*Senior authors

1. Effect of different dosages of oral vitamin D supplementation on vitamin D status in healthy, breastfed infants: A randomized trial Sina Gallo¹, Kathryn Comeau¹, Catherine Vanstone¹, Sherry Agellon¹, Atul Sharma², Glenville Jones3, Mary L’Abbé4, Ali Khamessan5, Celia Rodd¹²*, Hope Weiler¹* 1School of Dietetics and Human Nutrition, McGill University, Montréal, Québec 2Montreal Children’s Hospital, McGill University Health Centre, Montréal, Québec 3Departments of Biomedical & Molecular Sciences & Medicine, Queen’s University, Kingston, Ontario 4Department of Nutritional Sciences, University of Toronto, Toronto, Ontario 5Euro-pharm International Canada Inc., Montréal, Québec *Senior authors

2. Role of the Sponsor and Conflict of Interest Disclosures • Role of the Sponsor: The funding organizations had no role in design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript. • Canadian Institutes of Health Research, Nutricia Research Foundation and the Canadian Foundation for Innovation • Fonds de la Recherche en Santé du Québec doctoral scholarship • The Canada Research Chairs professor salary award • Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. AK is an employee of Europharm International Canada Inc. All other authors have no conflicts of interest.

3. Vitamin D Sources and Use: Infant Nutrition and Growth Maternal Sources: Sun, food and supplements Mother’s Skin 25(OH)D http://www.freedigitalphotos.net

4. Vitamin D Sources and Use: Infant Nutrition and Growth Maternal Sources: Sun, food and supplements Infant Sources: Maternal-fetal transfer, breast milk, supplements Infant Vitamin D Sources DBP Mother’s Skin CYP27A1 (liver) Infant Vitamin D Stores 25(OH)D http://www.freedigitalphotos.net

5. Vitamin D Sources and Use: Infant Nutrition and Growth Maternal Sources: Sun, food and supplements Infant Sources: Maternal-fetal transfer, breast milk, supplements Infant Vitamin D Sources DBP Mother’s Skin CYP27A1 (liver) Infant Vitamin D Stores 25(OH)D CYP27B1 (kidneys) DBP Healthy bone growth 1,25(OH)2D “Biologically Active” http://www.freedigitalphotos.net

6. Vitamin D Recommendations and Safety Levels: year 2006 Prevention of Rickets and for Healthy Infant Growth Recommendations Safety Levels (International Units) Health Canada; Canadian Paediatric Society Adequate Intake, Institute of Medicine USA; American Academy of Pediatrics Tolerable Upper Intake Level, Institute of Medicine USA Canadian Paediatric Society No observed adverse effect level, Institute of Medicine USA http://www.freedigitalphotos.net

7. Vitamin D Status based on Body Stores and Bone Health: year 2006 Rickets Arnaud 1976 “Severe”(Canada/US) Canadian Paediatric Society Recommendation ---------------------------- Based on adults and older children; but unclear in infants. Arnaud 1976 “Mild” (Canada/US) Cesur 2003 (Turkey) Molla 2000 (Kuwait) Dawodu 2005 (UAE) Garabedian 1983 (Belgium/France) Oginni 1996 Graff 2004 (Nigeria) Balasubraman 2003 (India) 25(OH)D concentrations (nmol/L)

8. The study was conducted with the objectives to: • establish a vitamin D dosage which would support 25(OH)D concentrations ≥ 75 nmol/L in 97.5% of breast fed infants • ≥ 50 nmol/L 75 nmol/L = 30 ng/mL 50 nmol/L = 20 ng/mL • further define the appropriate dosage using: • weight, length and head circumference growth; • the addition of mineral to growing bone. • Ethics: McGill University Institutional Review Board; • Health Canada Clinical Trials; • Trial Registration clinicaltrials.gov Identifier: NCT00381914.

9. Study Groups: Healthy Infants Assessed for eligibility (n=937) • Did not meet criteria (n= 275) • Contact declined or unavailable (n=185) • Other reason (n=345) Discontinued July 2008 - 81% of group pl. 25(OH)D >125 nmol/L after 2 mo Randomized (n=132) 400 IU/d (n=39) 800 IU/d (n=39) 1200 IU/d (n=38) 1600 IU/d (n=16) Follow-up 3 mo n=34 n=35 n=32 n=15 Follow-up 12 mo n=29 n=28 n=29 n=12 74% retention Analyzed as intent-to-treat

10. Trial Time Course • Infants were randomized to receive • 400, 800, 1200 or 1600 IU of vitamin D3 daily Baseline Visit 1 Visit 2 Visit 3 Visit 4 Visit 5 Visit 6 Recruitment 87% on vitamin D Birth • Baseline Characteristics • Mothers on average 33 y of age, 85% were white • High income (60% > Canadian average $75,000) • University educated mothers (89%) • Infants (58% males) • Born April-October (60%) 1 2 3 6 9 12 Age (months) http://www.freedigitalphotos.net

11. How many infants met the vitamin D status target of 75 nmol/L of 25(OH)D? 3 months 800 vs 400 IU OR 3.5 95% CI, 1.1-11 1200 vs 400 IU OR 9.7 95% CI, 1.9-49.7 *p<0.01 vs. 400 IU/d at same time; logistic regression at each time point

12. How many infants met the vitamin D status target of 75 nmol/L of 25(OH)D? 3 months 800 vs 400 IU OR 3.5 95% CI, 1.1-11 1200 vs 400 IU OR 9.7 95% CI, 1.9-49.7 *p<0.01 vs. 400 IU/d at same time; logistic regression at each time point

13. How many infants met the vitamin D status target of 75 nmol/L of 25(OH)D? 3 months 800 vs 400 IU OR 3.5 95% CI, 1.1-11 1200 vs 400 IU OR 9.7 95% CI, 1.9-49.7 *p<0.01 vs. 400 IU/d at same time; logistic regression at each time point

14. How many infants met the vitamin D status target of 50 nmol/L of 25(OH)D? Recommended Status Target IOM, AAP No differences among treatments over time by logistic regression at each time point

15. Assessment of Growth Mean ± 95% CI; No differences among treatments over time by repeated measures ANOVA

16. Assessment of Bone Health Mean ± SEM; No differences among treatments over time by repeated measures ANOVA accounting for race after adjustment for multiple comparisons

17. Vitamin D Recommendations and Safety Levels: year 2013 • 400 IU dosage: sufficient to achieve 50 nmol/L of 25(OH)D; • Higher dosages needed if target is 75 nmol/L of 25(OH)D; • No further benefits to growth or bone health of infants. Healthy Infant Nutrition: Public Policy & Position Statements for daily vitamin D supplementation dosages (International Units)

18. Vitamin D Recommendations and Safety Levels: year 2013 • 400 IU dosage: sufficient to achieve 50 nmol/L of 25(OH)D; • Higher dosages needed if target is 75 nmol/L of 25(OH)D; • No further benefits to growth or bone health of infants. Institute of Medicine Health Canada Canadian Paediatric Society American Academy of Pediatrics Canadian Paediatric Society Birth to 6 months Tolerable Upper Intake Level Institute of Medicine 6 to12 months Tolerable Upper Intake Level Institute of Medicine Healthy Infant Nutrition: Public Policy & Position Statements for daily vitamin D supplementation dosages (International Units)

19. Unanswered Questions: During and beyond infancy • Identify status targets • Benefits to bone health • Underpowered to detect early and longer-term benefits • Other health benefits • Needs of other population groups • Underrepresented darker skin pigmentation • Higher risk for deficiency • Remote geographic location • Infant born with low vitamin D stores

20. This work was made possible by the following: Funding Agencies Leading Institutions Recruitment and Monitoring Families and Infants • Recruitment • Five Pediatric Clinics West • Island & Greater Montreal Area • Lakeshore General Hospital • Safety Officer • Dr. J. Mitchell, M.D., F.R.C.P.C. http://www.freedigitalphotos.net