Vitamin-D levels in exclusively breast fed infants less than six months of age : Do they need supplementation ?

Introduction: There is growing interest in subclinical vitamin D deficiency for the non-skeletal health benefits. There is good evidence that breastfed babies not supplemented with vitamin D have low vitamin D concentration and are at risk of rickets. Hypothesis: Exclusively breastfed babies often have low levels of vitamin D and are at risk of having rickets if not supplemented with vitamin D. Objective: Measurement of serum 25(OH) D levels in exclusively breastfed babies. Method: It was an observational, cross sectional study. Thirty healthy infants less than 6 months of age, on exclusive breast feeding and not on any vitamin supplementation were randomly selected. Vitamin D, serum calcium, phosphorus and alkaline phosphatase levels were measured in all these babies and those with clinical features of rickets were subjected for x-ray evaluation. Results: Twenty eight (93.3%) had hypovitaminosis D (value <20ng/ml) including severe deficiency (<5ng/ml) in two babies (6.7%). The mean value of vitamin D level was 8.871± 4.78ng/ml. The mean ionic calcium value was 5.07± 0.67 and the mean inorganic phosphorus value was 6.25±1.18 mg/dl, which were normal for this age group. The alkaline phosphatase level was uniformly elevated with 28 (93.3%) babies having values >420U/L. There was no statistically significant relationship between vitamin D levels and the birth weight or nutritional status of the infant. Radiological changes were found in three (10%) babies. Conclusion: Vitamin D deficiency is highly prevalent in exclusively breastfed healthy infants and hence vitamin D should be supplemented. ___________________________________________ Associate Professor, Final Year student, JN Medical College, Sawangi (M), Wardha, Maharashtra India (Received on 23 August 2013: Accepted after revision on 27 September 2013) (


Introduction
Vitamin D, the generic term for a family of secosteroids with anti-rachitic activity, comprises a family of fat soluble vitamins and hormones that, when deficient in the diet, causes rickets from defective mineralization of growing bone and osteomalacia in non-growing bones 1 .Vitamin D2 (ergocalciferol), obtained from the influence of ultraviolet B radiations on plants and yeast and vitamin D3 (cholecalciferol), produced in skin by ultraviolet rays are the two main forms of vitamin D. Both forms are metabolized similarly in the body, first by hepatic 25 hydroxylation into inactive but stable 25(OH) D (Calcidiol) and then by renal 1hydroxylation into active but unstable 1, 25(OH)2 D (Calcitriol).Calcitriol exerts its effects by binding to vitamin D receptor, which belongs to the family of nuclear hormone receptors 2 .
Vitamin-D deficiency is associated with a higher risk of autoimmune diseases and several forms of malignancy, such as prostate, colon and breast cancer 3 .Rickets is an extreme form of vitamin D deficiency and represents the tip of vitamin D deficiency iceberg 4 .Serum 25 (OH) vitamin D level is the best available biomarker for the diagnosis of vitamin D deficiency 5 .Many breastfed infants, not supplemented with vitamin D during the first 6 months of life have serum vitamin D concentrations <50nmol/L and are therefore at increased risk of rickets.This is especially true for infants who have high skin pigmentation and little sun exposure 6 .Most of the available data are from western countries and urban Indian populations; hence we undertook this study to measure vitamin D level in exclusively breast fed babies in a rural population around Wardha city in Central India.

Hypothesis
Exclusively breastfed babies often have low levels of vitamin-D and are at risk of having rickets if not supplemented with vitamin-D.

Objectives
• The primary objective was the measurement of serum 25 (OH) D levels in exclusively breastfed babies below six months of age.The birth weight was recorded from the medical records whenever available or as told by the mother.Birth weight <2500g irrespective of gestational age was considered as low birth weight 7 .Baby born with a gestation of <37 completed weeks was considered preterm 8 .Exclusive breast feeding was defined as no food or liquid other than breast milk, not even water, given to infant from birth by mother, health care provider, or family member/supporter 9 .Clinical examination was done by author using standard methods.Weight was recorded with minimal clothes on an electronic weighing machine with a minimum reading of 5g.Length was measured using an infantometer with minimum reading of 0.1cm with accuracy + 0.5 cm.Head circumference and chest circumference were recorded with help of fiber tape using standard methods.Nutritional status was assessed using WHO growth charts 10 .
Blood samples of these infants were collected for estimation of serum 25 (OH) D, calcium, phosphorus and alkaline phosphatase levels.Biochemical tests for serum 25(OH) D levels were conducted by M/s Religare Laboratories using fully automated chemi luminescent immunoassay method.The serum ionic calcium was measured using calcium arsenazo method, serum phosphorus by colorimetry method and alkaline phosphatase by p-nitro phenyl phosphate kinetic method at the central laboratory of the hospital.The normal values of these parameters in relation to age of the baby were obtained from the Harriet Lane handbook 18 th edition 11 .Babies with overt clinical findings were subjected to radiological examinations.X -rays of both wrists, antero-posterior and lateral views, were taken.These were reported by a senior radiologist who was unaware of the vitamin D status of these babies.The vitamin D status was categorized as follows:

Results
Of the 30 babies fulfilling the inclusion criteria 14 (46.7%) were male and 16 (53.3%)were female giving a male: female ratio of 1: 1.4.The average age of the babies in the study was 2.7 months, the youngest being 1 month (three babies) and the oldest being 6 months (two babies).All the babies were born at term.Preterm babies were not recruited as all of them were on vitamin supplementation.All 30 babies were on exclusive breast feeding.The vitamin D status of the babies is shown in Table 1.The nutritional status of the babies as per WHO charts is shown in Table 3. Twenty percent of babies were below the 3 rd percentile.Eighty percent were between the 3 rd and 97 th percentile.
The vitamin D status in relation to nutritional status is shown in Table 4.

Discussion
In our study 93.3% had hypovitaminosis D (vitamin D level <20 ng/ml) and the mean vitamin D level was 8.871 + 4.78ng/ml.In the study by Jain V, et al of vitamin D deficiency in healthy breast fed term infants at 3 months, the prevalence rate was 86.5% 12 .
Agarwal N, et al in their study found the mean vitamin D level was 11.55±7.17ng/mlat 10 weeks and 16.96± 13.33ng/ml at 6 months of age 13 .In their study 55.7% infants at 10 weeks had moderate vitamin deficiency and at 6 months 44.3% were moderately deficient, 16.5% developing rickets 13 .Bhalala U, et al have found an 80% prevalence of subclinical hypovitaminosis D at 3 months 14 .A study by Seth et al. on vitamin D nutritional status of exclusively breast fed infants and their mothers found a mean vitamin D level of 11.6±8.3ng/mlwith 43.2% of infants having levels below 10ng/ml 15 .A Western study found 10% and 37% prevalence of vitamin deficiency during summer and winter respectively 16 , and another had 12% prevalence 17 .
Mean ionic calcium value in our study was 5.07± 0.67mg% (10.15±0.8),which is normal for age and is similar to values of Jain V et al (10± 0.8) and Bhalala U et al (10.13±0.78).Only one baby (3.33%) had hypocalcaemia versus 7.1% in Jain V et al study 12,14 .
Normal inorganic phosphorus and normal alkaline phosphatase levels range from 10 days to 24 months are 4.5 -6.7mg/dl and 145-420U/L respectively.The average inorganic phosphorus level in our study was 6.25±1.18mg/dland alkaline phosphatase level was 643±195U/L.These values are similar to those in other studies 12,14 .
The average alkaline phosphatase value was uniformly increased, with 28 (93.3%)babies having value >420U/L, which may suggest hypovitaminosis D. Bhalala et al in their study had found elevated alkaline phosphatase level in only 51% of patients with low vitamin D 14 .Ziegler EE et al in their study found an occasional elevated alkaline phosphatase value, but no significant differences in alkaline phosphatase activity between vitamin D deficient and sufficient subjects 18 , suggesting inconsistent relation between 25(OH) D and alkaline phosphatase levels 17 .
Of the 30 babies 20% were below the ).This result is in concordance to that obtained by Agrawal et al 13 .

Conclusions
• Vitamin D deficiency is highly prevalent in exclusively breastfed healthy infants.• Biochemical rickets is more common than clinical.• The vast majority of these babies do not have clinical or radiological evidence of rickets as it may take more time to manifest.• The birth weight has no relation with vitamin D status.

Suggestion
We propose that exclusively breast fed babies should be supplemented with vitamin D on a regular basis.

Limitations of study
• The sample size was small.
• Because of financial constraints and nonavailability of test, parathyroid hormone levels were not done.
• We could not correlate the gestational age and vitamin D status as we could not recruit preterm babies who were all on vitamin supplementation.
Further studies recruiting large numbers of babies with different gestational ages and birth weights with different exclusion criteria and parathyroid hormone levels should be carried out so that a strong case can be made for vitamin D supplementation. 2