD2 (range 20–135 nmol l−1) at follow-up. According to information from the questionnaire, there were no other sources of Vitamin D2 in the diet of the infants.
We examined the effects of infant supplementation with vitamin D drops on S-25OHD, and found that infants receiving a free supply of vitamin D supplementation in form of vitamin D drops together with a tailor-made information brochures significantly increased S-25OHD concentrations compared to a control group receiving usual care.
The only commercially available vitamin D drops for infants in Norway, which we used in this study, contain vitamin D2. We measured both S-25OHD2 and S-25OHD3, and the great majority of the infants in the intervention group (86%) had a substantial increase in S-25OHD2 during the study. As we could not identify any other sources of vitamin D2, this indicates a very good compliance that was in fact consistent with the mother's reports on the daily administration of the drops to infants.
Effect of the intervention
To our knowledge, this is the first randomized intervention study on vitamin D supplementation among healthy infants with immigrant background. We found that the vitamin D status of the infants was poor at baseline. After 7 weeks of supplementation the vitamin D status was significantly improved in the intervention group and only three infants (14%) had S-25OHD <50 nmol l−1. Also in the control group S-25OHD increased and reached a mean level in the vitamin D-sufficient range. One reason for this could be that the largest commercial supplier of cod liver oil in Norway offered one bottle of free cod liver oil to each infant at some child health clinics. In spite of this, 31% of the infants in the control group had S-25OHD <50 nmol l−1 at follow-up.
Not least, the intervention improved vitamin D status in the exclusively breastfed infants, as demonstrated in Figure 3. In the control group, more than half of the exclusively breastfed infants had S-25OHD <50 nmol l−1 at 3 months. Our results confirm that it is important for exclusively breastfed infants to take vitamin D supplementation.
As rickets has recently been reported among infants in certain immigrant groups in Norway, and given the high prevalence of S-25OHD below 50 nmol l−1 in infants found in this study, it is obvious that the official recommendations for vitamin D supplementation are not fully adhered to by the mothers. Thus, there is a need for measures to increase vitamin D status in these groups.
Strengths and possible limitations
It is a strength of this study that we were able to assess the effect of the intervention directly by measuring S-25OHD concentrations. This is the main circulating metabolite of vitamin D and its concentration in serum reflects the vitamin D stores in humans. All the serum samples were analyzed in two batches (one for baseline samples and one for follow-up samples) in the same lab and the concentrations of S-25OHD2 and S-25OHD3 were determined separately, enabling us to determine the proportion of vitamin D2 from the drops.
In Norway, vitamin D supplementation is recommended from the age of 4 weeks (The Norwegian Directorate of Health and Social Affairs, 2001; Nordic Nutrition Recommendations, 2004), and some infants in the control group had started to take cod liver oil supplements before the study started, resulting in higher S-25OHD concentrations at baseline. In spite of this, the intervention group ended at considerably higher S-25OHD concentrations at follow-up compared to the control group. This issue was also taken into account in the main analysis where the mean change of S-25OHD in the groups was adjusted for baseline levels. Another concern of this study is the choice of supplementation regimen (vitamin D2 drops). Vitamin D is available in two forms, ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3), and until recently researchers believed that vitamins D2 and D3 have essentially the same benefits in humans. However, a recent study concluded that vitamin D3 has greater bioefficacy than vitamin D2 and that vitamin D2 has reduced potency and shorter duration of action (Armas et al., 2004). We used vitamin D2 supplements in our study because that was the only vitamin D drop available in Norway
Another possible limitation of this study is a potential incomplete registration of mothers declining to participate. Recruitment of study subjects was undertaken by local health visitors and not by project staff. As this work was in addition to their daily routine assignments, it is possible that the health visitors did not register all mothers who declined to participate. However, comparison of the education levels of the participating mothers with data from Statistics Norway (www.ssb.no) suggests that the mothers included in this study are representative of mothers with Pakistani, Turkish and Somali background living in Norway.
There were no reports on adverse effect of the intake of vitamin D drops in our study. On the contrary, mothers reported that it was easy to administer.
On the basis of our results, provision of free vitamin D supplements appears to be an effective and feasible measure for combating vitamin D deficiency and rickets among infants with an immigrant background, especially those exclusively breastfed. Multilingual translated brochures on prevention of rickets and vitamin D deficiency should be a part of the intervention and be available at all health facilities along with free vitamin D drops.
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We thank all mothers, health visitors and members of the study advisory group Leif Brunvand, Aud Haugen, Else-Karin Grøholt and Kirsten Berge, our child health clinics coordinator, for their help with the study. We also thank Magne Thoresen, University of Oslo for conducting the independent statistical analysis of the primary outcomes. This study was funded by the Directorate for Health and Social Affairs. The views and opinions expressed do not necessarily reflect those of the Directorate for Health and Social Affairs.
Guarantor: HE Meyer.
Contributors: AAM carried out the data collection, performed data analysis and prepared the manuscript. HEM developed the study protocol, secured initial funding and assisted in the analysis of the data. K-IK and HEM supervised the study. All authors commented on the draft, contributed to the interpretation of the findings and approved the final version of the manuscript.
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Madar, A., Klepp, K. & Meyer, H. Effect of free vitamin D2 drops on serum 25-hydroxyvitamin D in infants with immigrant origin: a cluster randomized controlled trial. Eur J Clin Nutr 63, 478–484 (2009). https://doi.org/10.1038/sj.ejcn.1602982
- vitamin D deficiency
- 25-hydroxyvitamin D
- randomized controlled trial
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