Trial of Daily Vitamin D Supplementation in Preterm Infants WHAT’S KNOWN ON THIS SUBJECT: Despite widespread prevalence of vitamin D deficiency, there is a paucity of evidence on the appropriate supplemental dose in preterm infants. Various professional organizations empirically recommend different doses of vitamin D, ranging from 400 to 1000 IU per day. WHAT THIS STUDY ADDS: Daily vitamin D supplementation at a dose of 800 IU compared with 400 IU significantly reduces the prevalence of vitamin D deficiency in preterm infants. The clinical significance of achieving vitamin D sufficiency needs to be studied in larger trials.

abstract OBJECTIVE: To compare the effect of 800 vs 400 IU of daily oral vitamin D3 on the prevalence of vitamin D deficiency (VDD) at 40 weeks’ postmenstrual age (PMA) in preterm infants of 28 to 34 weeks’ gestation. METHODS: In this randomized double-blind trial, we allocated eligible infants to receive either 800 or 400 IU of vitamin D3 per day (n = 48 in both groups). Primary outcome was VDD (serum 25-hydroxyvitamin D levels ,20 ng/mL) at 40 weeks’ PMA. Secondary outcomes were VDD, bone mineral content, and bone mineral density at 3 months’ corrected age (CA). RESULTS: Prevalence of VDD in the 800-IU group was significantly lower than in the 400-IU group at 40 weeks (38.1% vs 66.7%; relative risk: 0.57; 95% confidence interval: 0.37–0.88) and at 3 months’ CA (12.5% vs 35%; relative risk: 0.36; 95% confidence interval: 0.14–0.90). One infant (2.4%) in the 800-IU group had vitamin D excess (100–150 ng/mL). Bone mineral content (mean 6 SD: 79.6 6 16.8 vs 84.7 6 20.7 g; P = .27) and bone mineral density (0.152 6 0.019 vs 0.158 6 0.021 g/cm2; P = .26) were not different between the 2 groups. CONCLUSIONS: Daily supplementation with 800 IU of vitamin D reduces the prevalence of VDD at 40 weeks’ PMA and at 3 months’ CA in preterm infants without showing any improvement in bone mineralization. However, there is a possibility that this dose may occasionally result in vitamin D excess. Pediatrics 2014;133:e628–e634

AUTHORS: Chandra Kumar Natarajan, DM,a M. Jeeva Sankar, DM,a Ramesh Agarwal, DM,a O. Tejo Pratap, DM,a Vandana Jain, MD,b Nandita Gupta, PhD,c Arun Kumar Gupta, MD,d Ashok K. Deorari, MD,a Vinod K. Paul, MD,a and Vishnubhatla Sreenivas, PhDe Divisions of aNeonatology and bPediatric Endocrinology, Department of Pediatrics and cDepartments of Endocrinology and Metabolism, dRadiodiagnosis, and eBiostatistics, All India Institute of Medical Sciences, New Delhi, India KEY WORDS DXA, preterm, supplementation, vitamin D ABBREVIATIONS ALP—alkaline phosphatase BMC—bone mineral content BMD—bone mineral density CA—corrected age CI—confidence interval DXA—dual-energy radiograph absorptiometry GA—gestational age NNT—number needed to treat PMA—postmenstrual age PTH—parathyroid hormone RR—relative risk UCa/Cr—urine calcium:creatinine ratio VDD—vitamin D deficiency 25(OH)D—25-hydroxyvitamin D Dr Natarajan conceptualized and designed the study and drafted the initial manuscript; Drs Sankar and Agarwal helped in designing the study and with data collection instruments and analysis and critically reviewed the manuscript; Dr Pratap helped in data collection and analysis; Drs Jain, A. K. Gupta, Deorari, and Paul helped in designing the study and critically reviewed the manuscript; Dr N. Gupta helped in designing the study and with analysis of the samples for vitamin D and parathyroid hormone; Dr Sreenivas helped in designing the study and with analysis and critically reviewed the manuscript; and all authors approved the final manuscript as submitted. This trial has been registered at Clinical Trial Registry of India (CTRI) (identifier CTRI/2012/02/002459). www.pediatrics.org/cgi/doi/10.1542/peds.2012-3395 doi:10.1542/peds.2012-3395 Accepted for publication Dec 13, 2013 Address correspondence to Ramesh Agarwal, DM, Newborn Health Knowledge Centre, Department of Pediatrics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029. E-mail: [email protected] (Continued on last page)

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Vitamin D, a fat-soluble vitamin, is essential for absorption of calcium from intestine, which, in turn, determines the skeletal health and other physiologic processes such as neuromuscular function. Inadequate serum vitamin D levels havebeenreportedtoresultinamultitude of health problems in children, including rickets and increased susceptibility to infections, among others.1–4 Vitamin D deficiency (VDD) is common among infants, children, and pregnant and lactating mothers.5–7 Preterm infants often have less vitamin D stores due to decreased transplacental transfer from deficient mothers and may also have a higher requirement.8,9 The prevalence of vitamin D insufficiency (serum 25-hydroxyvitamin D [25(OH)D] ,30 ng/ mL) in preterm infants was 97.4% in a study from India,7 whereas severe deficiency (serum 25[OH]D levels ,5 ng/mL) was found in 44% of the preterm infants in an Arab population.10 Vitamin D requirements in adults and older children are met from foods of animal origin, fortified foods, and/or through de novo synthesis in skin after UV-B exposure. Breast milk from mothers who are not receiving vitamin D supplementation or who have inadequate exposure to sunlight contains only 20 to 60 IU of antirachitic activity, which is insufficient to meet the requirements of the infant.11–14 Moreover, de novo synthesis of vitamin D in skin after UV-B exposure may not be safe.15 Therefore, newborn infants have to be supplemented with vitamin D. The American Academy of Pediatrics recommends that breastfed and partially breastfed infants should be supplemented with 400 IU/day of vitamin D.16 On the other hand, the European Society of Pediatric Gastroenterology, Hepatology and Nutrition recommends daily supplementation of 800 to 1000 IU/ day for preterm infants in the first months of life.8 The World Health Organization recommends 400 IU to 1000 IU/ day of vitamin D supplementation in low PEDIATRICS Volume 133, Number 3, March 2014

birth weight infants.17 Lack of evidence and the consequent disparate recommendations have resulted in varying practices among treating physicians. Therefore, we planned this randomized trial to determine the optimal dose of vitamin D supplementation in preterm infants.

appearing bottles containing 2 different strengths of vitamin D preparations as per random allocation. Amber-colored bottles containing identical-appearing drug suspensions ensured blinding of investigator and parents.

Theobjectiveofthisstudywastocompare the prevalence of VDD (serum 25[OH]D levels ,20 ng/mL2) at 40 weeks’ postmenstrual age (PMA) in preterm infants born at 28 to 34 weeks’ gestational age (GA) and randomly assigned to receive either 800 or 400 IU of vitamin D3/day.

Intervention

METHODS Subjects and Setting We conducted this randomized doubleblind trial in a level 3 neonatal unit in North India between August 2011 and March 2012. All preterm infants born between 28 and 34 weeks’ GA and receiving at least 100 mL/kg per day of enteral feedings by 2 weeks’ postnatal age were eligible for inclusion in the study. Infants with major malformations, those who received parenteral nutrition for $2 weeks, or those born to mothers receiving phenytoin therapy or with HIV infection were excluded. GA was ascertained by using either the first day of the last menstrual period, the first-trimester ultrasound, or Expanded New Ballard Score.18 Eligible infants were stratified by GA into 2 strata: 280/7 to 316/7 weeks and 320/7 to 346/7 weeks. Informed written consent was obtained from the parents before enrollment. The study was approved by the Ethics Committee of All India Institute of Medical Sciences (AIIMS).

The intervention was started after drawing a blood sample from the infants for estimation of serum 25(OH)D, parathyroid hormone (PTH), calcium, phosphorus, and alkaline phosphatase (ALP). The study drug preparations (Basic Human Health Care Private Ltd, Delhi, India) were tested for vitamin D3 content at Sigma Testing and Research Center, Delhi, a nationally accredited laboratory. It was found that the high-dose preparation (800 IU/mL) contained 859 IU/mL and the low-dose preparation (400 IU/ mL) contained 422 IU/mL of vitamin D3. The drug bottles were labeled with a unique study enrollment number. One milliliter of drug measured to the accuracy of 0.1 mL was administered once daily to the infant either directly or mixed in expressed breast milk. Mothers were counseled for exclusive breastfeeding at discharge. In those infants who were also receiving human milk fortifier in breast milk or formula feedings, the dose of the study drug was reduced as per the vitamin D content of human milk fortifier or formula, so that the total intake did not exceed 800 or 400 IU/day in the respective groups. We ensured compliance of the drug intake by asking the mother to maintain a daily diary of doses administered after discharge.

Randomization

Outcome Variables

Infants in both strata were randomly assigned to receive oral vitamin D3 at a dose of 800 or 400 IU/day. We used computer-generated random numbers to allocate infants to 1 of the study groups with a fixed block size of 4. Random allocation was concealed by assigning sequential numbers to identical-

The primary outcome variable was prevalence of VDD at the PMA of 40 6 2 weeks. Secondary outcomes included the following: VDD at the corrected age (CA) of 3 months; serum levels of calcium, phosphorus, ALP, and PTH at a PMA of 40 6 2 weeks and at 3 months’ CA; the proportion of infants with vitamin D e629

excess, nephrocalcinosis, or hypercalciuria (urine calcium:creatinine ratio [UCa/Cr] .0.8 mg/mg) at 40 weeks and at 3 months’ CA; and bone mineral content (BMC) and bone mineral density (BMD) measured by dual-energy radiograph absorptiometry (DXA) at 3 months’ CA. VDD was defined as serum 25(OH)D levels ,20 ng/mL,16 severe VDD as levels ,5 ng/mL, and vitamin D excess as levels of 100 to 150 ng/mL.2

that were normally distributed, whereas Wilcoxon rank-sum test was used for skewed data. Categorical variables were analyzed by using x2 test or Fisher’s exact test. We also calculated the relative risk (RR), risk difference, and number needed to treat (NNT) with 95% confidence intervals (CIs) for categorical variables and mean differences with 95% CIs for continuous variables. Sample Size

Outcome Measurement Serum 25(OH)D was assayed by an autoanalyzer (DiaSorin Liaison, Stillwater, MN) using a chemiluminescent tracer, with a measuring range of 4 to 150 ng/mL. Serum PTH levels were estimated by using electrochemiluminometric assay using a Cobas e411 (Roche Diagnostics, Basel, Switzerland) autoanalyzer, with a measuring range of 1.2 to 5000 pg/mL. Serum calcium, phosphorus, and ALP were estimated by colorimetric method using a Beckman Coulter Synchron-CX9 PRO clinical system (Beckman Coulter, Inc). Blood samples were analyzed for serum 25(OH)D and PTH on the same day. Sera were stored at 220°C for the subsequent analysis of calcium, phosphorus, and ALP. Whole-body BMC and BMD were measured with the Discovery Hologic device QDR, Wi series (Hologic), with infant-specific software using fan beam technology with a precision of 1%. Daily quality control of DXA was performed by using a spine phantom with an area of 54.7 cm2, a BMC of 51.4 g, and a BMD of 0.94 g/cm2. Statistical Analysis Statistical analysis was performed by using Stata 11.2 version (StataCorp, College Station, TX). Analysis was performed by intention to treat. Continuous data were expressed as means and SDs or medians with ranges, whereas categorical variables were expressed as proportions. Student’s t test was used for the analysis of continuous variables e630

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In an earlier study by Backström et al,19 mean (6SD) 25(OH)D levels at 6 weeks’ postnatal age were found to be 18.3 6 7.4 and 26.7 6 12.1 ng/mL in low-dose (#400 IU/day) and high-dose (960 IU/ day) groups, respectively. Assuming that the levels were normally distributed, we estimated that ∼70% to 75% of the infants in the low-dose group would be categorized as vitamin D–deficient (serum 25[OH]D ,20 ng/mL) at 6 weeks’ postnatal age. Assuming an estimate of prevalence of deficiency of 75% in the 400-IU group, we needed to enroll 40 infants per group to detect a 50% decrease in the prevalence of VDD after supplementation with 800 IU/day, with a power of 90% and an a error of 0.05. We planned to enroll 48 infants per group to account for loss to follow-up.

RESULTS Of the 1461 live births during the study period, 130 infants were born between 28 and 34 weeks’ gestation. After excluding 34 infants on the basis of prespecified criteria, we enrolled 96 infants in the study (Fig 1). Clinical and baseline characteristics of the study population were comparable between the 2 groups (Table 1). Study intervention was initiated in 94 infants because consent was withdrawn by 2parentssoonafter randomization.Of these 94 infants, 3 infants died before follow-up at 40 weeks (1 due to stage 3 necrotizing enterocolitis and 2 due to probable milk aspiration); another 4

infantswerelostto follow-up.Thus, atotal of 87 infants (95.6% of those who survived) were available for follow-up at 40 6 2 weeks. At 3 months’ CA, 80 infants (88.9% of those who survived) were available for analysis because 6 infants were lost to follow-up and 1 infant had died due to sudden infant death (Fig 1). Baseline Characteristics VDD at baseline in the 800-IU and the 400IU groupswas 79% and 83%, respectively (P = .57). Serum levels of 25(OH)D, PTH, calcium, phosphorus, and ALP were also comparable at baseline (Table 1). There was no difference in exclusive breastfeeding rates in both groups at 40 weeks’ PMA (78.6% vs 64.4%; P = .15) and at 3 months’ CA (67.5% vs 50.0%; P = .12). Regular oral vitamin D intake (#500 IU/day) by mothers was high at 40 weeks (97.6% vs 84.4%; P = .06), whereas at 3 months’ CA it decreased (56.4% vs 53.8%; P = .82) in both groups. Primary Outcome The proportion of infants with VDD was significantly lower in the 800-IU group than in the 400-IU group at 40 weeks (38.1% vs 66.7%; RR: 0.57; 95% CI: 0.37– 0.88; P , .01). The NNT was 4 (95% CI: 2– 12). Serum 25(OH)D levels at 40 weeks were significantly higher in the 800-IU group compared with the 400-IU group (median [range]: 22.6 [6.2–98.4] vs 15.7 [4–43.1] ng/mL; P , .001) (Table 2, Fig 2). Secondary Outcomes VDD at 3 Months’ CA Serum 25(OH)D levels increased further and the proportion of infants with VDD decreased from 40 weeks to 3 months’ CA in both groups. The prevalence of VDD was again lower in the 800-IU group (12.5% vs 35%; RR: 0.36; 95% CI: 0.14– 0.90; NNT: 4; 95% CI: 3–20) (Table 2). Markers of Vitamin D Status Serum levels of PTH, calcium, phosphorus, and ALP were not significantly

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80) at 3 months’ CA, and it was comparable in both groups. There was no difference in the proportion of infants with UCa/Cr .0.8 at 40 weeks (64% vs 45%) and at 3 months’ CA (15.4% vs 20.8%) (Table 3). None of the infants had nephrocalcinosis on renal ultrasound at 40 weeks and at 3 months’ CA (Table 3).

DISCUSSION This randomized trial of vitamin D supplementation was planned to determine the optimal dose of daily vitamin D supplementation required to achieve sufficient vitamin D levels in preterm infants and to study the effect of supplementation on bone mineralization.

Vitamin D Toxicity Profile

Despite the fact that the prevalence of VDD was comparable in infants in both groups at baseline, a significant decline in VDD was observed at 40 weeks and at 3 months in the 800-IU group. This improvement in VDD status at 40 weeks could be attributed solely to daily supplementation with 800 IU of vitamin D given that breast milk probably is a poor source of vitaminD due tothe high prevalence of VDD in lactating mothers in India6 and de novo synthesis from sun exposure is not a reliable source of vitamin D in infants. More than one-third of infants in the 800-IU group were still vitamin D–deficient at 40 weeks, raising concerns that even 800 IU of vitamin D daily may be inadequate in achieving sufficient vitamin D levels by 40 weeks, at least in some infants. This finding was probably due to the duration of vitamin D supplementation being not long enough, in the presence of low baseline serum 25(OH)D levels.16 The efficacy and safety of a bolus dose of vitamin D in rapidly increasing serum 25(OH)D levels .20 ng/mL need to be studied in preterm infants, even though the clinical significance of maintaining sufficient vitamin D levels is uncertain.

UCa/Cr was measured in 66% (58 of 87) of infants at 40 weeks and in 63% (50 of

To our knowledge, this study is the first trialof thiskindwithanadequatesample

FIGURE 1

Study flow.

different between the groups at 40 weeks and 3 months’ CA (Table 3). Bone Mineralization DXAwasperformed in 85%(68of 80) ofthe infants at 3 months’ CA. BMC was comparable between the groups (mean 6 SD: 79.6 6 16.8 vs 84.7 6 20.7 g; P = .27). BMD also did not differ significantly between the groups (Table 3).

hypercalciuria, or nephrocalcinosis. Serum 25(OH)D level was in the deficiency range at baseline (16.9 ng/mL) and in the insufficiency range at 40 weeks (26.7 ng/ mL) in this infant. The infant was not receiving any supplements other than the trial drug, and there was no inadvertent excess intake of the drug at any time. We could not check the actual vitamin D content in the drug that the infant was receiving.

Vitamin D Excess One infant in the 800-IU group had serum 25(OH)D of 141 ng/mL at 3 months’ CA. However, there was no hypercalcemia,

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TABLE 1 Baseline Characteristics Characteristic Maternal Age, y Parity, n Gestational hypertension, n (%) Gestational diabetes mellitus, n (%) Receiving vitamin D supplementation at 40 weeks, n (%) Receiving vitamin D supplementation at 3 months, n (%) Neonatal GA, wk Male gender, n (%) Small for GA, n (%) Apgar score at 5 minutes Birth weight, g Length, cm Occipitofrontal circumference, cm Respiratory distress syndrome, n (%) Bronchopulmonary dysplasia, n (%) Patent ductus arteriosus, n (%) Shock requiring inotropes, n (%) Feeding intolerance, n (%) Sepsis (clinical and culture positive), n (%) Mechanical ventilation, n (%) Continuous positive airway pressure, n (%) Parenteral nutrition, n (%) Vitamin D status VDD (,20 ng/mL), n (%) Vitamin D severe deficiency (,5 ng/mL), n (%) Serum calcium, mg/dL Serum phosphorus, mg/dL Serum ALP, IU/L Serum PTH, pg/mL Age at baseline sampling and initiation of intervention, d

800-IU Group (n = 48)

400-IU Group (n = 48)

P

28.1 6 5.4 4 (1–7) 13 (28) 5 (10) 41 (98)

28.3 6 4.7 4 (1–6) 10 (21) 8 (17) 38 (84)

.87 .76 .47 .55 .06

22 (56)

21 (54)

.82

32.4 6 1.9 26 (54) 17 (35) 9 (8–9) 1655 6 411 41 6 3 29.4 6 2.1 7 (14) 2 (4) 3 (6) 4 (8) 8 (17) 6 (13) 6 (13) 15 (31) 8 (17)

32.5 6 1.8 28 (58) 12 (25) 9 (4–9) 1694 6 513 41.6 6 4 29.5 6 2.3 7 (14) 1 (2) 3 (6) 2 (4) 6 (13) 5 (10) 3 (6) 14 (29) 7 (15)

.78 .68 .52 .68 .68 .39 .91 .99 .62 .99 .68 .39 .72 .49 .5 .78

37 (79) 7 (15) 9.6 6 1.2 6.1 6 1.6 173 (89–1037) 78.7 (5.2–379) 3 (1–14)

40 (83) 6 (13) 9.5 6 1.1 6 6 1.8 150 (88–563) 83.7 (4.4–440) 3 (1–12)

.57 .73 .83 .88 .06 .87 .69

Data are presented as n (%), medians (range), or means 6 SDs.

TABLE 2 VDD and Vitamin D Severe Deficiency at 40 Weeks’ PMA and 3 Months’ CA Variable 40 6 2 weeks’ PMA n VDD (,20 ng/mL) Vitamin D severe deficiency (,5 ng/mL) 3 months’ CA n VDD (,20 ng/mL) Vitamin D severe deficiency (,5 ng/mL)

800-IU Group

400-IU Group

42 16 (38) 0

45 30 (67) 2 (4.4)

0.57 (0.37–0.88) 0.21 (0.01–4.33)

.008 .50

40 14 (35) 1 (2.5)

0.36 (0.14–0.90) 0.33 (0.01 to 7.94)

.02 .99

40 5 (12.5) 0

RR (95% CI)

P

Data are presented as n (%) unless otherwise indicated.

size that investigated the prevalence of VDD in preterm infants after daily vitamin D supplementation with 2 different doses. Previous randomized trials in preterm infants were conducted in smaller numbers of infants and found e632

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almost similar vitamin D levels in highand low-dose groups.19–21 These studies reported only serum vitamin D levels and not the prevalence of VDD and had large losses to follow-up. In contrast, our study had a high follow-up rate of

95.6% and was powered for the primary outcome. Improvement in serum vitamin D levels did not result in significant improvement in bone mineralization variables at 3 months. We presume 1 of the following 2 factors to be the possible reason for this result: (1) our study was not powered to determine any meaningful difference in the bone mineralization and/or (2) the duration of vitamin D supplementation was not long enough to produce any significant difference in these variables. Our finding is consistent with those of others that showed that achieving vitamin D sufficiency does not translate into better BMC or BMD.19,20 Nevertheless, marginally higher BMC and BMD were observed in infants in the 400-IU group. This finding was probably due to a higher mean birth weight of the infants in the 400-IU group because there is a positive association between birth weight and BMC.22 Lack of improvement in BMC could also be due to the fact that our study included a few (n = 7) very low birth weight infants (,1000 g), who are likely to have maximum changes in BMC and BMD due to higher risk of metabolic bone disease.23,24 Even though daily supplementation with 800 IU significantly reduced the prevalence of VDD at 40 weeks and at 3 months’ CA, there is concern that 800 IU/day could result in vitamin D excess in a small proportion of infants. We found an incidence of 2.4% (95% CI: 0.06%–12.5%) in the intervention group. The infant with vitamin D excess was, however, asymptomatic. As far as we know, the vitamin D excess in this infant was not due to inadvertent excess intake. Exaggerated response to vitamin D supplementation could be explained by genetic polymorphisms in enzymes involved in vitamin D metabolism.25,26 However, we did not investigate the infant from this angle.

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FIGURE 2 Vitamin D levels: change from baseline to 40 weeks’ PMA and 3 months’ CA.

TABLE 3 Secondary Outcome Variables at 40 Weeks’ PMA and 3 Months’ CA Variable 40 6 2 weeks’ PMA n Serum calcium, mg/dL Serum phosphorus, mg/dL Serum ALP, IU/L Serum PTH, pg/mL UCa/Cra UCa/Cr .0.8a, n (%) Nephrocalcinosis, n Weight, g Length, cm Occipitofrontal circumference, cm 3 months’ CA n Serum calcium, mg/dL Serum phosphorus, mg/dL Serum ALP, IU/L Serum PTH, pg/mL UCa/Crb UCa/Cr .0.8b, n (%) Nephrocalcinosis, n Weight, g Length, cm Occipitofrontal circumference, cm BMC, gc BMD, g/cm2c

800-IU Group

400-IU Group

Mean Difference (95% CI)

P

42 9.9 6 0.9 6.1 6 1.2 236 (89–572) 22.3 (5.5–223) 1 (0.03–6) 16 (64) 0 2489 6 496 47.6 6 3.3 33.3 6 1.34

45 9.9 6 0.8 6.3 6 1.1 276 (118–556) 27.2 (3.8–363) 0.71 (0.14–5) 15 (45) 0 2468 6 508 46.6 6 4.0 33.7 6 2.4

20.02 (20.34 to 0.39) 20.17 (20.69 to 0.34) — — — — — 21 (2191 to 234) 1.0 (20.6 to 2.5) 0.4 (20.5 to 1.2)

.89 .50 .34 .23 .46 .16 — .84 .21 .36

40 10.1 6 0.4 6.0 6 1.1 266 (83–875) 27.8 (3.4–91.7) 0.3 (0.08–2.3) 4 (15.4) 0 4770 6 820 57.0 6 3.4 38.0 6 1.82 79.6 6 16.8 0.152 6 0.019

40 10.1 6 0.4 0.05 (20.14 to 0.25) 6.2 6 1.4 20.24 (20.82 to 0.34) 236 (86–740) — 31. 1 (4.5–135.4) — 0.51 (0.08–2.3) — 5 (20.8) — 0 — 4825 6 1053 256 (2472 to 361) 57.8 6 4.0 20.8 (22.4 to 0.9) 38.6 6 1.7 20.6 (20.2 to 1.4) 84.7 6 20.7 25.1 (214.1 to 4.0) 0.158 6 0.021 20.005 (20.015 to 0.004)

.58 .42 .33 .48 .27 .72 — .79 .35 .12 .27 .26

Data are presented as n (%), medians (range), or means 6 SDs unless otherwise indicated. —, could not be estimated because the data is skewed or categorical, or there are no events in both the groups. a n = 25 and 33 in the 800-IU and the 400-IU groups, respectively. b n = 26 and 24 in the 800-IU and the 400-IU groups, respectively. c n = 36 and 32 in the 800-IU and the 400-IU groups, respectively.

Also, routine monitoring for markers of vitamin D toxicity, such as hypercalciuria and nephrocalcinosis, did not reveal any significant clinical adverse effects in the 800-IU group. Despite improving vitamin D levels, UCa/Cr declined from 40 weeks to 3 months’ CA in both groups, indicating maturation of PEDIATRICS Volume 133, Number 3, March 2014

tubular function and thus improved tubular reabsorption of calcium in preterm infants.27 The role of UCa/Cr as a marker of vitamin D toxicity needs further evaluation. The major limitation of our study is that we did not quantify exposure to sunlight in infants. It has been observed that

comparable sun exposure results in almost similar vitamin D levels in term infants supplemented with either 250 or 500 IU of vitamin D.28 Higher sun exposure can theoretically explain the reduced prevalence of VDD in the 800-IU group. However, this seems unlikely given the comparable baseline variables such as birth weight and gestation, and geographical location of the enrolled infants. Another limitation of the study is that we did not document the dietary intake of vitamin D by the mother, even though we did measure the intake in the form of supplements. Because the transfer of vitamin D through breast milk of unsupplemented mothers without adequate sun exposure is negligible,11,29 a significant difference, if any, in the dietary intake of vitamin D by the mothers in the 2 groups is unlikely to have changed our results.

CONCLUSIONS In preterm infants of 28 to 34 weeks’ gestation with significant deficiency at baseline, daily supplementation of vitamin D in doses of 800 IU compared with 400 IU appears to reduce the prevalence of VDD at 40 weeks’ PMA and at 3 months’ CA. A small but possibly significant risk of vitamin D excess underscores the need to monitor serum vitamin D levels in neonates receiving high doses of vitamin D daily. Moreover, the clinical significance of maintaining sufficient vitamin D levels in the absence of any demonstrable effect on bone mineralization needs to be studied in trials with a larger sample size.

ACKNOWLEDGMENTS We acknowledge the contributions by Dr Tapas Kumar Som for help extended during recruitment of the subjects, Mr Lalit Gupta for performing DXA scans, and Mrs Shiji Binu, Mr Leslie James, and Mr Bijoy Jose for assistance in analysis of blood samples. e633

REFERENCES 1. Holick MF. Vitamin D deficiency. N Engl J Med. 2007;357(3):266–281 2. Misra M, Pacaud D, Petryk A, Collett-Solberg PF, Kappy M; Drug and Therapeutics Committee of the Lawson Wilkins Pediatric Endocrine Society. Vitamin D deficiency in children and its management: review of current knowledge and recommendations. Pediatrics. 2008;122(2):398–417 3. Karatekin G, Kaya A, Salihoglu O, Balci H, Nuhoglu A. Association of subclinical vitamin D deficiency in newborns with acute lower respiratory infection and their mothers. Eur J Clin Nutr. 2009;63(4):473–477 4. McNally JD, Leis K, Matheson LA, Karuananyake C, Sankaran K, Rosenberg AM. Vitamin D deficiency in young children with severe acute lower respiratory infection. Pediatr Pulmonol. 2009;44(10):981–988 5. Agarwal R, Virmani D, Jaipal ML, et al; Investigators of LBW Micronutrient Study Group, Departments of Pediatrics and Endocrinology. Vitamin D status of low birth weight infants in Delhi: a comparative study. J Trop Pediatr. 2012;58(6):446–450 6. Jain V, Gupta N, Kalaivani M, Jain A, Sinha A, Agarwal R. Vitamin D deficiency in healthy breastfed term infants at 3 months & their mothers in India: seasonal variation & determinants. Indian J Med Res. 2011;133 (3):267–273 7. Agarwal N, Faridi MM, Aggarwal A, Singh O. Vitamin D Status of term exclusively breastfed infants and their mothers from India. Acta Paediatr. 2010;99(11):1671–1674 8. Agostoni C, Buonocore G, Carnielli VP, et al; ESPGHAN Committee on Nutrition. Enteral nutrient supply for preterm infants: commentary from the European Society of Paediatric Gastroenterology, Hepatology and Nutrition Committee on Nutrition. J Pediatr Gastroenterol Nutr. 2010;50(1):85–91 9. Thandrayen K, Pettifor JM. Maternal vitamin D status: implications for the development of infantile nutritional rickets. Endocrinol Metab Clin North Am. 2010;39 (2):303–320 10. Dawodu A, Nath R. High prevalence of moderately severe vitamin D deficiency in

11.

12.

13.

14.

15.

16.

17.

18.

19.

preterm infants. Pediatr Int. 2011;53(2): 207–210 Hollis BW, Roos BA, Draper HH, Lambert PW. Vitamin D and its metabolites in human and bovine milk. J Nutr. 1981;111(7):1240–1248 Hollis BW, Wagner CL. Vitamin D requirements during lactation: high-dose maternal supplementation as therapy to prevent hypovitaminosis D for both the mother and the nursing infant. Am J Clin Nutr. 2004;80 (6 suppl):1752S–1758S Wagner CL, Hulsey TC, Fanning D, Ebeling M, Hollis BW. High-dose vitamin D3 supplementation in a cohort of breastfeeding mothers and their infants: a 6-month follow-up pilot study. Breastfeed Med. 2006; 1(2):59–70 Kreiter SR, Schwartz RP, Kirkman HN Jr, Charlton PA, Calikoglu AS, Davenport ML. Nutritional rickets in African American breastfed infants. J Pediatr. 2000;137(2):153–157 American Academy of Pediatrics Committee on Environmental Health. Ultraviolet light: a hazard to children. Pediatrics. 1999;104(2 pt 1):328–333 Wagner CL, Greer FR; American Academy of Pediatrics Section on Breastfeeding; American Academy of Pediatrics Committee on Nutrition. Prevention of rickets and vitamin D deficiency in infants, children, and adolescents. Pediatrics. 2008;122(5):1142–1152 World Health Organization. Guidelines on optimal feeding of low birth weight infants in low- and middle-income countries. Geneva, Switzerland: World Health Organization; 2011. Available at: www.who.int/maternal_child_ adolescent/documents/infant_feeding_low_ bw/en/index.html. Accessed October 25, 2012 Ballard JL, Khoury JC, Wedig K, Wang L, EilersWalsman BL, Lipp R. New Ballard Score, expanded to include extremely premature infants. J Pediatr. 1991;119(3):417–423 Backström MC, Mäki R, Kuusela AL, et al. Randomised controlled trial of vitamin D supplementation on bone density and biochemical indices in preterm infants. Arch Dis Child Fetal Neonatal Ed. 1999;80(3): F161–F166

20. Backström MC, Mäki R, Kuusela AL, et al. The long-term effect of early mineral, vitamin D, and breast milk intake on bone mineral status in 9- to 11-year-old children born prematurely. J Pediatr Gastroenterol Nutr. 1999;29(5):575–582 21. Koo WW, Krug-Wispe S, Neylan M, Succop P, Oestreich AE, Tsang RC. Effect of three levels of vitamin D intake in preterm infants receiving high mineral-containing milk. J Pediatr Gastroenterol Nutr. 1995;21(2):182–189 22. Martínez-Mesa J, Restrepo-Méndez MC, González DA, et al. Life-course evidence of birth weight effects on bone mass: systematic review and meta-analysis. Osteoporos Int. 2013;24(1):7–18 23. Fewtrell MS. Does early nutrition program later bone health in preterm infants? Am J Clin Nutr. 2011;94(6 suppl):1870S–1873S 24. Hovi P, Andersson S, Järvenpää AL, et al. Decreased bone mineral density in adults born with very low birth weight: a cohort study. PLoS Med. 2009;6(8):e1000135 25. Signorello LB, Shi J, Cai Q, et al. Common variation in vitamin D pathway genes predicts circulating 25-hydroxyvitamin D Levels among African Americans. PLoS ONE. 2011;6 (12):e28623 26. Levin GP, Robinson-Cohen C, de Boer IH, et al. Genetic variants and associations of 25-hydroxyvitamin D concentrations with major clinical outcomes. JAMA. 2012;308 (18):1898–1905 27. Aladangady N, Coen PG, White MP, Rae MD, Beattie TJ. Urinary excretion of calcium and phosphate in preterm infants. Pediatr Nephrol. 2004;19(11):1225–1231 28. Siafarikas A, Piazena H, Feister U, Bulsara MK, Meffert H, Hesse V. Randomised controlled trial analysing supplementation with 250 versus 500 units of vitamin D3, sun exposure and surrounding factors in breastfed infants. Arch Dis Child. 2011;96 (1):91–95 29. Wagner CL, Howard C, Hulsey TC, et al. Circulating 25-hydroxyvitamin D levels in fully breastfed infants on oral vitamin D supplementation. Int J Endocrinol. 2010; 2010:235035

(Continued from first page) PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275). Copyright © 2014 by the American Academy of Pediatrics FINANCIAL DISCLOSURE: The authors have indicated they have no financial relationships relevant to this article to disclose. FUNDING: The study drug was procured through Indian Council of Medical Research grant 5/7/305/08-RHN. POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose.

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