Vitamin D levels in Malawian infants from birth to 24 months
- Timothy K Amukele1,
- Dean Soko2,
- Pauline Katundu2,
- Melvin Kamanga2,
- Jin Sun3,
- Newton I Kumwenda3,
- Taha E Taha3
- 1Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
- 2Johns Hopkins University Research Project, Blantyre, Malawi
- 3Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA
- Correspondence to Dr Timothy Amukele, Department of Pathology, Johns Hopkins School of Medicine, Meyer B-125f, Baltimore, MD 21287, USA;
- Received 18 May 2012
- Revised 12 November 2012
- Accepted 15 November 2012
- Published Online First 7 December 2012
We measured longitudinal levels of vitamin D in unsupplemented Malawian infants at 0 (birth), 2, 12, 15, 18 and 24 months of age. Matched maternal plasma and breast milk vitamin D2 and D3 levels were also measured at delivery and 2 months postpartum. Vitamin D was measured using isotope-dilution liquid chromatography tandem-mass spectrometry. Vitamin D3 levels in children were 36% of adult levels at birth, 60% of adult levels at age 2 months, and at par with adult levels by 12 months of age. This adult-equivalent level is subsequently maintained through age 24 months and consisted of a 98% molar ratio of vitamin D3. Vitamin D levels in breast milk were below the limit of detection, 0.1 ng/ml. Breast milk of unsupplemented Malawian mothers is a poor source of vitamin D.
In addition to the role of vitamin D in childhood rickets, recent research demonstrates that maternal, cord blood, and childhood vitamin D levels are associated with respiratory,1–4 immunological,5–8 cognitive,9 and long-term bone health10 outcomes. However, what constitutes a normal or ‘ideal’ vitamin D level in children is unknown. For most biological substances, the normal reference range is determined by measuring levels found in a disease-free population. However mild hypovitaminosis D does not cause disease in the short term, and vitamin D levels are influenced by latitude, sun exposure, skin colour, clothing cover, the use of fortified foods, and sunscreen use.11
Given these challenges, there are two ways to begin to define biologically appropriate vitamin D levels. The first is to identify vitamin D levels that are associated with desirable health outcomes. The second is to determine vitamin D levels in a population with abundant sun exposure, where the vitamin D levels are determined primarily by biological regulation of vitamin D photosynthesis; not by climate, culture or access to fortified foods. Children in sub-Saharan Africa have several-fold higher vitamin D levels than those found in Western populations12 ,13 and show no clinical or serum response to exogenous vitamin D supplementation.14 ,15
In this exploratory, descriptive study, we measured longitudinal vitamin D2 and D3 levels in a cohort of unsupplemented Malawian children from birth to 24 months of age. Maternal vitamin D2 and D3 levels were also measured at delivery and at 2 months postpartum to establish adult levels. In addition to describing vitamin D levels over time, we examine the association of these longitudinal levels with child morbidity. These samples were collected as part of an earlier randomised clinical trial of short antiretroviral infant post-exposure prophylaxis to prevent mother-to-child transmission (MTCT) of HIV in Blantyre, Malawi16 (latitude 15°45′S, average temperature 22°C).
Vitamin D3 is made in human skin on exposure to ultraviolet radiation while vitamin D2 is only available to humans from enriched foods and vitamins. Although vitamin D3 can also be synthesised artificially, it is not used for food supplementation in Malawi or the surrounding countries. Hence vitamin D3 found in these populations is the direct result of solar-driven photosynthesis.
The NVAZ (nevirapine (NVP)/zidovudine (AZT)) study, a randomised clinical trial for prevention of MTCT of HIV-1 conducted in Malawi in 2000–2004, was approved by the College of Medicine, University of Malawi and the Johns Hopkins Bloomberg School of Public Health Institutional Review Boards. The current study is a secondary analysis of laboratory samples collected in that trial.16 Details of the study including recruitment, sample collection processing and storage have been described elsewhere.16 All women were breastfeeding their infants in the NVAZ study and no restrictions on duration of breastfeeding for HIV infected mothers were implemented at the time when the study was conducted.
In the current analysis, we measured longitudinal vitamin D levels in 21 HIV uninfected children born to HIV infected women. The 21 children included in the study were dark-skinned and samples were collected from the mother and baby at the same time of the year. We chose infants for whom plasma samples from the first and second visits (birth and 6–8 weeks, respectively), as well as serum samples for visits at 12, 15, 18 and 24 months, were available. The selection of infants was not based on any knowledge of the clinical status of the infant of the mother. We also included matched breast milk samples for all of the aforementioned visits; and matched maternal plasma samples at the first (delivery) and second (6–8 weeks postpartum) visits. Z-scores were calculated at each visit using standard anthropometric measurements obtained with the child lying supine.
Laboratory measurement of 25-hydroxy vitamin D, D2 and D3
Vitamin D is stable for up to 72 h in plasma and serum at room temperature before freezing,17 and is not affected by multiple freeze/thaw cycles.18 Total 25-hydroxy (OH) D, 25-OH D2 and 25-OH D3 fractions were measured in the samples from children and mothers by isotope-dilution liquid chromatography tandem-mass spectrometry (LC-MS/MS). This method for measuring vitamin D has been previously described.19 It has a recovery of 100% and a limit of detection of 0.1 ng/ml.
Variables considered in the current study of vitamin D
As part of the original parent NVAZ study, sociodemographic and clinical factors were collected at baseline (enrolment) and at subsequent visits as described previously.20 However, there were no measurements of factors that may have influenced the levels of vitamin D in the mothers, such as clothing cover to estimate the amount of body surface area that could be exposed to the sun.
Using Fisher's exact test for categorical variables and the Wilcoxon rank-sum test for continuous variables, we compared baseline covariates between those included or not included in this vitamin D study. In addition, we assessed the association of vitamin D3 status in children and mothers with overall child morbidity. Morbidity was defined as a composite of clinical illnesses, hospitalisations, medication use and malnourishment (based on WHO reference values20). We used generalised estimating equations to examine the association between repeated child measures of plasma vitamin D3 (as a continuous variable), baseline maternal vitamin D3 and child morbidity after adjusting for breastfeeding status.
The power of the study to detect a statistically significant (p≤0.05) difference in plasma/serum vitamin D (at the 0.05 level) was approximately 80% with a sample size of 21 infants with repeated measures at six time points (follow-up visits). Our power calculation assumed effect size of 0.421 and an expected correlation of 0.5 among the repeated measurements. All statistical analyses were conducted using SAS V.9.3.
Table 1 summarises selected baseline characteristics of mothers and children included in this analysis compared to the larger NVAZ cohort. The median body mass index (BMI) of mothers included in this study was significantly lower and the duration of breastfeeding of mothers included in this study was significantly longer than that in the overall NVAZ cohort. Nonetheless, the differences in BMI and length of breastfeeding are small and there is overlap in the interquartile ranges of the larger and smaller cohorts.
Mean levels of vitamin D2 for children and mothers were 0.6 ng/ml (median 0 ng/ml) and 1.5 ng/ml (median 1.5 ng/ml), respectively. Mean levels of vitamin D3 in infants and mothers over the same 24-month period were 30.9 ng/ml (median 29.1 ng/ml) and 35.3 ng/ml (median 34.7 ng/ml), respectively. Mean and median levels of D2 and D3 in breast milk were 0 ng/ml.
Table 2 shows the longitudinal vitamin D2 and D3 levels in children and mothers included in this study. Mean infant vitamin D2 levels at birth and age 2 months were 1.1 and 2.1 ng/ml, respectively. Mean child vitamin D2 levels were <0.2 ng/ml for the rest of the 24-month follow-up period. Mean maternal vitamin D2 levels at delivery and 2 months postpartum were 1.7 and 1.4 ng/ml, respectively.
Mean child vitamin D3 levels at birth, and 2, 12, 15, 18 and 24 months were 13.8, 19.7, 43.1, 34.0, 36.5 and 34.5 ng/ml, respectively. All children older than 2 months of age had vitamin D3 values that were >10 ng/ml. Mean maternal vitamin D3 levels at delivery and 2 months postpartum were 37.1 and 32.1 ng/ml, respectively. However, these vitamin D3 levels were not statistically different (p=0.15, Wilcoxon signed-rank test). There was a qualitative relationship between the level of vitamin D3 seen in individual maternal/infant pairs at birth and 6–8 weeks. We did not have enough mother–infant pairs to determine the strength of the association.
Vitamin D3 levels in children were on a par with adult levels by 12 months of age. These ‘adult’ vitamin D3 levels were maintained throughout the duration of follow-up. There were 66 individual vitamin D3 measurements in children aged ≥12 months, with a mean value of 35.4 ng/ml. Two per cent of these results (1/66) are below 20 ng/ml, and 5% of values (3 of 66) are above 70 ng/ml. There were no vitamin D results below 10 ng/ml.
Table 3 shows the frequency of overall morbidity in children included in this study. Overall morbidity was based on a composite measure of malnutrition and clinical morbidity at each visit. The frequency of malnutrition was consistently higher than reported clinical morbidity at each visit. Stunting (height-for-age Z-score ≤−2 SD) represented the highest frequency of malnutrition. Eleven (52%) of the 21 children in this study were stunted at age 12 months.
The levels of vitamin D3 in the child over time or the mother at baseline were not associated with overall child morbidity after adjusting for the practice of breastfeeding at each visit (data not shown).
To the best of our knowledge, this is the first report of longitudinal vitamin D levels in unsupplemented sub-Saharan children and their mothers. Other published reports from sub-Saharan Africa on vitamin D levels in children were either cross-sectional or did not include associated adult vitamin D levels.12–14 No clinical cases of rickets were observed in any of the infants in this study and vitamin D levels were not associated with infant morbidity.
Vitamin D3 levels in our infants are 36% of adult levels at birth, 60% of adult levels at 2 months, and on a par with adult levels by 12 months of age. This adult-equivalent vitamin D level is subsequently maintained through age 24 months and consists of a 98% molar ratio of vitamin D3 which is photosynthetically derived. In previous studies, cord blood vitamin D levels have been used as a basis for determining the vitamin D status in infants.22–24 Our data suggest that this practice may be based on incorrect assumptions about normal neonatal vitamin D levels. Breast milk does not appear to be a significant source of vitamin D supplementation for this cohort of Malawian children, which is consistent with earlier reports in other populations.25
The mean vitamin D level in children during the 12–24 months was 34.7 ng/ml, with a ±2SD range of 31.8–37.6 ng/ml. This value likely represents a physiologically ideal level of vitamin D for two reasons. First, this level of vitamin D coincides with levels required to optimise various vitamin D-associated health outcomes and physiological parameters, including bone mineral density,26 fracture risk in the elderly,26 colon cancer incidence,26 calcium absorption27 and urinary calcium excretion.28 Second, taking the imprecision of vitamin D assays into account,29 ,30 the levels of vitamin D seen in our cohort are similar to those found in other vitamin D-replete, unsupplemented, sub-Saharan infants.12–14 Of note, in one of these studies based in Oyem, Gabon (latitude 1°00′N, elevation 900 metres), there was no significant difference in the vitamin D levels of the treatment group receiving 1000 IU vitamin D daily and the control group.
This study has several potential limitations. First, there is a large gap in time between the 2-month and 12-month samples which obviates our ability to precisely determine the kinetics of the increase in infant vitamin D levels. Second, although the longitudinal nature of the observations increases the power of the study, it is based on a relatively small cohort. Third, the 24-month mortality rate for HIV-negative children in the original NVAZ cohort was 15.5%.31 This analysis of infant vitamin D levels over 24months necessarily included only the children who survived to age 24 months. Finally, all mothers included in this study were early-stage HIV positive patients. Although there is no consistent relationship between HIV-positivity and vitamin D status,32 ,33 we cannot exclude the possibility that the HIV status of these mothers is relevant to our findings.
This descriptive study provides longitudinal data on vitamin D levels in an unsupplemented sub-Saharan African cohort. It represents important baseline information for further larger studies to confirm these findings and assess the role of vitamin D in child growth and development.
Contributors TA and TT contributed to the planning of the work. All authors contributed to the conduct and reporting of the work. TA is responsible for the overall content as guarantor.
Competing interests None.
Ethics approval College of Medicine, University of Malawi and the Johns Hopkins Bloomberg School of Public Health Institutional Review Boards.
Provenance and peer review Not commissioned; externally peer reviewed.