1. 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

2. 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

3. 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

4. Do Preterm Infants Need Vitamin D? Calcium Absorption
3-day metabolic studies in 117 preterm infants
Vitamin D supplementation of 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

5. 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

6. 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.

7. 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

8. 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

9. 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

10. 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

11. 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

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

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

14. 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

15. 107.3 nmol/L 25(OH)D

16. 120.4 nmol/L 25(OH)D

17. 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
( , )

18. 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

19. 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

20. 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?

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

22. 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

23. 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..

24. 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

25. 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

26. Delving Deeper
Hanson C et al 2015

27. Delving Deeper
Hanson C et al 2015

28. Delving Deeper
Hanson C et al 2015

29. Delving Deeper
Hanson C et al 2015

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

31. 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