Department of Pediatrics1, Department of Public Health2 Functional Indicators of Vitamin D Adequacy for Very Low Birth Weight Infants Sarah N. Taylor1, Amy Wahlquist2, Carol L. Wagner1, Viswanathan Ramakrishnan2, Myla Ebeling1, Bruce W. Hollis1 Department of Pediatrics1, Department of Public Health2

Special Vitamin D Needs for Preterm Infants Vitamin D Deficiency in Pregnancy Preterm Birth Vitamin D Deficiency in Preterm Infant Newborn 25(OH)D 60-70% mothers

Special Vitamin D Needs for Preterm Infants 80% of fetal calcium and phosphorus accrued in 3rd trimester Metabolic Bone Disease of Prematurity Multi-factorial including vitamin D deficiency High oxidative stress Pro-inflammatory state Immune dysfunction

Do Preterm Infants Need Vitamin D? Calcium Absorption 3-day metabolic studies in 117 preterm infants Vitamin D supplementation of 1200-2000 IU/day Improved intestinal calcium absorption Metabolic studies in 103 preterm infants No association of vitamin D supplementation with intestinal calcium absorption No measurement of serum 25(OH)D status Senterre and Salle 1982; Bronner et al 1992

Do Preterm Infants Need Vitamin D? Bone Mineralization 2 studies using DEXA scan RCT of up to 400 IU/day vs. 960 IU/day No differences in bone mineralization by DEXA scan Low dose group achieved serum 25(OH)D 35 ng/ml Therefore, likely that both groups were vitamin D sufficient Observational study of pre- and post-discharge nutrition demonstrated phosphorus and male status were associated with sub-optimal bone mineralization Backstrom et al 1999; Kurl et al 2003

Purpose of Study Identify whether vitamin D status, as indicated by circulating 25(OH)D concentration, is associated with calcium homeostasis and bone mineralization in very low birth weight infants If so, is a specific threshold of 25(OH)D status related to optimizing these processes.

Methods Observational study of vitamin D status, calcium/phosphorus homeostasis, and bone mineralization Subject inclusion criteria Very low birth weight (VLBW) (<1500 g or 3.3 lbs) Appropriate for gestational age (AGA) infants Born at a single tertiary obstetrics/neonatal center Maternal race identification as black or white Twin and triplet pregnancies included

Methods At birth, 1-month age and term age (at expected due date 40 weeks gestation) Measurement of serum 25(OH)D, calcium, phosphorus, intact parathyroid hormone, alkaline phosphatase 25(OH)D and iPTH measurement by radioimmunoassay (Diasorin, Stillwater, MN) At 1-month and term age Measurement of urinary calcium and phosphorus excretion Normalized by creatinine excretion measurement At term age Measurment of bone mineral content and mineralization by dual energy X-ray (DEXA) scan

Methods Median (interquartile range) for each point value Association by Spearman’s Correlation Coefficients Scatter plot of montonic relationship Piecewise regression model To estimate the threshold of 25(OH)D status associated with leveling (plateau) of an indicator (PTH, femur bone mineralization) y is the outcome, x is 25(OH)D status, λ is the unknown threshold of 25(OH)D at the level where the trajectory potential plateaus α and ά are y-intercepts prior and β and β1 are the slopes threshold Identify 2 linear components and whether a threshold and 95% confidence interval within the range of the observed data

Results 97 infants enrolled 89 included in analysis (3 SGA, 3 exited or died in first week, 2 had fetal drug exposures with severe bone effect) Median (interquartile range) Birth weight: 1105 (900,1280) grams Birth Gestational age: 28 (26,29) weeks Daily vitamin D intake For postnatal month 1: 349 (234,517) IU/day For course of study: 609 (519,678) IU/day 62% black, 38% white 46% male

NM-not measured Birth 1 Month Term Age median IQR 25(OH)D, nmol/L 36.1 (26.9, 51.4) 80.8 (63.0, 101.0) 114.5 (89.8, 163.3) Serum calcium, mg/dl 9.0 (8.3, 9.6) 9.9 (9.7, 10.1) 10.0 (9.8, 10.3) Serum phosphorus, mg/dl 5.5 (4.5, 6.3) 6.7 (5.6, 7.0) (6.2, 7.0) PTH, pmol/L 295.2 (199,733.7) 232.9 (154.7,368.7) 197.1 (122.1, 273) Alkaline phosphatase, IU/L 231.0 (189.0, 308.0) 315.5 (263.0, 402.0) 338.0 (275.0, 406.0) Urine calcium excretion (mg/mg creat) NMb NM 0.5 (0.3, 0.9) 0.3 (0.2, 0.7) Urine phosphorus excretion (mg/mg) 0.8 (0.5, 1.3) 0.9 (0.5, 1.6) Lumbar spine BMC, g 0.819 (0.585, 1.267) Lumbar spine BMD, g/cm2 0.078 (0.067, 0.095) Femur BMC, g 1.673 (1.484, 1.883) Femur BMD, g/cm2 0.140 (0.133, 0.152) NM-not measured

r= -0.1 NS r=0.25 p=0.02 r=-0.17 NS r=0.23 p=0.04

r=-0.4 p<0.01 r=0.31 p<0.01 r=0.28 p=0.02 r=-0.0009 NS r=-0.002 NS r=-0.16 NS

r=-0.25 NS r=-0.41 p=0.003 r=-0.39 p=0.006 r=0.0005 NS r=0.36 p=0.005 r=-0.38 p=0.004 r=0.2 NS r= 0.12 NS r=0.29 p=0.01 r=0.26 p=0.02

107.3 nmol/L 25(OH)D

120.4 nmol/L 25(OH)D

25(OH)D (nmol/L) Before threshold Functional Indicator Threshold 95% CI Slope PTH 107.3 (80.5, 134.0) -0.6 (-0.9, -0.2) Femur BMC 120.4 (88.5, 152.4) 0.006 (0.002, 0.01) Femur BMD 116.4 (83.5, 149.3) 0.0003 (0.00006, 0.0006)

Bischoff-Ferrari et al Am J Med 2004 Summary PTH negatively correlated with 25(OH)D status at one-month and at term age Threshold of 107 nmol/L 25(OH)D identified as the level above which PTH plateaued. Term age femur BMC and BMD positively correlated with 25(OH)D (cortical bone) Threshold of 120 nmol/L and 116 nmol/L 25(OH)D, respectively identified as the levels above which bone mineralization plateaued. Bischoff-Ferrari et al Am J Med 2004 Heaney RP et al JACN 2003 Vieth R et al JCEM 2006

Lower circulating calcium Calcium normalization 25(OH)D Negatively Associated with Urinary Phosphorus Excretion and Positively Associated with Urinary Calcium Excretion Insufficient 25(OH)D Sufficient 25(OH)D Lower circulating calcium Calcium normalization Increased parathyroid hormone Decreased parathyroid hormone Increased calcium Increased phosphorus excretion Calcium remains normal Decreased phosphorus excretion Increased normal/high calcium Increased urinary calcium excretion High 25(OH)D

Next Steps Test identified thresholds in second population Have 68 subject cohort Does the positive association of 25(OH)D with calcium and phosphorus at 1-month represents A role of 25(OH)D in absorption? Improved delivery of all 3 nutrients? What are the active vitamin D metabolites for preterm infants? Radioimmunoassay did not measure epimers Should epimers be measured in preterm infants?

MUSC Pediatric Nutritional Sciences Center Financial Support: NIH/NCRR (K23 RR021891) NIH Clinical and Translational Science Award (CTSA) grant (UL1TR001450)

Randomize Trials of Preterm Infant Vitamin D Study Population Vitamin D doses (IU/day) 25(OH)D status achieved, ng/ml Fort et al 2016 (n=100) 23-27 weeks 0 (~200) Median 22 200 (~400) Median 39 800 (~1000) Median 84.5 Taylor et al 2016 (n=38) <1500 g, <34 weeks 0 (~520) Median 29 400 (~ 942) Median 33 Backstrom et al 1999 (n=16) <33 weeks 200 IU/kg (max of 400) Mean 32 960 Mean 45 Koo et al 1995 (n= 62) <1500 g Mean 161 Mean 26 Mean 361 Mean 766 Mean 31 Pittard et al 1991 (n=27 ) <2500g 400 Mean 51 800 Mean 49

Why? Poor absorption of oral vitamin D? Not likely High fat stores? Not likely Difference when evaluate IU/kg? No Assay differences? Potentially Hydroxylation by other enzymes? Perhaps Variation in vitamin D-binding protein? Hmm..

Assays Radioimmunoassay High performance liquid chromatography Liquid chromatography- mass spectrometry Differences in measurement of 25(OH)D epimers Commonly increased in pregnant women and neonates Potential bioactivity variation Other active metabolites? Hanson C et al 2016; Cooke DJ et al 2016; Aghajafrari et al 2015

Definitions 3-epi isomer 25(OH)D3 24, 25(OH)D3 vitamin D metabolite measured by some assays Lower affinity for vitamin D receptor, may have less biologic activity 24, 25(OH)D3 25(OH)D metabolite Potentially first step in inactivation of vitamin D Or, potentially action relating to bone/cartilage mineralization or PTH suppression

Delving Deeper Hanson C et al 2015

Delving Deeper Hanson C et al 2015

Delving Deeper Hanson C et al 2015

Delving Deeper Hanson C et al 2015

Plasma 3-epi-25(OH)D3 Measurement in Preterm Infants Ooms N et al Pediatr Res 2016

Free Vitamin D Decrease in vitamin D-binding protein from birth to NICU discharge Significant association of free vitamin D and bone mineralization at NICU discharge Vitamin D-binding protein has large number of polymorphism- Great potential for genetic variation in vitamin D health Hanson C et al JPEM 2015; FU L et al JSBMB 2016